Mechanistic Studies of Radical Sam Enzymes in Cystobactamid

MECHANISTIC STUDIES OF RADICAL SAM ENZYMES IN CYSTOBACTAMID,

ANAEROBILIN AND THIAMIN BIOSYNTHESIS

A Dissertation

YUANYOU WANG

Submitted to the Office of Graduate and Professional Studies of Texas A&M University in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

Chair of Committee, Tadhg P. Begley Committee Members, Frank M. Raushel Coran M. H. Watanabe Craig D. Kaplan Head of Department, Simon W. North

May 2019

Major Subject: Chemistry

ABSTRACT

The radical S-adenosylmethionine (SAM) enzymes represent a large class enzyme that catalyzes a wide range of reactions, including chemically challenging reactions in critical pathways across all domains of life. Work here describes the detailed mechanistic studies of three of these enzymes in unusual methylation, ring cleavage, and rearrangement reactions.

CysS is a cobalamin-dependent radical SAM methyltransferase that catalyzes the iterative methylations on the 3-methoxy-4-aminobenzoic acid moieties of the antibiotic cystobactamids. We were able to reconstitute the activity of CysS in vitro and demonstrate that it can catalyze sequential methylations from a methyl group to form not only ethyl, isopropyl groups but also sec-butyl, t-butyl groups by a radical mechanism for the first time. To further elucidate the mechanism, different strategic substrate analogs were designed to trap the organic radical species, cobalamin intermediates and probe the radical substitution step. Based on all the isotope labeling studies and substrate analogs experiments, we were able to build a detailed mechanistic model to reveal how

CysS uses cobalamin, SAM, [4Fe− 4S] cluster to perform the unusual iterative methylations.

ChuW can catalyze a radical-mediated reaction to methylate and break the protoporphyrin ring of heme to release iron in an anaerobic heme degradation pathway.

We were able to design a cyclopropyl heme analog to trap radical generated on the

porphyrin ring by forming a porphyrin compound with cyclopropyl ring cleaved. Several tetrapyrrole ring and SAM adducts were also observed, which gives us further insight into the mechanism of the enzymatic porphyrin cleavage.

ThiC is a non-canonical radical enzyme that catalyzes one of the most complicated rearrangements in all of the mechanistic enzymology, which is the conversion of the 5-aminoimidazole ribonucleotide (AIR) to form 4-amino-5- hydroxymethyl-2-methylpyrimidine phosphate (HMP-P) in thiamin biosynthesis. Here, we were able to trap a four-carbon fragment from the ribose part of AIR by methionine during the enzymatic reaction. Using isotope labeling substrates and cofactors, we were able to show the intermediate leaks out after the first hydrogen atom abstraction but before the loss of C3′ of AIR.

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DEDICATION

To my parents, who brought me up to be what I am...

ACKNOWLEDGEMENTS

Ph.D. is an exciting journey towards unknown territory. However, it would be impossible to truly enjoy the incredible scenery without the people who inspired and motived me along the trip.

First and foremost, I would like to thank Dr. Tadhg Begley for being a remarkable advisor. It’s Dr. Begley who leads me to experience the beauty of the logic of chemistry thinking and its application into complicated biological questions, especially in the arrow pushing enzymatic mechanism and chemical logic in the reconstitution of biosynthesis pathway. I am grateful for the freedom and patience Tadhg gave me to work on some very challenging projects. I not only got the best enzymology and organic chemistry training from Tadhg but also was inspired by his enthusiasm and critical thinking for science during the years.

I would also like to thank my committee members, Dr. Frank Raushel, Dr. Coran

Watanabe and Dr. Craig Kaplan, for serving on my graduate committees. I am grateful for their insightful comments and encouragement during my seminar, presentations and preliminary examinations.

My sincere thanks also go to all my collaborators for the three projects I have been worked on. I would like to thank Prof. Rolf Müller Müller, Bastien, Sascha for providing the plasmids for the CysS project, Prof. Catherine Drennan, Jennifer, Percy for the crystallography collaboration, Prof. William Lanzilotta for the protein plasmids and all the discussion for the ChuW project. I also like to thank Prof. Coran Watanabe for

her help in the activation of the peptide carrier protein and Prof. Squire Booker for providing me with the cobalamin uptake plasmid.

During my Ph.D. study, there are many people who give me tremendous support.

I would like to thank Sameh for teaching me the “trick” in organic synthesis and how to obtain a “clean” NMR spectrum, Seth and Deepika from Barondeau lab for helping me to run the size exclusion column in their glove box, Dmytro for providing me with initial building blocks for the photoaffinity project, Jian for helping me to make different constructs for CysS, ChuW and microbiology experiments, Berkir for training me on the

ITC instrument, Angad for helping me to start with the radical SAM enzymes, Lisa for mentoring me in the initial molecular biology experiments.

Thanks to DJ for helping me troubleshooting during the years and Prem for suggestion from many aspects, Rung-yi and Niklamal for insightful suggestions in my early graduate studies, Yiquan and Yindrila for MS instrument, experiment training,

Nitai and Lina for their enthusiasm and encouragements in science, Sanjoy, Isita for their suggestion and discussion in flavin enzymes, Sumedh, for many stimulating discussions and advice on the radical SAM enzymes. I also enjoy the time with the energetic new students Anushree, Avick, Indranil, Saad, Sohan, Hannah in Begley lab.

Last but not the least, I would like to thank my family: my parents and my cousin for both spiritually and physically support and “tolerating” me throughout the graduate study.

CONTRIBUTORS AND FUNDING SOURCES

Contributors

This work was supervised by a dissertation committee consisting of Professor

Tadhg P. Begley (advisor), Professor Frank M. Raushel, and Professor Coran M. H.

Watanabe of the Department of Chemistry and Professor Craig D. Kaplan of the

Department of Biochemistry and Biophysics.

The CysS plasmids and the initial small molecules for the substrate screening of

CysS were obtained from Professor Rolf Müller in Helmholtz Center for Infection

Research. The CysG1 plasmids were provided by Bastien Schnell, Dr. Sascha Baumann in Professor Rolf Müller lab.

The origin ChuW plasmids, information about the substrates specificity of ChuW were provided by Professor William Nicholas Lanzilotta in the University of Georgia.

The labeling AIR substrate analogs were synthesized by Dr. Sameh Abdelwahed.

The crystallography study of CysS is collaborated with Prof. Catherine

Drennan’s lab.

All other work conducted for the dissertation was completed by the student independently.

Funding Sources

Graduate study was supported by a fellowship from Chemistry department at

Texas A&M University.

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This work was also made possible in part by the Robert A. Welch Foundation under Grant Number A-0034 and National Institutes of Health(NIH) under Grant

Number DK44083.

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NOMENCLATURE

5′-dA 5′-deoxyadenosine

ACP Acyl carrier protein

ABB 4-amino-3-butoxybenzoic acid

AIR Aminoimidazole ribotide

AMB 4-amino-3-methoxybenzoate acid

APB 4-amino-3-isopropoxybenzoic acid

B12 Cobalamin

CIP Calf intestine phosphatase

CO Carbon monoxide

CoA Coenzyme A

EPR Electron paramagnetic resonance

IRN Imidazole ribonucleotide

HMP-P 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate

HMP 4-amino-5-hydroxymethyl-2-methylpyrimidine

HPLC High performance liquid chromatography

LC-MS Liquid chromatography mass spectrometry

NMR Nuclear magnetic resonance

IPTG Isopropyl β-D-1-thiogalactopyranoside

LB Lysogeny broth

MeCbl Methylcobalamin

NADPH Nicotinamide adenine dinucleotide phosphate hydrate

NRP Nonribosomal peptide

PK Polyketides

SAM S-adenosyl-L-methionine

SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis

Tris•HCl Tris(hydroxymethyl)aminomethane hydrochloride

TABLE OF CONTENTS

Page

ABSTRACT ...... ii

DEDICATION ...... iv

ACKNOWLEDGEMENTS ...... v

CONTRIBUTORS AND FUNDING SOURCES ...... vii

NOMENCLATURE ...... ix

TABLE OF CONTENTS ...... xi

LIST OF FIGURES ...... xv

1. INTRODUCTION ...... 1

1.1. Radical S-Adenosylmethionine (SAM) Enzymes ...... 1 1.1.1. Non-canonical radical SAM enzymes ...... 4 1.1.2. Radical SAM methyltransferases ...... 5

2. RECONSTITUTION OF B12-DEPENDENT RADICAL SAM METHYLTRANSFERASE CYSS CATALYZED t-BUTYLATION BY ITERATIVE METHYLATION ...... 10

2.1. Introduction ...... 10 2.2. Result and discussion ...... 13 2.2.1. Reconstitution of CysS activity with minimal substrates ...... 13 2.2.2. Reconstitution of CysS activity with native substrates ...... 21 2.3. Conclusion ...... 23 2.4. Experimental methods ...... 24 2.4.1. overexpression and purification of CysS ...... 24 2.4.2. Iron and sulfide quantification of CysS ...... 25 2.4.3. In vitro reconstitution assay with CysS: ...... 26 2.4.4. LC-MS parameters for the analysis of the CysS reaction mixture ...... 31 2.4.5. Quantification of enzymatic products in the CysS reaction mixture ...... 32 2.4.6. General procedure for the synthesis of the 4-nitrobenzoic acids (2-32a, b, d) ...... 33 2.4.7. General Procedure for synthesis of 4-aminobenzoic acids (33a-d) ...... 35 xi

2.4.8. General Procedure for synthesis of S-pantetheinyl-4-aminobenzothioate (2-24a-d) ...... 38 2.4.9. Synthesis of AMB-CoA analog 2-27 ...... 40 2.4.10. Overexpression and purification of CysG1 ...... 41 2.4.11. Chemoenzymatic synthesis of the 4-amino-3-methoxybenzoate (AMB) CysG1 thioester 2-28 ...... 42 2.4.12. Reconstitution of the CysS-catalyzed methyl transfer reaction ...... 43

3. MECHANISTIC STUDIES OF SEQUENTIAL METHYLTIONS BY CYSS ...... 45

3.1. Introduction ...... 45 3.2. Result and discussion ...... 49 3.2.1. Measure methyl transfer rate with a radical clock probe ...... 49 3.2.2. Trapping a Cbl(I) intermediate by brominated substrate analog ...... 52 3.2.3. Identification of physiological cobalamin cofactor for CysS ...... 63 3.2.4. Measurement of the competitive and intrinsic isotope effect ...... 64 3.2.5. Efforts to obtain a crystal structure of CysS ...... 67 3.3. Conclusion ...... 68 3.4. Experimental methods ...... 71 3.4.1. Quantification of enzymatic products in the CysS catalyzed cyclopropyl substrate analog reaction ...... 71 3.4.2. Model reaction for generating the Cbl crosslinking species 2-50 ...... 72 3.4.3. Generation and derivatization of the Cbl crosslinking species in CysS catalyzed reaction ...... 72 3.4.4. Measurement of the competitive isotope effect of CysS ...... 72 3.4.5. Measurement of the intrinsic isotope effect of CysS ...... 73 3.4.6. Synthesis of (R)-S-pantetheinyl-4-amino-3-(cyclopropylmethoxy)- benzothioate ...... 74 3.4.7. Synthesis of (R)-S-pantetheinyl-4-amino-3-(1-cyclopropylethoxy)- benzothioate ...... 77 3.4.8. Synthesis of S-(2-acetamidoethyl) 4-amino-3-(2-bromoethoxy)- benzothioate ...... 79 3.4.9. Synthesis of (R)-S-pantetheinyl-4-amino-3-(methoxy- d3)-benzothioate..81 3.4.10. Synthesis of (R)-S-pantetheinyl-4-amino-3-(methoxy- d2)- benzothioate ...... 83 3.4.11. Synthesis of S-(2-acetamidoethyl) 4-amino-3-hydroxybenzothioate (2- 52) ...... 85 3.4.12. Synthesis of (R)-S-pantetheinyl-4-amino-3-hydroxybenzothioate (2- 46) ...... 85

4. TRAPPING RADICAL INTERMEDIATES IN AN ANEROBIC HEME DEGRADATION PATHWAY ...... 87

4.1. Introduction ...... 87

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4.2. Result and discussion ...... 90 4.2.1. Reconstitution of ChuW catalyzed heme degradation ...... 90 4.2.2. Design of cyclopropyl heme analog as a radical trap for ChuW catalyzed reaction ...... 93 4.2.3. Cyclopropyl heme analog as a substrate for ChuW ...... 95 4.2.4. Detection of protoporphyrin ring opening product in ChuW catalyzed cyclopropyl heme 4-6 degradation ...... 99 4.2.5. Detection of SAM adducts in ChuW catalyzed cyclopropyl heme degradation ...... 103 4.2.6. ChuW catalyzed cyclopropyl protoporphyrin degradation ...... 108 4.2.7. Proposed mechanisms for the ring opening product of ChuW catalyzed cyclopropyl substrates reaction ...... 116 4.2.8. Proposed mechanisms for the ring opening product of ChuW catalyzed heme degradation ...... 121 4.3. Conclusion ...... 122 4.4. Experimental methods ...... 122 4.4.1. Synthesis of cyclopropyl heme analog ...... 122 4.4.2. Overexpression and purification of ChuW ...... 125 4.4.3. In vitro activity assay with ChuW: ...... 126 4.4.4. HPLC parameters for the analysis of the ChuW reaction mixture ...... 127 4.4.5. LC-MS parameters for the analysis of the ChuW reaction mixture ...... 127

5. TRAPPING A FOUR-CARBON FRAGMENT IN THIC CATALYZED REARRANGEMENT IN THIAMINE BIOSYNTHESIS ...... 128

5.1. Introduction ...... 128 5.2. Result and discussion ...... 132 5.2.1. Identification of bound metabolites co-purifying with ThiC ...... 132 5.2.2. Detection of a four-carbon fragment in ThiC reaction by LC-MS/MS ...136 5.2.3. Identification of the methionine as the trapping agent for the four-carbon fragment of the AIR ...... 139 5.2.4. Determination of possible reference compounds for the detected shunt product in ThiC catalyzed reaction ...... 142 5.3. Conclusion ...... 146 5.4. Experimental methods ...... 147 5.4.1. Overexpression and purification of AtThiC ...... 147 5.4.2. Enzymatic synthesis of AIR from AIRs: ...... 148 5.4.3. Enzymatic assay of ThiC reaction ...... 148 5.4.4. Alkaline phosphate treatment of ThiC reaction ...... 149 5.4.5. HPLC parameters for the analysis of the ThiC reaction mixture ...... 149 5.4.6. LC-MS parameters for the analysis of the ThiC reaction mixture ...... 149

6. SUMMARY AND OUTLOOK ...... 151

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6.1. Summary ...... 151 6.2. Outlook ...... 151 6.2.1. B12 dependent radical SAM methyltransferase-CysS ...... 151 6.2.2. ChuW in anaerobic heme degradation pathway ...... 155 6.2.3. HMP-P synthase ThiC in thiamin pyrimidine biosynthesis ...... 156

REFERENCES ...... 158

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LIST OF FIGURES

Page

Figure 1.1 Reductive cleavage of SAM to the 5′-dA radical by [4Fe−4S] cluster .....1

Figure 1.2 Complex radical reactions catalyzed by radical SAM enzymes...... 3

Figure 1.3 Domain structures of different classes of RSMTs...... 6

Figure 1.4 Selected secondary metabolites whose biosynthesis include class B RSMTs that have been reconstituted in vitro...... 8

Figure 1.5 Class C radical SAM methyltransferases catalyzed modification...... 9

Figure 2.1 Mechanistic proposals for the formation of the t-butyl group in representative natural products...... 11

Figure 2.2 Proposed formation of branched alkoxy groups of cystobactamids by CysS-catalyzed iterative methylations of a methyl ether...... 12

Figure 2.3 (A) UV spectrum of purified CysS (B) SDS/PAGE analysis of purified CysS ...... 13

Figure 2.4 Identification of two substrates for CysS ...... 14

Figure 2.5 LC-MS detection of the CysS reaction products using methyl ether 2-21 and ethyl ether 2-22 ...... 15

Figure 2.6 LC-MS analysis of CysS-catalyzed iterative methylations of methyl ether 2-23...... 17

Figure 2.7 LC-MS detection of 5′-dA and SAH in the CysS-catalyzed iterative methylations of the methyl ether 2-23 ...... 19

Figure 2.8 MS analysis of a reaction mixture in which CH3-SAM is replaced with CD3-SAM showing CD3 incorporation into the ethyl and isopropyl ethers of 2-24a and 2-24b, respectively ...... 20

Figure 2.9 Proposal for CysS-catalyzed iterative methylations to form branched alkoxy groups ...... 21

Figure 2.10 Synthesis of the 4-amino-3-methoxybenzoate (AMB) CysG1 thioester ...... 22

Figure 2.11 The CysS-catalyzed reaction of the AMB-CysG1 thioester ...... 23

Figure 2.12 LC-MS detection of 2-24a in the CysS reaction using methyl ether 2-23 as substrate...... 27

Figure 2.13 LC-MS detection of 2-24b in the CysS reaction using methyl ether 2-23 as substrate...... 28

Figure 2.14 LC-MS detection of 2-24c in the CysS reaction using methyl ether 2-23 as substrate...... 29

Figure 2.15 LC-MS detection of the CysS reaction products using ethyl ether 2-24a (A), (B) or isopropyl ether 2-24b (C)...... 29

Figure 2.16 LC-MS detection of the CysS reaction products using isopropyl ether 2-24b as substrate ...... 30

Figure 2.17 LC-MS detection of 5¢-dA produced in the CysS reaction products using (A) methyl ether 2-23 or (B) CD3-methyl ether CD3-2-23 as substrate...... 31

Figure 2.18 MS analysis of AMB-CysG1 thioester 2-28 ...... 43

Figure 3.1 Selected secondary metabolites whose biosynthesis include class B RSMTs that have been reconstituted in vitro ...... 46

Figure 3.2 Proposed mechanism for CysS-catalyzed iterative methylations...... 48

Figure 3.3 CysS catalyzed reaction with cyclopropyl analog ...... 51

Figure 3.4 (A) Strategy of developing the brominated substrate analog to trap Cbl(I) species (B) Model reaction for cobalamin cross-linking species through Cbl(I) ...... 53

Figure 3.5 UV spectrum of monitoring the chemical reduction of from Cbl(III) to Cbl(I) ...... 54

Figure 3.6 Upper panel: MS of the substrate cobalamin adduct 2-50 in the model reaction; lower panel: MS/MS of the substrate cobalamin adduct 2-50 in model reaction ...... 55

Figure 3.7 CysS-catalyzed reaction with brominated analog 2-49 ...... 56

Figure 3.8 LC-MS detection of the CysS-catalyzed reaction with brominated analog 2-49 and the model reaction...... 57

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Figure 3.9 LC-MS detection of the CysS-catalyzed reaction with brominated analog 2-49 and the model reaction. (A) MS spectrum at 24 min. (B) Mass fragments of 805.84...... 57

Figure 3.10 UV spectrum of CysS catalyzed reaction with (blue) and without (red) exposure to light...... 58

Figure 3.11 LC-MS detection of 2-52 in the CysS reaction using brominated analog 2-49 as substrate...... 59

Figure 3.12 LC-MS detection of the CysS reaction products using brominated analog 2-49 as substrate ...... 60

Figure 3.13 HPLC spectrum of derivatization of CysS catalyzed reaction using brominated analog 2-49 as substrate by cyanide ...... 61

Figure 3.14 Proposed orientation of adduct and cofactors in the CysS active sites ....62

Figure 3.15 CysS catalyzed methylation with (A) CD3Cbl, SAM (top) or CD3 SAM, CH3Cbl (bottom) in the presence of reducing system...... 64

Figure 3.16 The strategy of intrinsic isotope effect measurement ...... 66

Figure 3.17 (A) LipA in the biosynthesis of lipoic acid; (B) The conversion of dethiobiotin to biotin catalyzed by biotin synthase (BioB)...... 67

Figure 3.18 Proposed model for CysS catalyzed sequential methylation...... 69

Figure 3.19 calibration curves for the ring opening products and the methylated product ...... 72

Figure 4.1 The heme uptake operon in Escherichia coli O157:H7 ...... 88

Figure 4.2 ChuW-catalyzed degradation of heme anaerobically ...... 89

Figure 4.3 UV spectrum of the time course of the ChuW catalyzed heme degradation...... 91

Figure 4.4 LC-MS analysis of ChuW catalyzed heme degradation. Upper panel: EICs of 5′-dA [M+H]+ (252.11±0.02). Lower panel: EICs of SAH [M+H]+ (385.12±0.02)...... 92

Figure 4.5 HPLC analysis of ChuW catalyzed heme degradation...... 92

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Figure 4.6 LC-MS analysis of ChuW catalyzed heme degradation. Upper panel: EICs of anaerobilin [M+H]+ (577.28±0.02). Lower panel: mass spectrum of anaerobilin at 15 min...... 93

Figure 4.7 Synthetic route of cyclopropyl heme analog ...... 95

Figure 4.8 UV spectrum of the time course of the ChuW catalyzed cyclopropyl heme degradation...... 96

Figure 4.9 LC-MS analysis of ChuW catalyzed cyclopropyl heme 4-6 degradation. EICs of 5′-dA [M+H]+ (252.11±0.02)...... 96

Figure 4.10 HPLC analysis of ChuW catalyzed cyclopropyl heme degradation under UV of 500 nm...... 97

Figure 4.11 LC-MS analysis of ChuW catalyzed cyclopropyl heme degradation ...... 98

Figure 4.12 Mass spectrum of ChuW catalyzed cyclopropyl heme degradation 13 product at 15 min with (A) CD3-SAM or (B) CH3-SAM...... 99

Figure 4.13 HPLC analysis of ChuW catalyzed cyclopropyl heme degradation...... 100

Figure 4.14 HPLC analysis of stability of products in ChuW catalyzed cyclopropyl heme degradation over time under 400 nm ...... 101

Figure 4.15 LC-MS analysis of ChuW catalyzed cyclopropyl heme degradation. ...102

Figure 4.16 LC-MS analysis of ChuW catalyzed cyclopropyl heme degradation. Upper panel: EICs of [M+2H]2+ (502.22±0.02). Lower panel: mass spectrum of the porphyrin cross-linking species 4-10 at 12.5 min...... 103

Figure 4.17 LC-MS analysis of ChuW catalyzed cyclopropyl heme degradation. Upper panel: EICs of [M+H]+ (902.40±0.02). Lower panel: massspectrum of the degradation porphyrin cross-linking species 4-11 at 13.5 min...... 104

Figure 4.18 Mass spectrum of the porphyrin cross-linking species (503.22) when 13 ChuW used (upper panel) CD3-SAM, (lower panel) CH3-SAM as cofactor...... 105

Figure 4.19 Mass spectrum of the porphyrin cross-linking species 4-10 (1003.45) 13 when ChuW used (upper panel) CD3-SAM, (lower panel) CH3-SAM as the cofactor...... 105

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Figure 4.20 Mass fragmentation of the porphyrin cross-linking species (502.23) when ChuW used (upper panel) CH3-SAM, (middle panel) CD3-SAM, 13 (lower panel) CH3-SAM as cofactor...... 106

Figure 4.21 Mass spectrum of the degradation porphyrin cross-linking species 4-11 (902.40) when the ChuW used (upper panel) CD3-SAM, 13 (lower panel) CH3-SAM as cofactor...... 107

Figure 4.22 The general scheme of SAM radical generated in the Class C radical SAM enzyme...... 108

Figure 4.23 Mass spectrum of 5'-dA when ChuW used (upper panel) CD3-SAM, 13 (lower panel) CH3-SAM as the cofactor...... 108

Figure 4.24 LC-MS analysis of ChuW catalyzed cyclopropyl protoporphyrin degradation. EICs of 5'-dA [M+H]+ (252.10±0.02)...... 109

Figure 4.25 HPLC analysis of ChuW catalyzed cyclopropyl protoporphyrin 4-12 degradation under 455 nm UV...... 110

Figure 4.26 LC-MS analysis of ChuW catalyzed cyclopropyl protoporphyrin degradation...... 111

Figure 4.27 LC-MS analysis of ChuW catalyzed cyclopropyl protoporphyrin degradation. Upper panel: EICs of the reduced form of ring cleavage product [M+H]+ (607.33±0.02). Lower panel: mass spectrum of the reduced form of ring cleavage product 4-13 at 12 min...... 111

Figure 4.28 LC-MS analysis of ChuW catalyzed cyclopropyl protoporphyrin degradation. EICs of the porphyrin cross-linking species [M+H]+ (502.23±0.02)...... 112

Figure 4.29 Mass spectrum of the porphyrin cross-linking species (502.23) when the ChuW used (upper panel) CH3-SAM, (middle panel) CD3-SAM, (lower panel) D4-SAM as cofactor...... 113

Figure 4.30 LC-MS analysis of ChuW catalyzed cyclopropyl protoporphyrin degradation. Upper panel: EICs of the degradation product of porphyrin cross-linking species [M+H]+ (902.40±0.02)...... 114

Figure 4.31 LC-MS analysis of ChuW catalyzed cyclopropyl protoporphyrin degradation using titanium citrate as reducing agent...... 115

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Figure 4.32 Mass spectrum of the cross-linking species in ChuW catalyzed cyclopropyl protoporphyrin degradation using titanium citrate as reducing agent...... 116

Figure 4.33 Proposed mechanism 1 for how cyclopropyl heme substrate analog 4-6 trap radical intermediate by formation of porphyrin ring opening product in the ChuW catalyzed reaction...... 118

Figure 4.34 Proposed mechanism 2 for how cyclopropyl heme substrate analog 4-6 trap radical intermediate by formation of porphyrin ring opening product in the ChuW catalyzed reaction...... 119

Figure 4.35 Proposed mechanism for the formation of the crosslinking species in ChuW catalyzed cyclopropyl heme degradation...... 120

Figure 4.36 Proposed mechanism of ChuW catalyzed heme degradation...... 121

Figure 5.1 ThiC catalyzed rearrangement from AIR to HMP-P ...... 129

Figure 5.2 Proposed mechanism for ThiC catalyzed AIR 5-1 to HMP-P 5-2 rearrangement...... 131

Figure 5.3 HPLC analysis of bound metabolites in ThiC under 250 nm. UV spectrum of different bound metabolites metabolites...... 133

Figure 5.4 HPLC analysis of bound metabolites in the ThiC mutant C474S under 250 nm...... 134

Figure 5.5 Upper panel: UV spectrum of guanosine and adenosine...... 135

Figure 5.6 LC-MS analysis of ThiC catalyzed AIR to HMP rearrangement...... 136

Figure 5.7 Isotope labeling pattern in 204.10 shunt product in ThiC catalyzed reaction...... 137

Figure 5.8 Isotope labeling pattern in 204.10 compound from different isotope labeling SAM...... 140

Figure 5.9 Isotope labeling pattern in 204.10 compound from different isotope labeling SAM...... 141

Figure 5.10 Isotope labeling pattern in 204.10 compound when excess of D8- methionine was added to the ThiC catalyzed reaction...... 142

Figure 5.11 Possible structures for the shunt product...... 143

Figure 5.12 The distance between sulfur atom of SAH and the 5′C of the AIR in ThiC crystal structure (4S28) ...... 143

Figure 5.13 Mass fragmentation of the shunt product (204.1053) when ThiC catalyzed reaction with SAM, AIR, dithionite and methionine (upper panel), D8-methionine (lower panel)...... 144

Figure 5.14 Possible structures for the shunt product...... 145

Figure 5.15 Mass fragmentation of the shunt product (204.1053) in ThiC catalyzed reaction when substrate is (A) 2′-13C-AIR; 13 2 (B) 1′,2′,3′,4′,5′- C5-AIR; (C) 4′- H-AIR...... 145

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1. INTRODUCTION

1.1. Radical S-Adenosylmethionine (SAM) Enzymes

Radical SAM enzymes, first classified as a superfamily in 2001,1 utilize a

[4Fe−4S] cluster and SAM to initiate a diverse set of radical reactions that are otherwise difficult or impossible to catalyze by polar mechanisms. Radical SAM enzymes usually involve H-atom abstraction from an inactivated C−H bond via generation of a 5′- deoxyadenosyl radical (5′-dA•) intermediate by reductive [4Fe−4S] cluster (Figure 1.1).

Compared with iron(IV)-oxo-derived radicals, radical generated by hydrogen abstraction from 5′-dA is more persistent because iron-bound oxygen species is not available.2 Since the high intrinsic reactivity of organic radicals, radical SAM enzymes can catalyze a wide range of reactions, including oxidation, methylation, double-bond addition, and fragmentation reactions.3

O NH O 2 NH O 2 N O 1+ N N N S Fe NH2 2+ S Fe NH2 S N S Fe S N Fe S N N Fe S H2C Fe S O O S Fe S Fe OH OH OH OH S-Adenosyl methionine (SAM) 5’-deoxyadenosyl radical (5ʹ-dA•) 1-1 1-2

Figure 1.1 Reductive cleavage of SAM to the 5′-dA radical by [4Fe−4S] cluster

For example, many radical SAM enzymes are involved in complicated cofactor biosynthesis (Figure 1.2). The thiamin pyrimidine synthase (ThiC) catalyzes a complex rearrangement of aminoimidazole ribotide (AIR) to form hydroxymethylpyrimidine phosphate (HMP-P) in thiamin biosynthesis.4 Hydroxybenzimidazole synthase (BzaF) carries out a similar rearrangement of AIR to form 5- hydroxybenzimidazole (HBI) in cobalamin biosynthesis.5 Aminofutalosine synthase (MqnE) catalyzes the addition of the adenosyl radical to the double bond of 3-[(1-carboxyvinyl)oxy]benzoic acid to form

6-7 aminofutalosine in the menaquinone biosynthesis. F0-synthase (CofG/CofH or FbiC) cleaves tyrosine and couples it to pyrimidinedione to give the deazaflavin chromophore

8 in coenzyme F420 biosynthesis. MoaA and MoaC catalyze the rearrangement of GTP into a cyclic pyranopterin monophosphate (cPMP) in molybdopterin biosynthesis.9-10

A NH H 2 H H H N H O ThiC N 2- O N H OPO3 2- solv H CO O3PO H H H N H H OH NH2 solv HO OH 1-6 Hsolv 1-5 AIR HMP-P 1-3 1-4 B H N H O H O N BzaF N OH Hsolv 2-O PO 3 H OH NH2 N H HO OH H 1-5 AIR H 1-3 1-7

C NH2 NH2 N N N N COOH COOH N N N N MqnE H C O 2 O CO2 O COO OH OH OH OH O 1-8 1-2 1-9

D O O O H2N NH NH OH CofG/CofH HN COOH NH HN N O HO N N O NH3 HO 2 H HO or FbiC HO 1-10 1-11 1-12 1-13 1-14 HO HO OH OH

HO HO

O N PPPO N O O O OH H H MoaA MoaC O O N HO P NH OH N NH O O N N NH2 H2N H H 1-15 1-16

Figure 1.2 Complex radical reactions catalyzed by radical SAM enzymes. (A) ThiC in thiamin biosynthesis (B) BzaF in cobalamin biosynthesis (C) MqnE in menaquinone biosynthesis (D) CofG/CofH in F420 biosynthesis (E) MoaA-MoaC in molybdopterin biosynthesis.

The radical SAM superfamily has grown rapidly since 2001 and now contains almost 113,000 unique sequences that represent more than 65 distinct reactions.11

Around 50,000 of the sequences relate to proteins have unknown function. Based on the radical SAM enzyme’s capability of catalyzing unusual reactions, it’s considered as a new treasure for the discovery of novel enzymatic transformations and biochemical pathways.

1.1.1. Non-canonical radical SAM enzymes

For most of the racial SAM enzymes that have been identified, their sequences contain a consensus CX3CX2C motif where each of the three cysteines coordinates with one of the three irons in [4Fe–4S] cluster. The fourth iron coordinates with the amino and carboxylate groups of SAM in a bidentate fashion. However, there are several enzymes that do not contain CX3CX2C motif but still generate a 5′-deoxyadenosyl radical to catalyze the reactions, such as ThiC (CX2CX4C), PhnJ (CX2CX21CX5C), QueE

4, 12-13 (CX14CX2C).

Besides the difference of the conserved sequence for binding the [4Fe–4S] cluster, ThiC also lacks the classic SAM binding motifs.14 From the ThiC crystal structure with SAH or 5′-dA and L-methionine, it is suggested that in ThiC active site,

SAM anchors to an additional active site metal through its amino and carboxylate groups instead of the iron in [4Fe–4S] cluster.15 It’s still not clear whether the captured conformation is in the catalytic cycle and whether this extra iron helps ThiC to catalyze such complicated rearrangement. What’s more, the [4Fe–4S] cluster-binding loop of

ThiC is at the dimer interface of the C-terminal instead of N-terminal for the classic

radical enzymes. This swapped structure is most resembled the adenosylcobalamin

(AdoCbl)-dependent enzymes (like glutamate mutase), which indicates an evolutionary relationship between the two super-families.

Dph2 is another non-canonical radical SAM enzyme that uses reduced [4Fe-4S] cluster and SAM, but generates a 3-amino-3-carboxypropyl radical instead of a 5′- deoxyadenosyl radical in the diphthamide biosynthesis. It is shown that Dph2 adopts a completely unrelated tertiary structure for [4Fe-4S] cluster binding and C�,Met of SAM is

16 closer to the unique Fe than the SMet in the classical radical SAM enzymes. This structure orientation helps to explain why SAM is reductively cleaved to form 3-amino-

3-carboxypropyl radical instead of a 5′-deoxyadenosyl radical.

1.1.2. Radical SAM methyltransferases

Before radical SAM enzyme has been classified as a superfamily, SAM was best known as a natural methyl donor in many important biological processes, including regulation of gene expression, protein modification, RNA processing.17 In classical methyltransferase (MTase) reactions, substrates need strong nucleophiles such as oxygen, nitrogen or sulfur to attack the methyl group of SAM, which is electrophilic due

18 to the adjacent sulfonium of SAM. However, this nucleophilic SN2 methylation mechanism cannot apply to the saturated carbon atoms or electrophilic substrates.

Alternatively, nature uses radical mechanisms to catalyze these unusual reactions.

To date, all the known enzymes that catalyze radical-mediated methylation belong to the radical SAM superfamily. Sequence analysis reveals that the currently identified radical SAM methyltransferases (RSMTs) can be grouped into several classes

(Figure 1.3).19-20 Class A RSMTs have a single conserved radical SAM domain and use two conserved cysteine residues to methylate sp2-hybridized carbon atoms. Class B

RSMTs include a cobalamin binding domain (CBD) at the N-terminal as well as a radical SAM domain at the C-terminus. Class C RSMTs has high sequence similarity with HemN at the C-terminus in addition to the classic TIM barrel radical SAM domain.

Currently, RlmN and Cfr in the class A RSMTs are the most well-studied methyltransferases in radical SAM family.20 There are more and more reconstitutions and mechanistic studies for Class B RSMTs while there are few reconstituted examples for Class C RSMTs.

Class A Radical SAM RlmN, Cfr

Cobalamin GenK, ThnK Class B Radical SAM (B12) PoyC, CysS

NosN, TbtI Class C Radical SAM HemN YtkT, ChuW

Figure 1.3 Domain structures of different classes of RSMTs.

As the data shown in 2017, the number of total radical SAM sequences are nearly

255 000. Among them, around 7000 radical SAM proteins contain CBDs based on

bioinformatics.21-22 Currently, class B RSMTs represents the largest class of RSMTs, which can catalyze a wide range of reaction including phosphinates, unactivated sp3- hybridized carbons, and sp2-hybridized carbons. Van der Donk lab first proposed that methylation can be performed from methylcobalamin (MeCbl) to the substrate radical

23 for these B12 dependent methyltransferases more than ten years ago. However, the poor solubility upon overexpression and the substrate complexity have hampered the reconstitution and detailed in vitro studies of these enzymes for a long time. Recently,

Booker’s lab is able to develop a strategy to enhances the uptake of cobalamin into the cytoplasm and improves the solubility of the target enzymes significantly by co- overexpression of Cbl uptake protein.24 Some of the earlier pioneer work in this area includes PhpK for the phosphinic acid P-methylation,25 GenK and Fom3 for the alcohol

C-methylation,26-29 ThnK for the iterative C-methylation to form the ethyl group,30 TsrM for the aromatic C-methylation,31-32 PoyC for the valine residue C-methylation,33-34

CysS for sequential methylations in cystobactamid biosynthesis (Figure 1.4). 35

2- H3C O PO3 H H

Fosfomycin (Fos) Thienamycin 1-19 1-18 O P R Gentamicin C H3C 1 O NHAc 1-17 CH3 N N-acetylphosphinothricin H Thiostrepton 1-21 1-20

Figure 1.4 Selected secondary metabolites whose biosynthesis include class B RSMTs that have been reconstituted in vitro. Carbon atoms that are introduced by class B RSMTs are highlighted by red.

Class C RSMTs share high sequence similarity with HemN at the C-terminus in addition to the classical TIM barrel radical SAM domain. Class C RS can methylate sp2- hybridized carbon centers, but do not have the two conserved cysteines like RlmN, Cfr in the sequence. In the crystal structure of HemN, there are two simultaneously bound

SAM molecules in the active site.36 Since, many residues in Class C RSMTs are conserved in HemN, including the residues for SAM binding, it has proposed that Class

C RSMTs uses one molecular of SAM to generate 5′-dA radical and another molecule to provide methyl group. This suggests a possible mechanism in which the 5′dA• generated by the first SAM abstracts a hydrogen atom from the methyl group of the second SAM, which then can attack the unsaturated carbon to initiate the methylation.20 Qi’s group showed an alternative mechanism for NosN that SAM is first converted to 5′-

methylthioadenosine (MTA) and then was used as a direct methyl donor after it was abstracted a hydrogen atom by 5′-dA radical, which is different than what Booker’s group observed.37-38 Due to the poor accessibility of the complicated substrates and a lack of knowledge of the step along the biosynthetic pathway, there are few systems reconstituted for the Class C RSMTs. Recent studies also revealed that Class C RSMTs can not only methylate sp2-hybridized carbon but also form cyclopropane rings or ester linkage (Figure 1.5). These include enzymes that catalyze the formation of cyclopropane rings formation (Jaw5 in jawsamycin biosynthesis, YtkT in yatakemycin biosynthesis,

C10P and C10Q in CC-1065 biosynthesis),39-41 methylation of sp2-hybridized carbon

(TbtI in thiomuracin biosynthesis, MenK/MqnK in menaquinone biosynthesis),42-43 methylation and ester linkage (NosN in nosiheptide biosynthesis).38

N S Jaw5

1-22 TbtI O 1-23 N O N H NosN N H O 1-26 O YtkT MenK/MqnK C10P,C10Q 1-25 1-24

Figure 1.5 Class C radical SAM methyltransferases catalyzed modification (highlight in red) in the core structure of selected secondary metabolites.

2. RECONSTITUTION OF B12-DEPENDENT RADICAL SAM

METHYLTRANSFERASE CysS CATALYZED t-BUTYLATION BY ITERATIVE

METHYLATION*1

2.1. Introduction

Natural products with branched alkoxy groups play an important role in the development of bioactive compounds. In addition, the t-butyl group has fascinated organic chemists for more than a century and has played a major role in mechanistic studies on carbocation chemistry, organic substitution reactions, and the design and characterization of theoretically interesting molecules such as the remarkable tetra t- butyl tetrahedrane.44 Although numerous t-butyl group substituted terpenes, polyketides and peptides have been identified, experimental studies on the biosynthesis of t-butyl groups are still at an early stage and many of the mechanistic proposals in the literature have not been adequately experimentally tested.45

For the ginkgolides and several other t-butyl substituted terpenes, the t-butyl group is formed by a double bond methylation using S-adenosylmethionine (Figure

2.1A).46 Formation of the t-butyl group in the coumarin swietenone is proposed to involve carbocation insertion into a CH bond to give a cyclopropyl intermediate, which

* Reprinted in parts with permission from “Biosynthesis of Branched Alkoxy Groups: Iterative Methyl Group Alkylation by a Cobalamin-Dependent Radical SAM Enzyme” by Wang, Y.; Schnell, B.; Baumann, S.; Müller, R.; Begley, T. P. 2017, J. Am. Chem. Soc. 139,1742-1745. Copyright 2017 American Chemical Society. Reprinted from Methods Enzymol, 606, Wang, Y.; Schnell, B.; Muller, R.; Begley, T. P., Iterative Methylations Resulting in the Biosynthesis of the t-Butyl Group Catalyzed by a B12-Dependent Radical SAM Enzyme in Cystobactamid Biosynthesis, 199-216, Copyright (2018), with permission from Elsevier.

then undergoes acid mediated ring-opening (Figure 2.1B).47 The biosynthesis of pivalic acid, a starter unit in the biosynthesis of t-butyl substituted polyketides, is mediated by a

48-49 vitamin B12-dependent mutase (Figure 2.1C). Recently, a B12/radical SAM mediated conversion of isopropyl glycine to t-butyl glycine in the polytheoamide propeptide was reported (Figure 2.1D).33-34 A polyketide synthase is also found to catalyze coupled decarboxylation and methylation reactions to form pivaloyl-ACP from dimethylmalonyl-

ACP in the apratoxin A biosynthesis(Figure 2.1E).50 However, all of these enzymes catalyze a single methyl transfer to form the t-butyl group from an isopropyl group.

A CO2H C CO2H H PCM S IcmF S CoA CoA HO2C HO2C O O Ado-CH H3C SHR2 2-9 Ado-CH2 3 2-10 2-1 O S CoA S CO2H CoA HO O O O Ado-CH Ado-CH2 O 2-11 3 2-12 O O HO2C OH D O O 2-2 H PoyC H Ginkgolides R N R N N R' N R' B H H O O H H H 2-13 2-14 O O O O O O O O O E 2-3 2-4 2-5 H O O AprA O ACP ACP S OH S O OH Oxidation O O O O O O O O 2-15 2-16

2-8 2-7 2-6 Swietenone

Figure 2.1 Mechanistic proposals for the formation of the t-butyl group in representative natural products.

The cystobactamids are a novel class of isopropyl substituted antibacterial compounds produced by myxobacteria.51 The biosynthetic gene cluster has been identified and sequence analysis suggested that CysS is a cobalamin-dependent radical

SAM methyltransferase, potentially involved in the iterative methylations of the 3- methoxy-4-aminobenzoic acid moieties of cystobactamid 2-19 (Figure 2.2). Some minor derivatives exhibit methyl, ethyl, isopropyl, and sec-butyl groups (Stephan Hüttel and

R.M., unpublished results) supporting the hypothesis of CysS being an enzyme iteratively adding methyl-groups to its substrate.

NH2 MeO O O H N O N O H H N HN SR SR H CysS O N O O H N H2N 2 R OH O O N 1 O COOH O O 2 R R2 2-17 2-18 2-19

R1 = Et, i-Pro, 2-butyl R2 = Et, i-Pro, 2-butyl

Figure 2.2 Proposed formation of branched alkoxy groups of cystobactamids by CysS- catalyzed iterative methylations of a methyl ether.

Here we successfully reconstitute CysS in vitro and demonstrate that this enzyme can assemble isopropyl, sec-butyl, and t-butyl groups by sequential methylations of a methyl group. To our knowledge, this is the first example of an isopropyl, sec-butyl, and a t-butyl group biosynthesis from a methyl group using radical chemistry.

2.2. Result and discussion

2.2.1. Reconstitution of CysS activity with minimal substrates

CysS was cloned into a pET28b vector and co-expressed with a plasmid encoding the suf operon ([4Fe-4S] biosynthesis)52 in Escherichia coli BL21 (λDE3). The protein was then purified, under anaerobic conditions, by Ni-NTA affinity chromatography. The UV−visible spectrum of purified CysS revealed a 420 nm shoulder, typical of a bound Fe/S cluster (Figure 2.3). Iron and sulfide analysis yielded

2.5 irons and 2.8 sulfides per monomer of CysS, demonstrating partial cluster formation in the overexpressed protein.

A B UV spectrum CysS Ladder

0.6

0.5 75kDa 0.4

0.3

0.2 Absorbance 0.1

0 250 300 350 400 450 500 550 SDS-PAGE Gel wavelength(nm)

Figure 2.3 (A) UV spectrum of purified CysS (B) SDS/PAGE analysis of purified CysS.

Several p-aminobenzoic acid (PABA) analogs were tested as substrates for CysS.

None gave the desired methylated product as indicated by LC-MS analysis. Further analysis of the cystobactamid biosynthesis cluster suggested the coenzyme A or the acyl

carrier protein thioester of 2-17 (CysG)51 as possible CysS substrates. To test this proposal, N-acetylcysteamine thioester 2-20 was synthesized and incubated with CysS,

SAM, MeCbl, and flavodoxin/flavodoxin reductase/NADPH (Figure 2.4). LC-MS analysis of the resulting reaction mixture demonstrated the formation of the ethyl ether

2-21(Figure 2.5A). This was further confirmed by co-elution of the reaction product with a synthesized sample of 2-21 (Figure 2.5D). When the ethyl ether 2-21 was incubated with CysS, the isopropyl ether 2-22 was detected by LC-MS analysis (Figure 2.5C).

A O O O O S O S O S N N N H H H CysS SAM MeCbl CysS SAM MeCbl

Reducing system Reducing system O O O NH NH2 NH2 2 2-20 2-21 2-22

O O O O B O S OH O S OH N N N N H H CysS SAM MeCbl H H OH OH

Reducing system R O O a: R = Et b: R = i-Pro NH NH 2 2 c: R = t-Bu 2-23 2-24 d: R = sec-Bu

Figure 2.4 Identification of two substrates for CysS. A competition reaction with a 1:1 mixture demonstrated that 2-23 is 47 times more reactive than 2-20.

Intens . x105 A O O 0.8 O S O S N N H H 0.6 CysS SAM MeCbl

Reducing system 0.4 O O

NH2 NH2 0.2 18 19

0.0 0 5 10 15 20 25 30 35 Time [min] Full WAM87NAC_1-E,2_01_20438.d: EIC 283.1111±0.02 +All MS no CysS_1-E,3_01_20439.d: EIC 283.1111±0.02 +All MS no Fld_1-E,7_01_20444.d: EIC 283.1111±0.02 +All MS no MeCbl_1-E,6_01_20443.d: EIC 283.1111±0.02 +All MS no SAM_1-E,5_01_20441.d: EIC 283.1111±0.02 +All MS no WAM87NAC_1-E,4_01_20440.d: EIC 283.1111±0.02 +All MS Intens . B x105

1.5 NH2

N N 1.0 N N O 0.5 OH OH

0.0 0 5 10 15 20 25 30 35 Time [min] Full WAM87NAC_1-E,2_01_20438.d: EIC 252.1091±0.02 +All MS no CysS_1-E,3_01_20439.d: EIC 252.1091±0.02 +All MS no Fld_1-E,7_01_20444.d: EIC 252.1091±0.02 +All MS no MeCbl_1-E,6_01_20443.d: EIC 252.1091±0.02 +All MS no SAM_1-E,5_01_20441.d: EIC 252.1091±0.02 +All MS no WAM87NAC_1-E,4_01_20440.d: EIC 252.1091±0.02 +All MS Intens . C x105 1.0 O O O S O S 0.8 N N H H CysS SAM MeCbl 0.6 O Reducing system O 0.4 NH2 NH2 0.2 19 20

0.0 20 21 22 23 24 25 26 27 28 Time [min] Full WAM87NACethyl_1-E,1_01_20664.d: EIC 297.1267±0.02 +All MS No CysS WAM87NACethyl_1-E,2_01_20665.d: EIC 297.1267±0.02 +All MS no Fld WAM87NACethyl_1-E,6_01_20669.d: EIC 297.1267±0.02 +All MS no MeCbl WAM87NACethyl_1-E,5_01_20668.d: EIC 297.1267±0.02 +All MS no SAM WAM87NACethyl_1-E,4_01_20667.d: EIC 297.1267±0.02 +All MS no WAM87NACethyl_1-E,3_01_20666.d: EIC 297.1267±0.02 +All MS

D O O O S O S N N H H CysS SAM MeCbl

O Reducing system O

NH2 NH2 19 20

Figure 2.5 LC-MS detection of the CysS reaction products using methyl ether 2-21 and ethyl ether 2-22. Red trace is the full reaction where all the components are present. Orange, cyan, blue, black and green traces respectively are for reaction mixtures where either CysS, MeCbl, SAM, substrate, or reducing system flavodoxin/flavodoxin reductase/NADPH) is absent. (A) EIC of compound 2-21 [M+H]+ (283.11±0.02) (B) EIC of 5¢-dA [M+H]+ (252.10±0.02) (C) EIC of compound 2-22 [M+H]+ (297.13±0.02) (D) EIC of compound 2-22 co-elution with an authentic sample [M+H]+ (283.11±0.02). Red trace is the full enzymatic reaction. Purple trace is compound standard 2-22. Green trace is the co-elution of the enzymatic product and the synthetic standard.

Pantetheinyl thioester 2-23 was a better substrate for CysS and iterative methylations to give the ethyl, isopropyl, and the butyl ethers were detected by LC-MS analysis (Figure 2.6). Small amounts of the ethers 2-24a and 2-24b were detected in the absence of the reducing agent suggesting that some of the purified enzyme contained the reduced [4Fe−4S] cluster. To confirm the structures of 2-24a-c, authentic samples of these compounds were synthesized. The enzymatic products matched the synthetic standards in terms of retention time, exact mass, and fragmentation pattern (Figure 2.12,

2.13). In addition, CysS catalyzed the conversion of synthetic 2-24a to 2-24b-d and the conversion of synthetic 2-24b to 2-24c, d (Figure 2.15). The second component in the extracted ion chromatogram for the t-butyl ether 2-24c (Figure 2.6C, D) was identified as the sec-butyl ether 2-24d by co-migration with an authentic standard of 2-24d (Figure

2.16).

Intens . x105

2.0 A O O O S OH 1.5 N N H H OH 1.0 O

0.5 NH2 22a

0.0

Intens . 16 18 20 22 24 26 28 30 Time [min] x105 No Fld pant_1-F,2_01_20899.d: EIC 442.2006±0.02 +All MS No Fld pant_1-F,2_01_20899.d: EIC 10000.0000±0.002 +All MS No Fld pant_1-F,2_01_20899.d: EIC 10000.0000±0.005 +All MS Full WAM87pant_1-E,7_01_20895.d: EIC 442.2006±0.02 +All MS 5 Full WAM87pant_1-E,7_01_20895.d:B EIC 10000.0000±0.002 +All MS Full WAM87pant_1-E,7_01_20895.d: EIC 10000.0000±0.005 +All MS O O O S OH 4 N N H H OH 3

O 2 NH2 22b

Intens . 0 16 18 20 22 24 26 28 30 Time [min] No Fld pant_1-F,2_01_20899.d: EIC 456.2163±0.2 +All MS No Fld pant_1-F,2_01_20899.d: EIC 10000.0000±0.002 +All MS No Fld C pant_1-F,2_01_20899.d: EIC 10000.0000±0.005 +All MS Full WAM87pant_1-E,7_01_20895.d: EIC 456.2163±0.2 +All MS 1500 Full WAM87pant_1-E,7_01_20895.d: EIC 10000.0000±0.002 +AllO MS O Full WAM87pant_1-E,7_01_20895.d: EIC 10000.0000±0.005 +All MS O S OH N N H H 1000 OH

500 NH2 22c

0 16 18 20 22 24 26 28 30 Time [min] No Fld pant_1-F,2_01_20899.d: EIC 470.2319±0.001 +All MS, Smoothed (2.17,1,GA) No Fld pant_1-F,2_01_20899.d: EIC 10000.0000±0.002 +All MS No Fld pant_1-F,2_01_20899.d: EIC 10000.0000±0.005 +All MS Full WAM87pant_1-E,7_01_20895.d: EIC 470.2319±0.002 +All MS, Smoothed (2.27,1,GA) Full WAM87pant_1-E,7_01_20895.d:D EIC 10000.0000±0.002 +All MS Full WAM87pant_1-E,7_01_20895.d: EIC 10000.0000±0.005 +All MS O O O S OH N N H H OH

NH2 22c

16 20 24 28

Retention time (minutes)

Figure 2.6 LC-MS analysis of CysS-catalyzed iterative methylations of methyl ether 2- 23. Red trace is for the complete reaction mixture. Green trace is for reaction mixtures where the reducing system (flavodoxin/ flavodoxin reductase/NADPH) is absent. Ethyl ether 2-24a, isopropyl ether 2-24b were not formed in the control reactions lacking CysS, SAM, or MeCbl. (A) Extracted ion chromatograms (EICs) of the ethyl ether 2-24a [M + H]+ (442.20 ± 0.02). (B) EICs of the isopropyl ether 2-24b [M + H]+ (456.22 ± 0.02). (C) EICs of the t-butyl ether 2-24c [M + H]+ (470.23 ± 0.02). (D) EICs of [M + H]+ (470.23 ± 0.02) showing co-migration with a synthesized sample of 2-24c. Cyan trace is the t-butyl ether standard. Blue trace is co-elution of the enzymatic product and synthetic standard. The second component in the extracted ion chromatogram for the t- butyl ether 2-24c (panels C, D) was identified as the sec-butyl ether 2-24d.

Various [4Fe−4S] cluster reducing agents were tested in addition to the flavodoxin/flavodoxin reductase/NADPH. NADPH/methyl viologen, a commonly used electron source for cobalamin-dependent radical SAM enzymes, gave similar activity.

However, dithionite or the combination of methyl viologen and dithionite gave a significantly lower activity.53 Buffer thiols inactivate the substrate by trans thioesterification and need to be avoided.

Quantitative analysis of the enzymatic reaction mixture (CysS, methyl ether 2-

23, flavodoxin/flavodoxin reductase/NADPH, reaction run to completion) by LC-MS showed that 1 equiv of enzyme undergoes >2 turnovers, generating around 2.0 equiv of

5′-dA, 2.0 equiv of SAH, 1.4 equiv of ethyl ether 2-24a and 0.3 equiv of isopropyl ether

2-24b (Figures 2.6 and 2.7). The t-butyl ether 2-24c was detected only when the concentration of the isopropyl ether 2-24b was >23 µM. The ratio of 5′-dA to SAH was close to 1, suggesting that two molecules SAM were consumed for each methylation reaction and that the uncoupled production of 5′-dA is low. This is consistent with SAM functioning as the source of both the adenosyl radical and the methyl group and was further supported by LC-MS analysis of a reaction mixture containing CD3-SAM which demonstrated CD3 incorporation into the ethyl ether 2-24a and the isopropyl ether 2-24b

(Figure 2.8).

Intens . x105 A NH2 3 N N

N N 2 O 5’dA 1 OH OH

0 Intens . x105 6 8 10 12 14 16 18 20 Time [min] Full WAM87pant_1-E,7_01_20895.d: EIC 252.1091±0.02 +All MS NH2 No CysS pant_1-E,8_01_20896.d: EIC 252.1091±0.02 +All MS No Fld pant_1-F,2_01_20899.d: EIC 252.1091±0.02 +All MS No MeCbl pant_1-F,1_01_20898.d: EIC 252.1091±0.02 +All MS 3 B No SAM pant_1-E,9_01_20897.d: EIC 252.1091±0.02 +All MS No substrate _1-E,3_01_20891.d: EIC 252.1091±0.2 +All MS N N

N N HOOC S 2 O NH2 OH OH 1 SAH

6 8 10 12 14 16 18 20 Time [min] Full WAM87pant_1-E,7_01_20895.d: EIC 385.1289±0.02 +All MS No CysS pant_1-E,8_01_20896.d: EIC 385.1289±0.02 +All MS No Fld pant_1-F,2_01_20899.d:6 8 10 12 14 EIC 385.1289±0.02 +All MS No MeCbl pant_1-F,1_01_20898.d:16 EIC 385.1289±0.02 +All18 MS 20 No SAM pant_1-E,9_01_20897.d: EIC 385.1289±0.02 +All MS No substrate _1-E,3_01_20891.d: EIC 385.1289±0.02 +All MS Retention time (minutes)

Figure 2.7 LC-MS detection of 5ʹ-dA and SAH in the CysS-catalyzed iterative methylations of the methyl ether 2-23. Red trace is for the complete reaction mixture. Green trace is for reaction mixtures where the reducing system (flavodoxin/flavodoxin reductase/NADPH) is absent. (A) EICs of 5ʹ-dA [M + H]+ (252.11 ± 0.02). (B) EICs of SAH [M + H]+ (385.13 ± 0.02). Yellow, green, cyan, and black traces are for reaction mixtures where either CysS, reducing system, MeCbl or substrate is absent. The product ratio was determined by calibrating the signal intensity with known concentrations of standards

Figure 2.8 MS analysis of a reaction mixture in which CH3-SAM is replaced with CD3-

SAM showing CD3 incorporation into the ethyl and isopropyl ethers of 2-24a and 2-24b, respectively. Panels A and C: Mass spectra of 2-24a and 2-24b formed from CH3-SAM. Panels B and D: Mass spectra of 22a and 22b formed from CD3-SAM.

A mechanistic proposal for the CysS-catalyzed reaction, based on the proposed mechanisms for GenK and ThnK is shown in Figure 2.9. After initial formation of methylcobalamin by SAM mediated methylation, reductive cleavage of SAM by the

[4Fe− 4S] +1 cluster generates the 5′-deoxyadenosyl radical. This abstracts a hydrogen atom from the methyl group of the substrate 2-23 to give radical 2-25, which then undergoes a radical substitution with methyl cobalamin to give the ethyl ether 2-24a. An analogous methyl transfer, by a radical substitution mechanism, has precedence in cobalamin model chemistry.54 Regeneration of MeCbl from Cbl(II) can be achieved by reduction to Cbl(I) by the [4Fe−4S]+1 cluster followed by SAM-mediated methylation.

Repetition of this sequence results in the successive formation of the isopropyl, t-butyl, and sec-butyl ethers of 2-23. 20

Co(I) Co(III) 2-23,e- O SR Co(III) O SR Co(III) CH3 Met Me CH3 CH3 H S S H Ade R' Ade H R' O O C C H O H O H SAH 2-23 2-23 NH2 Me NH OH OH OH OH 2 S R' Ade CH2 Ade SAM SAH O O

[4Fe-4S]2+ [4Fe-4S]2+ [4Fe-4S]1+ OH OH [4Fe-4S]2+ OH OH SAM

O SR O SR Co(III) Co(II) SAM, e- Co(II) Co(I) CH3 CH3 Me Me H C H S S O R' Ade Ade O H H O R' O 2-25 2-24a NH NH2 2 2-24a, 5'dA CH3 Ade CH3 Ade OH OH O O OH OH SAM SAM 2+ OH OH [4Fe-4S] [4Fe-4S]2+ OH OH [4Fe-4S]1+ [4Fe-4S]2+ 5'dA 5'dA

Co(III) O SR Co(III) O SR O SR - CH3 2-24a,SAM, e CH3 CH CH CH3 3 3 S Ade H CH3 CH3 R' O O O O H H CH3 2-24a 2-24b NH 2-24c NH SAH NH2 Me 2 2 OH OH S R' Ade SAH O 2+ 1+ O O [4Fe-4S] [4Fe-4S] OH OH COOH R: S OH R': SAM N N H H OH NH2

Figure 2.9 Proposal for CysS-catalyzed iterative methylations to form branched alkoxy groups. The mechanism assumes two different SAM binding sites. It is also possible that SAM binds to a single site and that the position of the sulfonium moiety is altered by a protein conformational change.

2.2.2. Reconstitution of CysS activity with native substrates

The most logical substrate for CysS is the 4-amino-3-methoxybenzoate thioester with the peptidyl carrier protein of the polypeptide synthase.51 This was synthesized as shown in Figure 2.10. Condensation of 2-26 with coenzyme A yielded the CoA ester 2-

27. The phosphopantetheinyl thioester moiety of 2-27 was then transferred to CysG1 in a reaction catalyzed by phosphopantetheinyl transferase.

Figure 2.10 Synthesis of the 4-amino-3-methoxybenzoate (AMB) CysG1 thioester 2-28. Adapted with permission from 55.

When AMB-CysG1 thioester was incubated with CysS, MeCbl, SAM and methyl viologen, dimethylated 2-29a and trimethylated products 2-29b were detected by

LC-MS (the formation of trimethylated products required higher enzyme concentrations)

(Figure 2.11). This further demonstrated that the peptide carrier protein thioester of 2-28 is the native substrate of CysS.55

Figure 2.11 The CysS-catalyzed reaction of the AMB-CysG1 thioester. (A) MS of the dimethylated product (APB-CysG1 thioester 2-29a, calculated monoisotopic mass: 73217.8Da, experimental mass: 73218.0Da), (B) MS of the trimethylated product (ABB- CysG1 thioester 2-29b, calculated monoisotopic mass: 73231.7Da, experimental mass: 73232.7Da), (C) The reaction catalyzed by CysS. Adapted with permission from 55.

2.3. Conclusion

Here we describe the successful in vitro reconstitution of CysS and demonstrate that this enzyme can assemble isopropyl, sec-butyl, and t-butyl groups by sequential methylations of a methyl group. To our knowledge, this is the first example of an isopropyl, sec-butyl, and a t-butyl group biosynthesis from a methyl group using radical chemistry. This biosynthetic strategy in principle enables the host myxobacterium to

biosynthesize a combinatorial antibiotic library of 25 cystobactamid analogs. Analogous methyl-based combinatorial libraries could potentially be generated in natural products such as kedarcidin and quinomycin SW-163s, which have ethyl, isopropyl, and sec-butyl substituents on a thiomethyl group.56-57

2.4. Experimental methods

2.4.1. overexpression and purification of CysS

A 15mL overnight culture was grown in LB medium in the presence of 40 µg/mL kanamycin and 30 µg/mL chloramphenicol. This was then added to 1.5 L of LB medium containing 40 µg/mL kanamycin and 30 µg/mL chloramphenicol. The cultures were incubated at 37°C with shaking (180 rpm) until the OD600 reached 0.45. The culture was then incubated at 4°C without shaking for 1 hour. Expression was induced by the addition of 100 µM IPTG followed by ferrous ammonium sulfate (120 mg) and cysteine

(120 mg) and the culture was grown at 15 °C with shaking (110 rpm) overnight. The cells were harvested by centrifugation and stored in liquid nitrogen until further use. All steps for protein purification were carried out in an anaerobic chamber (COY laboratories). Cell pellets (~12 g) were thawed and resuspended in 30 mL lysis buffer

(100 mM Tris-HCl, pH 7.5) in the presence of lysozyme (0.2 mg/mL) and benzonase

(100 units). This mixture was cooled in an ice-bath with continuous stirring for 1-2 hrs.

The suspension was then sonicated to complete cell lysis for 5 × 120 s using a Misonix

Sonicator XL-2000 (setting = 9). Cell debris was removed by centrifugation, and the lysate was loaded onto a Histrap column pre-equilibrated with lysis buffer. The column was washed with 5-10 column volumes of wash buffer (100 mM Tris-HCl, 30 mM

Imidazole, pH 7.5). The CysS protein was then eluted using elution buffer (100 mM

Tris-HCl, 300 mM NaCl, 250 mM imidazole, pH 7.5). The brown-colored fractions, containing the desired protein, were pooled and buffer exchanged to final buffer (100 mM Tris-HCl, pH 7.5 at 25 °C, 30% glycerol) using Econo-Pac 10DG size exclusion chromatography. The purified CysS was divided into aliquots in eppendorf tubes, frozen in liquid nitrogen, and stored at −80 °C, submerged in liquid nitrogen, until future use.

Each tube has a pin hole in the cap to allow for gas release during thawing. Protein concentration was measured by the absorbance at 280 nm (A280) when the protein was exposed to air or the [4Fe-4S]2+ cluster was reduced to [4Fe-4S]1+ by dithionite

(prevent the interference from absorbance from [4Fe-4S]2+) with an extinction coefficient calculated using the ProtParam tool of the ExPASy proteomics server.

Typical yields were approx. 5 mg per liter.

2.4.2. Iron and sulfide quantification of CysS

Iron content was analyzed using the ferene assay (Cooper et al., 2013). The following reagents were prepared: A, 1.35 g SDS, 30 mL water, 450 µL saturated sodium acetate; B, 540 mg L-ascorbic acid, 18 mg sodium metabisulfite, 11.2 mL water,

800 µL saturated sodium acetate; C, 18 mg ferene in 1 mL water. Guanidine hydrochloride (8M, 300 µL), Reagent A (300 µL), and Reagent B (300 µL) were added to 300 µL of a CysS solution. After mixing by inversion, the samples were incubated for

15 min at 37 °C and then 15 µL of Reagent C was added. The samples were incubated for 5 min at 25 °C and centrifuged for 5 min at 25 °C. The absorbance at 593 nm of the supernatant was recorded using a Varian Cary Bio 300 UV-Visible Spectrophotometer.

The iron content was finally determined by comparing the reading to a standard curve that was generated under identical conditions using FeCl3 in 2% HNO3 with a concentration range from 0 to 200 µM.

Sulfide content was determined using the methylene blue assay.6 To 300 µL of the assay solution containing CysS, 1 ml of 1% (w/v) zinc acetate was added followed by 50 µL of 3 M NaOH. This mixture was agitated gently and 250 µL of 0.1 % N, N- dimethyl-p-phenylenediamine (DMPD) monohydrochloride in 5 M HCl and 50 µL of 23 mM FeCl3 in 1.2 M HCl were added. The resulting solution was mixed vigorously for

30 mins. The samples were then centrifuged for 10 min at 25 °C. The supernatant was collected and the absorbance at 670 nm was recorded using a Varian Cary Bio 300 UV-

Visible Spectrophotometer. The sulfide content was determined by comparing this reading to a standard curve that was generated under identical conditions using a fresh solution of sodium sulfide (Na2S) in 0.1 M NaOH with a concentration range from 0 to

100 µM.

2.4.3. In vitro reconstitution assay with CysS:

All CysS enzymatic reactions were carried out in an anaerobic chamber containing 95% nitrogen and 5% hydrogen. A typical enzymatic reaction was performed in 100 mM phosphate buffer, pH 7.5 containing 55 µM of CysS with 0.75 mM MeCbl,

2.5 mM SAM, 0.9 mM substrate with different reducing systems, flavodoxin, flavodoxin reductase system (25 µM FldA, FPR; 50 µM FAD, FMN; 3 mM NADPH); 1 mM methyl viologen, 4 mM NADPH; 10 mM dithionite, 10 mM dithiothreitol (DTT); 1 mM methyl viologen, 4 mM NADPH, 50 mM DTT; 1 mM methyl viologen, 4 mM NADPH,

50 mM DTT. The reaction was incubated at room temperature for 12-15 hours. For LC-

MS analysis the enzyme was removed by ultrafiltration using a 10kDa cut-off filter

(VWR).

A O O O S OH N N H H OH

O NH 2

Intens . +MS, 26.3-26.7min #(1563-1587) x105 442.2005 1.25 B 1.00

0.75

0.50

443.2030 0.25 424.1920 444.2003 459.3175 425.1947 453.3440 0.00 420 425 430 435 440 445 450 455 460 m/z

Intens . Intens . +MS2(442.5024), 35eV, 26.7min #(1586) +MS2(442.3994), 26.6-26.6min #(1575-1579) x106 x105 164.0729 164.0713 1.25 C D

1.5 1.00

0.75 1.0

0.50

0.5

0.25

136.0396 136.0396 0.00 0.0 100 120 140 160 50 180 100 200 150 220 200 240 250 260 300 m/z350 400 450 500 m/z Figure 2.12 LC-MS detection of 2-24a in the CysS reaction using methyl ether 2-23 as substrate. Red trace is the enzymatic full reaction. Green trace is the co-elution of the enzymatic product and synthetic standard of the ethyl ether 2-24a (A) EIC of ethyl ether 2-24a [M+H]+ (442.20±0.02). (B) Mass spectrum of ethyl ether 2-24a (exact mass: 442.2021, Accuracy: <1 ppm). (C) Mass fragments of the ethyl ether 2-24a produced in the enzymatic reaction. (D) Mass fragments of synthetic standard of the ethyl ether 2- 24a.

A O O O S OH N N H H OH

O NH 2

Intens . +MS, 27.2-27.6min #(1615-1643) x105 456.2171 3 B

478.1990 2

453.3466 494.1723 438.2054 454.8506 464.3367 0 430 440 450 460 470 480 490 m/z

Intens . Intens . +MS2(456.2188), 27.2-27.3min #(1611-1619) +MS2(456.5179), 35eV, 27.3min #(1618) x104 x105 178.0867 178.0865 6 C D 4 5

4 3

3 2

136.0390 1 136.0391 1

0 0 200 400 600 200 800 400 1000 600 1200 800 1400 1000 m/z 1200 1400 m/z Figure 2.13 LC-MS detection of 2-24b in the CysS reaction using methyl ether 2-23 as substrate. Red trace is enzymatic full reaction. Blue trace is synthetic standard of isopropyl ether 2-24b (A) EIC of isopropyl ether 2-24b [M+H]+ (456.21±0.02). (B) Mass spectrum of isopropyl ether 2-24b (exact mass: 456.2171, Accuracy: 1.7 ppm). (C) Mass fragments of the isopropyl ether 2-24b produced in the enzymatic reaction. (D) Mass fragments of synthetic standard of isopropyl ether 2-24b.

Intens . +MS, 28.2-28.2min #(1675-1679) 5 x10 A 470.2306 492.2121 1.25

1.00

0.75

0.50

456.2144 478.1958 0.25 452.2190

0.00 450 455 460 465 470 475 480 485 490 m/z

Intens . Intens . +MS2(470.5902), 28.1-28.2min #(1670-1674) +MS2(470.4227), 28.0-28.2min #(1664-1671) x104 x105 B 136.0392 C 136.0416 3 192.1045 6

192.1021

2 4

1 2 325.2722 464.1837 261.1270 0 0 200 400 200 600 400 800 600 1000 800 1200 1000 1400 m/z1200 1400 m/z Figure 2.14 LC-MS detection of 2-24c in the CysS reaction using methyl ether 2-23 as substrate. (A) Mass spectrum of t-butyl ether 2-24c (exact mass: 470.2322, Accuracy: 3.4 ppm). (B) Mass fragments of t-butyl ether 2-24c produced in the enzymatic reaction. (C) Mass fragments of synthetic standard of t-butyl ether 2-24c.

Intens . x106 A

1.00 O O O O 0.75 O S OH O S OH N N N N H H CysS SAM MeCbl H H OH OH 0.50 Reducing system O O 0.25 NH2 NH2

0.00 0 5 10 15 20 25 30 35 Time [min] Ethyl_1-D,2_01_22981.d: EIC 456.2163±0.02 +All MS Figure 2.15 LC-MS detection of the CysS reaction products using ethyl ether 2-24a (A), (B) or isopropyl ether 2-24b (C). (A) EIC of isopropyl ether 2-24b [M+H]+ (456.21±0.02). (B), (C) EIC of butyl ethers 2-24c, 2-24d [M+H]+ (470.23±0.02).

O O O O O S OH O S OH N N N N H H H H CysS SAM MeCbl OH OH Reducing system R O O c: R = t-Bu NH2 d: R = sec-Bu NH2

Intens . x105 C 1.0 O O O O O S OH 0.8 O S OH N N N N H H H H CysS SAM MeCbl OH OH 0.6 Reducing system R O 0.4 O c: R = t-Bu NH2 d: R = sec-Bu NH2 0.2

0.0 0 5 10 15 20 25 30 35 Time [min] isopropyl_1-D,3_01_22982.d: EIC 470.2319±0.005 +All MS, Smoothed (2.55,1,GA) Figure 2.15 Continued

O O O S OH N N H H OH

NH2

Figure 2.16 LC-MS detection of the CysS reaction products using isopropyl ether 2-24b as substrate. The second peak was co-migrated with an authentic standard of sec-butyl ether 2-24d. Red trace is EIC of mass (470.23±0.02) for enzymatic full reaction. Green trace is synthetic standard of sec-butyl ether 2-24d.

In t e n s . +MS, 17.5-17.7min #(1040-1052) x104 252.1106 8 A

2 253.1129

250.0950 0 250 252 254 256 258 260 m/z

In t e n s . +MS, 17.6-17.7min #(1049-1053) x104 253.1169

6 B

4 252.1107

254.1180 250.0956 0 248 250 252 254 256 258 m/z

Figure 2.17 LC-MS detection of 5¢-dA produced in the CysS reaction products using (A) methyl ether 2-23 or (B) CD3-methyl ether CD3-2-23 as substrate.

2.4.4. LC-MS parameters for the analysis of the CysS reaction mixture

LC-ESI-TOF-MS was performed using an Agilent 1260 HPLC system equipped with a binary pump and a 1200 series diode array detector followed by a MicroToF-Q II mass spectrometer (Bruker Daltonics) using an ESI source in positive mode. Analysis was performed on a LC-18-T column (15 cm x 3 mm, 3 µm particles, Supelco).

LC conditions:

A- 5 mM Ammonium acetate buffer, pH 6.6

B- 75 % Methanol and 25 % Water.

LC method:

0min – 100% A, 2 min – 100% A, 4 min – 80% A 20% B, 27 min – 100% B, 29 min – 100% B, 30 min – 100% A, 40 min – 100% A.

The mass spectrometer parameters for small molecules: Capillary, 4500 V; end plate offset, 500 V; nebulizer gas, 3.0 bar; dry gas, 10.0 L/min; dry gas temperature,

200°C; funnel 1 RF, 250.0 Vpp; funnel 2 RF, 300.0 Vpp; isCID, 0.0 eV; hexapole RF,

200 Vpp; quadrupole, Ion energy, 3.0 eV; low mass, 150 m/z; collision cell, collision energy, 8.0 eV; collision RF, 150.0 Vpp; transfer time, 80.0 µs; prepulse storage, 5.0 µs.

Data were processed with DataAnalysis ver. 4.0 (Bruker Daltonics).

2.4.5. Quantification of enzymatic products in the CysS reaction mixture

The CysS assay, using methyl ether 2-23 was carried out under the above conditions with flavodoxin, flavodoxin reductase or methyl viologen, NADPH as reducing systems. Calibration curves were constructed by measuring the peak intensity of 5′-dA, SAH, synthetic ether 2-21, 2-24a and 2-24b with known concentration using

LC-MS. Then the calibration curves were used to measure the concentration of 5′-dA,

SAH, 2-21, 2-24a and 2-24b in the enzymatic reaction. Control reactions without CysS were used to assess the quantity of non-enzymatically produced SAH and this amount was subtracted out of the enzymatic reaction to give the final result. With our current enzyme preparation, 55 µM of CysS with 0.75 mM MeCbl, 2.5 mM SAM, 0.9 mM substrate and flavodoxin, flavodoxin reductase system (25 µM FldA, FPR; 50 µM FAD,

FMN; 3 mM NADPH) produced 111 µM of 5′-dA, 109 µM of SAH, 78 µM of ethyl ether 2-24a, 15 µM of isopropyl ether 2-24b. The relative rate of 2-20 and 2-23 was measured by the amount of product formations of 2-21 and 2-24 by CysS with 1:1 ratio of 2-20 to 2-23. 11 µM CysS was incubated with 0.3 mM MeCbl, 0.5 mM SAM, 0.5

mM 18, 0.5 mM 21, and 1 mM methyl viologen, 3 mM NADPH produced 0.4 uM of 2-

21, 15.8 µM of 2-24a, 3.2 µM of 2-24b.

2.4.6. General procedure for the synthesis of the 4-nitrobenzoic acids (2-32a, b, d)

O OH O OH 1.KOH ROH

2. HCl R F O NO a: R = Et NO2 2 b: R = i-Pro 2-32 2-31 d: R = sec-Bu

Potassium hydroxide (5.0 mmol) was added to a solution of 3-fluoro-4- nitrobenzoic acid (2.0 mmol) 31 in excess of alcohol (10 ml) and the reaction mixture was slowly heating to reflux for several hours and monitored by TLC (Hexane and

EtOAc solvent system). Then it was acidified using 2N HCl and extracted with ethyl acetate. The extracts were combined, washed with water, dried with MgSO4 and evaporated to give 4-nitro-benzoic acids 2-32a, b and d.

3-ethoxy-4-nitrobenzoic acid (2-32a)

Compound 2-32a was prepared by the above procedure using ethanol in 63% yield. 1H NMR (400 MHz, MeOD) δ 7.80 (m, 2H), 7.68 (d, J = 9.8 Hz, 1H), 4.26 (q, J =

7.0 Hz, 2H), 1.43 (t, J = 7.0 Hz, 3H). 13C NMR (101 MHz, MeOD) δ 152.59, 136.72,

125.79, 122.58, 116.71, 106.43, 66.77, 14.72.

3-isopropoxy-4-nitrobenzoic acid (2-32b)

Compound 2-32b was prepared by the above procedure using isopropyl alcohol in 90% yield. 1H NMR (400 MHz, MeOD) δ 7.81 (s, 1H), 7.76 (d, J = 8.3 Hz, 1H), 7.66

(d, J = 8.3 Hz, 1H), 4.80 (m, 1H) 1.36 (d, J = 6.0 Hz, 6H). 13C NMR (101 MHz, MeOD)

δ 172.96, 151.57, 136.51, 125.75, 122.57, 118.13, 74.13, 22.03, 20.85.

3-(sec-butoxy)-4-nitrobenzoic acid (2-32d)

Compound 2-32d was prepared by the above procedure using 2-butanol in 37% yield. 1H NMR (400 MHz, MeOD) δ 7.87 – 7.72 (m, 2H), 7.66 (d, J = 8.3 Hz, 1H), 4.68

– 4.51 (m, 1H), 1.82 – 1.60 (m, 2H), 1.33 (d, J = 6.1 Hz, 3H), 0.99 (t, J = 7.4 Hz, 3H).

13C NMR (101 MHz, MeOD) δ 167.76, 151.82, 136.52, 125.75, 122.40, 117.79, 106.38,

78.82, 30.03, 19.21, 9.67.

3-(tert-butoxy)-4-nitrobenzoic acid (2-32c)

O OH O OH

KOtBu THF

F O

NO2 NO2 2-31 2-32c

3-fluoro-4-nitrobenzoic acid 2-31 (0.5 mmol) was dissolved in 5 ml anhydrous

THF in a flame-dried flask under argon and cooled to 0 °C. A 1.0 M solution of potassium tert-butoxide (1.5 mmol) was added dropwise. This solution was stirred and allowed to warm from 0 °C to room temperature overnight and then diluted with ethyl acetate and washed with saturated aqueous NH4Cl. The aqueous layer was back- extracted with CH2Cl2. The organic layers were combined, dried over MgSO4, and concentrated. Purification of the crude mixture by flash chromatography (silica gel, 15:1

CH2Cl2: MeOH) afforded 2-32c in 90% isolated yield. 1H NMR (400 MHz, MeOD) δ

7.90 (s, 1H), 7.80 (d, J = 8.3 Hz, 1H), 7.74 (d, J = 8.3 Hz, 1H), 1.43 (s, 9H). 13C NMR

(101 MHz, MeOD) δ 167.63, 149.76, 125.76, 125.37, 124.96, 84.11, 29.08.

2.4.7. General Procedure for synthesis of 4-aminobenzoic acids (33a-d)

O OH O OH

Pd/C R R H2 O O a: R = Et NH NO2 2 b: R = i-Pro 2-32 2-33 c: R = t-Bu d: R = sec-Bu

Using 10% Pd/C as catalyst, 50 mL of 4-nitrobenzoic acid (2.0 mmol) in methanol was pumped through the H-Cube®. The pressure of the system was set to full hydrogen mode, and the temperature to 35 °C. The flow rate was 0.8 mL/min. The fraction was analyzed using TLC, which showed complete conversion to the product, and the solvent was reduced to dryness, affording the 4-aminobenzoic acids 2-33a-d.

4-amino-3-ethoxybenzoic acid (2-33a)

Compound 2-33a was prepared by the above procedure from 3-ethoxy-4- nitrobenzoic acid 2-32a in 98% yield. 1H NMR (400 MHz, MeOD) δ 7.57 – 7.30 (m,

2H), 6.71 (d, J = 8.2 Hz, 1H), 4.09 (q, J = 7.0 Hz, 2H), 1.43 (t, J = 7.0 Hz, 3H). 13C

NMR (101 MHz, MeOD) δ 170.56, 146.83, 143.50, 125.45, 120.08, 114.36, 113.49,

65.07, 15.11.

4-amino-3-isopropoxybenzoic acid (2-33b)

Compound 2-33b was prepared by the above procedure from 3-isopropoxy-4- nitrobenzoic acid 2-32b in 98% yield. 1H NMR (400 MHz, MeOD) δ 7.54 – 7.40 (m,

2H), 6.70 (s, 1H), 4.58 (m, 1H), 1.34 (d, J = 6.0 Hz, 6H). 13C NMR (101 MHz, MeOD) δ

171.77, 145.15, 126.99, 115.39, 113.88, 112.36, 72.35, 26.60, 22.32.

4-amino-3-(tert-butoxy) benzoic acid (2-33c)

Compound 2-33c was prepared by the above procedure from 3-(tert-butoxy)-4- nitrobenzoic acid 2-32c in 99% yield. 1H NMR (400 MHz, MeOD) δ 7.56 (s, 1H), 7.54

(d, J = 8.3 Hz, 1H), 6.71 (d, J = 8.3 Hz, 1H), 1.39 (s, 9H). 13C NMR (101 MHz, MeOD)

δ 170.48, 148.85, 142.48, 127.64, 125.04, 119.07, 114.84, 81.01, 29.10.

4-amino-3-(sec-butoxy) benzoic acid (2-33d)

Compound 2-33d was prepared by the above procedure from 3-(sec-butoxy)-4- nitrobenzoic acid 2-32d in 97% yield. 1H NMR (400 MHz, MeOD) δ 7.51 – 7.35 (m,

2H), 6.69 (d, J = 8.2 Hz, 1H), 4.36 (m, 1H), 1.86 – 1.57 (m, 2H), 1.30 (d, J = 6.1 Hz,

3H), 1.00 (t, J = 7.5 Hz, 3H).

S-(2-acetamidoethyl) 4-amino-3-methoxybenzothioate (2-20)

O O OH O S N H EDC, HOBT, TEA, SNAC

O rt. overnight O NH 2 NH2 2-34 2-20

To a solution of triethylamine (TEA) (2.80 mmol) in dichloromethane (10 mL) was added 4-amino-3-methoxybenzoic acid 2-34 (1.40 mmol), (3- dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) (1.40 mmol), 1- hydroxybenzotriazole (HOBt) (1.40 mmol) and N-acetylcysteamine (SNAC) (1.4 mmol). The reaction mixture was stirred overnight under argon. The organic layer was 36

washed with saturated NaHCO3 solution, 0.1 N HCl solution and brine. It was then dried over anhydrous sodium sulfate and concentrated under vacuum. Purification of the crude mixture by flash chromatography (silica gel, 1:1 to 2:1 of hexanes:EtOAc) afforded 2-20

1 in 20% isolated yield. H NMR (400 MHz, CDCl3) δ 7.53 (d, J = 8.2, 1H), 7.36 (s, 1H),

6.63 (d, J = 8.2 Hz, 1H), 3.89 (s, 3H), 3.51 (dd, J = 12.2, 5.9 Hz, 2H), 3.18 (t, J = 6.3 Hz,

13 2H), 1.95 (s, 3H). C NMR (101 MHz, CDCl3) δ 190.29, 170.29, 146.21, 142.34,

126.60, 122.85, 112.78, 108.72, 55.63, 40.08, 28.31, 23.21.

S-(2-acetamidoethyl) 4-amino-3-ethoxybenzothioate (2-21)

O O OH O S N H EDC, HOBT, TEA, SNAC

O rt. overnight O NH 2 NH2 2-33a 2-21

Compound 2-21 was prepared from 4-amino-3-methoxybenzoic acid 2-33a in the same manner as the above procedure in 30% yield. 1H NMR (400 MHz, CDCl3) δ 7.49

(d, J = 8.2 Hz, 1H), 7.33 (s, 1H), 6.62 (d, J = 8.2 Hz, 1H), 4.09 (q, J = 7.0 Hz, 2H), 3.48

(dd, J = 12.3, 5.9 Hz, 2H), 3.15 (t, J = 6.3 Hz, 2H), 1.93 (s, 3H), 1.42 (t, J = 7.0 Hz, 3H).

13 C NMR (101 MHz, CDCl3) δ 190.30, 170.29, 145.49, 142.45, 126.56, 122.69, 112.80,

109.63, 77.34, 77.03, 76.71, 64.05, 40.09, 28.30, 23.21, 14.77.

2.4.8. General Procedure for synthesis of S-pantetheinyl-4-aminobenzothioate (2-

24a-d)

O O O OH O S OH N N H H 1. DCC, THF, NHS OH

R 2. Pantetheine, THF R O O a: R = Et b: R = i-Pro NH2 NH2 c: R = t-Bu 2-33 2-24 d: R = sec-Bu

To a stirred solution of 4-aminobenzoic acid 2-33 (2.0 mmol) in 10 mL of dry

THF was added N-hydroxysuccinimide (NHS) (2.0 mmol), followed by N, N'-

Dicyclohexylcarbodiimide (DCC) (2.0 mmol) at room temperature. The resulting mixture was stirred overnight at room temperature. The white precipitate was filtered and then washed with EtOAc. The filtrate was concentrated under reduced pressure. The residue was triturated with CH2Cl2 to give the NHS ether of 2-33 as a pale brown powder. Pantethine (0.075 mmol), sodium bicarbonate (1.9 mmol), and 1,4-dithiothreitol

(DTT) (0.08 mmol) were dissolved in 3 mL of water, and left to stand for 10 minutes to allow reduction of the pantethine to pantetheine to take place. The NHS ester (0.54 mmol) was dissolved in 7 mL of tetrahydrofuran (THF) and added to the 3 mL water mixture. The reaction mixture was stirred overnight and then the solvent was evaporated under reduced pressure. The crude product was then dissolved in water and the insoluble residue was removed by filtration. The resulting solution was lyophilized and purified by flash chromatography (silica gel, 20:1 to 10:1 CH2Cl2: MeOH). The fraction was further purified by Prep HPLC using a SPLC-18DB column (10 x 250 mm, 5 µm, Supelco) at a flow rate of 2 mL/min on an Agilent 100 HPLC system with a quaternary pump and an automatic injector. 38

(R)-S-pantetheinyl-4-amino-3-methoxybenzothioate (2-33)

Compound 2-33 was prepared by the above procedure from 4-amino-3-

1 methoxybenzoic acid 2-34. H NMR (400 MHz, D2O) δ 7.52 (dd, J = 8.3, 1.9 Hz, 1H),

7.35 (d, J = 1.9 Hz, 1H), 6.79 (d, J = 8.3 Hz, 1H), 3.91 (s, 1H), 3.88 (s, 3H), 3.47 – 3.29

(m, 6H), 3.17 (t, J = 6.3 Hz, 2H), 2.44 (t, J = 6.5 Hz, 2H), 0.83 (d, J = 13.9 Hz, 6H). 13C

NMR (101 MHz, D2O) δ 193.17, 175.04, 174.02, 146.63, 126.36, 123.04, 114.04,

109.43, 75.86, 68.49, 55.92, 38.93, 38.58, 35.50, 35.33, 28.03, 22.30, 20.46, 19.11.

(R)-S-pantetheinyl-4-amino-3-ethoxybenzothioate (2-24a)

Compound 2-24a was prepared by the above procedure from 4-amino-3-

1 ethoxybenzoic acid 2-33a. H NMR (400 MHz, D2O) δ 7.56 (dd, J = 8.3, 1.9 Hz, 1H),

7.41 (d, J = 1.8 Hz, 1H), 6.84 (d, J = 8.3 Hz, 1H), 4.15 (q, J = 7.0 Hz, 2H), 3.91 (s, 1H),

3.54 – 3.25 (m, 6H), 3.20 (t, J = 6.2 Hz, 2H), 2.44 (t, J = 6.5 Hz, 2H), 1.40 (t, J = 7.0 Hz,

3H), 0.83 (d, J = 13.8 Hz, 6H). 13C NMR (101 MHz, D2O) δ 193.40, 175.05, 174.08,

145.68, 143.76, 126.41, 123.11, 114.25, 111.11, 75.86, 68.48, 65.24, 38.90, 38.58,

35.51, 35.34, 28.06, 20.45, 19.09, 14.02.

(R)-S-pantetheinyl-4-amino-3-isopropoxybenzothioate (2-24b)

Compound 2-24b was prepared by the above procedure from 4-amino-3-

1 isopropoxybenzoic acid 2-33b. H NMR (400 MHz, D2O) δ 7.54 (dd, J = 8.4, 1.8 Hz,

1H), 7.45 (d, J = 1.8 Hz, 1H), 6.82 (d, J = 8.4 Hz, 1H), 4.62 (m, 1H), 3.88 (s, 1H), 3.48 –

3.21 (m, 6H), 3.17 (t, J = 6.2 Hz, 2H), 2.41 (t, J = 6.5 Hz, 2H), 1.30 (d, J = 6.1 Hz, 6H),

13 0.79 (d, J = 14.0 Hz, 6H). C NMR (101 MHz, D2O) δ 193.36, 175.05, 174.07, 144.96,

144.19, 126.43, 123.38, 114.65, 114.13, 75.86, 73.18, 68.48, 38.90, 38.59, 35.50, 35.35,

28.07, 21.21, 20.47, 19.11

(R)-S-pantetheinyl-4-amino-3-(tert-butoxy)benzothioate (2-24c)

Compound 2-24c was prepared by the above procedure from 4-amino-3-(tert- butoxy)benzoic acid 2-33c. 1H NMR (400 MHz, MeOD) δ 7.65 (dd, J = 8.4, 2.0 Hz,

1H), 7.59 (d, J = 2.0 Hz, 1H), 6.88 (d, J = 8.5 Hz, 1H), 3.92 (s, 1H), 3.49 – 3.32 (m, 6H),

3.20 (t, J = 6.2 Hz, 2H), 2.44 (t, J = 6.5 Hz, 2H), 1.41 (s, 9H), 0.83 (d, J = 14.1 Hz, 6H).

13C NMR (101 MHz, MeOD) δ 193.19, 175.05, 174.06, 148.38, 140.87, 125.93, 125.41,

122.52, 115.25, 82.68, 75.84, 68.48, 38.89, 38.61, 35.51, 35.36, 28.08, 27.86, 20.50,

19.14.

(R)-S-pantetheinyl-4-amino-3-(sec-butoxy)benzothioate (2-34d)

Compound 2-22d was prepared by the above procedure from 4-amino-3-(sec- butoxy)benzoic acid 2-33d. 1H NMR (400 MHz, MeOD) δ 7.48 (dd, J = 8.3, 1.8 Hz,

1H), 7.38 (d, J = 1.7 Hz, 1H), 6.71 (d, J = 8.3 Hz, 1H), 4.40 (m, 1H), 3.92 (s, 1H), 3.58 –

3.37 (m, 6H), 3.16 (t, J = 6.6 Hz, 2H), 2.44 (t, J = 6.6 Hz, 2H), 1.82-1.64 (m, 2H), 1.33

(d, J = 6.1 Hz, 3H), 1.03 (t, J = 7.5 Hz, 3H), 0.94 (s, 6H). 13C NMR (101 MHz, MeOD) δ

189.63, 174.62, 172.51, 144.87, 144.07, 125.29, 122.32, 112.41, 111.11, 75.98, 75.79,

69.01, 39.11, 38.95, 35.03, 34.36.95, 28.76, 27.51, 19.89, 19.53, 18.15, 8.63.

2.4.9. Synthesis of AMB-CoA analog 2-27

4-amino-3-methoxybenzoic acid 2-34 (1.4 mg, 8.7 µmol), coenzyme A (sodium salt hydrate, 9 mg, 13 µmol), and benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP, 6.7 mg, 13 µmol) were dissolved in 500 µL of a

tetrahydrofuran/ddH2O (1:1, v/v). N,N-Diisopropylethylamine (DIPEA, 15 µL, 87

µmol) was added, the reaction mixture was stirred at 25 °C for 2 h. and then lyophilized.

The dried product was resuspended in 10% aqueous acetonitrile and purified by preparative HPLC. The product-containing fractions were combined and lyophilized

1 yielding AMB-CoA 2-27 as a white solid. H NMR (400 MHz, D2O) δ 8.45 (s, 1H), 8.10

(s, 1H), 7.37 (dd, J = 8.3, 1.6 Hz, 1H), 7.21 (d, J = 1.6 Hz, 1H), 6.70 (d, J = 8.3 Hz, 1H),

6.06 (d, J = 5.6 Hz, 1H), 4.75, 4.55 (m, 1H), 4.23 (m, 2H), 4.00 (s, 1H), 3.85 (s, 3H),

3.82 – 3.43 (m, 6H), 3.18 (m, 2H), 2.43 (m, 2H), 0.89 (s, 3H), 0.75 (s, 3H). 13C NMR

(101 MHz, D2O) δ 192.92, 174.78, 174.09, 155.40, 152.65, 146.42, 143.10, 139.66,

126.07, 122.86, 113.91, 108.95, 86.60, 74.01, 71.97, 55.84, 38.96, 38.43, 35.44, 28.04,

23.28, 20.94, 18.25.

2.4.10. Overexpression and purification of CysG1

CysG1 (adenylation domain and peptidyl-carrier-protein of CysG module 5,

(Baumann et al., 2014) was cloned into a pHisTEV plasmid (derived from pET30a).

Single colony transformants of E. coli BL21(DE3) carrying pHisTEV-CysG1 were grown aerobically for 12-15 h at 37 °C in 10 mL of LB medium supplemented with 50

µg/mL kanamycin (LB-kan). Cells from the starter culture were added to 1 L of LB-kan, and the culture was grown aerobically at 37 °C until the A600 was 0.6-0.8. Expression was induced by the addition of 1 mM IPTG and the culture was grown aerobically at 15

°C for an additional ~ 20 h. Cells were harvested by centrifugation. The medium was discarded and the cell paste was stored at –80 °C until use. Cells were thawed, resuspended in 20 mL of Lysis Buffer (50 mM potassium phosphate, pH 7.8, 150 mM

NaCl), and then lysed by sonication on ice for 6 × 20 s using a Misonix Sonicator 3000

(24 W, 1.5 s pulse, 1.5 s pause). Cell debris was removed by centrifugation and the lysate was loaded onto a Histrap column pre-equilibrated with lysis buffer. The column was washed with 5-10 column volumes of wash buffer (100 mM Tris-HCl, 30 mM

Imidazole, pH 7.5). The protein was then eluted using elution buffer (100 mM Tris-HCl,

300 mM NaCl, 250 mM imidazole, pH 7.5). Brown colored fractions containing the desired protein were pooled and buffer exchanged to final buffer (100 mM Tris-HCl, pH

7.5 at 25 °C, 30% glycerol) via Econo-Pac 10DG size exclusion chromatography. The purified enzyme was stored in liquid nitrogen. Protein concentration was measured by the absorbance at 280 nm (A280) with an extinction coefficient calculated using the

ProtParam tool of the ExPASy proteomics server.

2.4.11. Chemoenzymatic synthesis of the 4-amino-3-methoxybenzoate (AMB)

CysG1 thioester 2-28

A typical enzymatic reaction for synthesizing the AMB CysG1 thioester 2-28 was performed in 100 mM phosphate buffer, pH 7.5 containing the Svp phosphopantetheinyl transferase (20 µM), CysG1 (43 µM), AMB-CoA (0.3 mM) and

MgCl2 (10 mM). The reaction was allowed to proceed aerobically at room temperature for about 6 hrs and was quenched by desalting using a Bio-spin 6 column (Bio-Rad).

Then the sample was injected onto a reverse phase HPLC Zorbax 300SB-C8 3.5µ,

3×150 mm column operating at room temperature coupled to an ESI-TOF mass spectrometer operating in positive ionization mode. LC-MS analysis of the reaction mixture demonstrated the expected around 490 Daltons mass increase to CysG1

resulting from the formation of the phosphopantetheinyl-AMB thioester 2-28 (Figure

2.18).

Figure 2.18 MS analysis of AMB-CysG1 thioester 2-28. A: CysG1 thioester 2-28 (calculated monoisotopic mass: 73189.7 Da, experimental mass: 73190.6 Da) and B: CysG1 without modification (calculated monoisotopic mass: 72700.5 Da, experimental mass: 72699.3 Da).

2.4.12. Reconstitution of the CysS-catalyzed methyl transfer reaction

AMB-CysG1 thioester (53 µM), prepared as described above, was buffer exchanged into anaerobic 100 mM phosphate buffer, pH 7.5 using a Bio-spin 6 column in a glove box and then treated with 0.28 mM MeCbl, 0.56 mM SAM and 0.6 mM methyl viologen, 1.8 mM NADPH and 22 µM CysS or 44 µM CysS (The formation of trimethylated products required higher enzyme concentrations). The reaction was

allowed to proceed anaerobically at room temperature for about 14 hrs. The protein in the reaction mixture was desalted using 30K Amicon Ultra-0.5 mL centrifugal filters.

LC-ESI-TOF-MS on protein samples was performed using an Agilent 1260

HPLC system equipped with a binary pump and a 1200 series diode array detector followed by a MicroToF-Q II mass spectrometer (Bruker Daltonics) using an ESI source in positive mode. The analysis was performed on a Zorbax 300SB-C8 column (3 ×150 mm column, 3.5 µm particles, Agilent).

LC conditions: A- 0.1 % formic acid in H2O, B- 0.1 % formic acid in acetonitrile

LC method: 0 min – 90% A, 10 min – 80% A, 25 min – 10% A 90% B, 27 min –

90% A, 30 min – 90% A.

The mass spectrometer parameters for protein analysis: Capillary, 4500 V; end plate offset, 500 V; nebulizer gas, 3.0 bar; dry gas, 10.0 L/min; dry gas temperature,

220°C; funnel 1 RF, 400.0 Vpp; funnel 2 RF, 250 Vpp; quadrupole, Ion energy, 4.0 eV; low mass, 200 m/z; collision cell, collision energy, 8.0 eV; collision RF, 1000.0 Vpp; transfer time, 120.0 µs; prepulse storage, 10.0 µs. Data were processed with

DataAnalysis ver. 4.1 (Bruker Daltonics)

3. MECHANISTIC STUDIES OF SEQUENTIAL METHYLTIONS BY CysS*2

3.1. Introduction

Methylation has played a crucial role in many biological processes. The classic nucleophilic substitution is well established to generate the methylated product. There is not much known for the methylations at non-nucleophilic centers in numerous natural products, which have been recently found to be catalyzed by the enzymes in the radical

SAM superfamily. Among them, cobalamin-dependent radical SAM methyltransferases can catalyze unusual methylations on diverse classes of substrates, including sp2- hybridized, sp3-hybridized carbon atoms, and phosphinate phosphorous atoms.20

Although, they are widespread in nature and have been first discovered in 1995,58 it was not until recently more and more enzymes in this class have been reconstituted in vitro, such as PhpK for the phosphinic acid P-methylation59, GenK and Fom3 for the alcohol

C-methylation26, 28, ThnK for the iterative C-methylation to form the ethyl group30, TsrM for the aromatic C-methylation32, PoyC for the valine residue C-methylation33 (Figure

3.1). However, details of mechanistic studies are still hindered because of the poor activity of the protein and substrates complexity.

* Wang, Y.; Begley, T. P. Mechanistic studies of cobalamin dependent radical SAM enzyme CysS. Manuscripts in Preparation.

1 O 2 O O O CH3 P R PhpK Fom3 H H C P R P P 3 RO OH RO OH O O O NHAc NHAc O

2-60 2-61 2-62 2-63

H OH 3 H3C OH 4 HO HO NH H H H H 2 NH2 H2N O GenK H N HO NH 2 O ThnK 2 HO NH2 SR SR O O O N N OH O OH O O OH OH HO HO O O NHMe NHMe

2-64 2-65 2-66 2-67

5 O O 6 OH OH

NH2 NH2 H PoyC H TsrM R N R N CH N R' N R' 3 H N N H O H H O

2-68 2-69 2-70 2-71

Figure 3.1 Selected secondary metabolites whose biosynthesis include class B RSMTs that have been reconstituted in vitro. Carbon atoms that are introduced by class B RSMTs are highlighted by red.

After it is shown that CysS can perform sequential methylations on a methyl group to form ethyl, isopropyl, sec-butyl, and t- butyl groups on p-aminobenzoate thioester by a radical mechanism in vitro, other combinations on methyl group of p- aminobenzoate thioester in cystobactamids analogs were also explored. At first, only isopropoxylation was discovered on the 3-hydroxy-4-aminobenzoic acid moieties in the antibiotic cystobactamids from Cystobacter sp.51 Later, ethyl and sec-butyl groups in addition to the isopropyl group were also found in the new cystobactamids derivatives. 60

Some of these new derivatives display significantly improved antibacterial activities in the low µM range against Gram-negative pathogens.60 Studying the mechanism of how

these modifications occur in nature will help us better understand the structure–activity relationships of antibiotic discovery.

A mechanism is proposed for CysS in Figure 3.2. After initial formation of methylcobalamin by SAM mediated methylation, a second SAM is reductively cleaved by the [4Fe− 4S]+1 cluster to generate the 5′-dA •. This 5′-dA • abstracts a hydrogen atom from the methyl group of the substrate 2-23 to give the substrate radical 2-25, which then undergoes a radical substitution with methylcobalamin to give the ethyl ether

2-24a. Regeneration of MeCbl from Cbl(II) can be achieved by reduction to Cbl(I) by the [4Fe−4S]+1 cluster followed by SAM-mediated methylation. Repetition of this sequence results in the successive formation of the isopropyl, t-butyl, and sec-butyl ethers of 2-23.

Co(I) Co(III) 2-23,e- O SR Co(III) O SR Co(III) CH3 Met Me CH3 CH3 H S S H Ade R' Ade H R' O O C C H O H O H SAH 2-23 2-23 NH2 Me NH OH OH OH OH 2 S R' Ade CH2 Ade SAM SAH O O

[4Fe-4S]2+ [4Fe-4S]2+ [4Fe-4S]1+ OH OH [4Fe-4S]2+ OH OH SAM

Figure 3.2 Proposed mechanism for CysS-catalyzed iterative methylations.

Because of the poor activities from the inclusion bodies and substrate complexities, detailed mechanism studies were not available for B12-dependent radical

SAM methyltransferase, except for few stereochemistry studies26-29. Here, we are able to use a radical clock probe to trap the substrate radical and estimate the radical substitution of a methyl group from cobalamin in the mechanism. A brominated substrate analog is designed to trap the Cbl(I) intermediate, which is the critical species for the regeneration of MeCbl during each turnover. The resulting crosslinking species also provides useful information on structural orientations of cofactors in the enzyme active site. Competitive

and intrinsic isotope effects were also obtained to investigate the transition step of hydrogen abstraction step during the reaction.

3.2. Result and discussion

3.2.1. Measure methyl transfer rate with a radical clock probe

During the CysS catalytic cycle, it is proposed that the substrate radical can undergo a radical substitution with methylcobalamin to give the methylated product.

Since all the classic B12-dependent methyltransferases go through nucleophilic attack of the substrate to get methylated, the racial mechanism proposal was considered speculative for a long time. Even though such methyl group transfer from MeCbl to a carbon-based radical exists in cobalamin model studies and radical substitutions are widely used in organic synthesis, there is no such precedent of a radical species get methylated by MeCbl for the B12-dependent methyltransferases in nature until more and more B12-dependent radical SAM enzymes were discovered recently. For GenK, both nucleophilic and radical mechanism are proposed for the methyl group transfer from the

MeCbl to the substrate and currently there is no experimental evidence that can distinguish them. But for ThnK, Fom3, and PoyC, it’s more likely that radical mechanism is responsible for the methylation of the substrates.

In order to probe the radical substitution step for methylation, a cyclopropyl analog 2-41 was designed. The ring opening by the cyclopropylcarbinyl radical is a well- known fast radical reaction, which has been used as a radical trap and a measurement of the unknown rate in many competition reactions. The reported cyclopropylcarbinyl radical ring opening rate constant of (methoxymethyl) –cyclopropane 2-47 is 2 × 107 s−1.

When cyclopropyl analog was incubated with CysS, SAM, MeCbl, MV and NADPH,

LC-MS analysis of the CysS reaction mixture showed the formation of one of the new products had a mass corresponding to the methylated product 2-43, which was confirmed by the synthetic standard (Figure 3.3A, B). A large amount of 5′-dA formation was also observed in the same reaction when compared with the no substrate control, which further demonstrated the cyclopropyl analog was accepted as a substrate for CysS. When the reaction mixture was treated with 1% TFA, the ring opening derived product aminophenol compound 2-46 was detected by LC-MS (Figure 3.3C). The ratio of the methylated compound 2-43 and aminophenol compound 2-46 was quantified by using the authentic standard as 12.0. If we use the literature reported value (2 × 107 s−1) to estimate the value of the ring opening rate constant of the cyclopropyl substrate 2-41

(kring), we can obtain the rate constant of the methyl group transfer from MeCbl to substrate radical (kme) based on the ratio of the final products 2-43 and 2-46. The

8 −1 estimated kme is around 2.4 × 10 s , which has never been estimated before. Since the cyclopropyl analog is a methoxybenzothioate derivative instead of a simple methoxy group as in the reference reaction and there might be a conformational restriction at the enzyme active site to create stereoelectronic effects, the exact ring opening rate constant of cyclopropyl analog would be lower than 2 × 107 s−1. Thus, the rate constant of the methyl group transfer by radical substitution could be lower than 2.4 × 108 s−1.

Nevertheless, the methyl transfer rate constant is comparable with the ring opening rate constant of cyclopropyl analog 2-43. In solution chemistry, it’s reported that a ((6-iodo-

2-methylhexan-3-yl)oxy)diphenyl(trimethylstannyl)silane species can go through an

intramolecular homolytic substitution reaction at silicon (SHi) to form a cyclic five- membered alkoxysilanes and a trimethylstannyl radical at a rate constant as 1 × 107 s−1.61

The radical substitution rate constant could be larger when the bond orientation is restrained in the enzyme active site.

Intens . 061716 Full p228O4 HPLC_1-A,4_01_28827.d: +MS, 29.5-29.9min #1753-1777 x104 1+ O 1.25SR 482.2319 A E O SR 1.00 O 0.75 NH2 O 2-43 NH2 0.50 1+ 483.2360 B 2-43 0.25 1+ 484.2334 475.3258 480.2137 0.00 Intens . 472 474 476 Full O4HPLC HCl_1-A,3_01_28917.d: +MS, 23.0-23.3min #1370-1386478 480 482 484 486 488 m/z

C 2500 F 414.1708

2000 O SR

1500 D O SR

1000 OH NH2 OH 500 2-46 2-46 NH2 412.7828 405.2319 419.0198 0 400.0 402.5 405.0 407.5 410.0 412.5 415.0 417.5 420.0 m/z 10 15 20 25 30

Retention time (minutes)

Figure 3.3 CysS catalyzed reaction with cyclopropyl analog, extracted ion chromatograms (EICs) of the methylated cycropropyl product 2-43 [M + H]+ (482.23 ± 0.02) for (A) Synthetic standard; (B) CysS catalyzed reaction; EICs of the ring opening derived product 2-46 [M + H]+ (414.17 ± 0.02) for (C) CysS catalyzed reaction; (D) Synthetic standard; (E) Mass spectrum of the methylated cyclopropyl product 2-43 in CysS catalyzed reaction (Exact mass: 482.2319, Accuracy: <1 ppm); (F) Mass spectrum of the ring opening derived product 2-46 in CysS catalyzed reaction (Exact mass: 414.1693, Accuracy: 3.6 ppm); (G) Strategy for measuring the methyl transfer rate using the ring opening rate of cyclopropylcarbinyl radical.

G O O O SR O SR O SR R: S N N OH H H OH CysS !'e

MeCbl O O SAM 5’dA O NH NH2 NH2 2 2-41 2-42 2-43 ! #$%& O SR O SR O SR !′#$%& Acid O O OH 2-47 2-48 O O R NH2 NH2 NH2 7 −1 !′#$%& = 2 × 10 s 2-44 2-45 2-46

Figure 3.3 continued

3.2.2. Trapping a Cbl(I) intermediate by brominated substrate analog

Since the rate of the methyl transfer from Cbl to the substrate is very fast, we can expect that the carbon atom from the methoxyl group of the substrate should be in close vicinity of the carbon from the MeCbl. As we proposed Cbl(I) species is formed for the regeneration of the MeCbl (Figure 3.1) and Cbl(I) is known as a super nucleophile, if we replace the hydrogen on the methoxy carbon with a good leaving group, like a bromide,

Cbl(I) can have a nucleophilic substitution to form a cobalamin substrate cross-linking species (Figure 3.4A). In order to explore this idea, brominated substrate analog 2-49 was synthesized and was first tested in the model reaction to investigate whether Cbl(I) can react with brominated substrate analog 2-49 to generate the cross-linking species.

Cbl(I) was first chemically reduced from Cbl(III) by using an excess of NaBH4 anaerobically, indicated by the UV spectrum (Figure 3.5). When brominated substrate analog 2-49 was added to the reduced Cbl(I) reaction mixture, the color immediately 52

changed from light gray to pink, which suggested a Cbl(III) species was formed. Further

HPLC and LCMS analysis of the reaction mixture revealed the new species had a similar

UV spectrum of Cbl(III) and had a MS corresponding to the Cbl crosslinking species 2-

50 (Figure 3.6).

A O SR O SR O SR

H H H H H H C C C O O CH3 O CH3 NH NH 2 CH3 NH2 2

Co(III) Co(II) Co(I)

O SR O SR

H H Bromo substrate trap H H C Br C O C O CH H H 2 NH2 NH2 Co(III) Co(I)

B O O S N H O Br O S O N H NH2 NaBH4 2-49 OHCbl (III) Cbl (I) O NH MeOH 2 Co(III)

2-50 Figure 3.4 (A) Strategy of developing the brominated substrate analog to trap Cbl(I) species (B) Model reaction for cobalamin cross-linking species through Cbl(I)

NaBH4 NaBH4 OHCbl (III) Cbl (II) Cbl (I) H2O H2O

2 HOCbl 1.8

1.6 Add NaBH4 10 min

1.4 Add NaBH4 15 min 1.2 more NaBH4 1

0.8 more NaBH4 7 min Absorbance(AU) 0.6 more NaBH4 16min 0.4 more NaBH4 40 min 0.2

0 more NaBH4 1h 250 300 350 400 450 500 550 600 650

Wavelength (nm)

Figure 3.5 UV spectrum of monitoring the chemical reduction of from Cbl(III) to Cbl(I)

In t e n s . +MS, 24.2-24.3min #(1438-1442) x104 805.8431 2.0 806.3440 805.8431 Accuracy: 0.4 ppm 1.5

806.8444 1.0 2-50

0.5 807.3443

807.8424

0.0 806 808 810 812 814 816 m/z

In t e n s . +MS2(806.3682), 24.1-24.3min #(1435-1443) x104 665.2925

0.8

0.6

359.0989 0.4

147.0903 0.2

971.4816 486.7388 0.0 200 400 600 800 1000 1200 1400 m/z Figure 3.6 Upper panel: MS of the substrate cobalamin adduct 2-50 in the model reaction; lower panel: MS/MS of the substrate cobalamin adduct 2-50 in model reaction

Then, brominated substrate analog 2-49 was incubated with CysS, OHCbl, MV,

NADPH, a new peak was detected by LC-MS, which has a MS corresponding with the

Cbl substrate crosslinking species 2-50 (Figure 3.7B). This peak is absent when CysS,

Cbl, 2-49 was excluded in the enzymatic reaction (Figure 3.7A). Also, the new peak had the same retention time, MS and MS/MS spectrum as the compound 2-50 formed in the model reaction (Figure 3.8, 3.9).

A B Intens . Full BNAC_1-B,1_01_26584.d: +MS, 23.7-24.0min #1412-1428 x106 2+ 2+ 805.8488806.3504 1.5 Full reaction 2+ 806.8493 1.0 2+ 807.34772+ 807.8464 0.5 805 806 807 808 Full reaction- SAM 0.0 Full reaction- substrate 803 804 C 809 810 m/z

Full reaction- CysS

Full reaction- OHCbl

Full reaction- reducing system

10 15 20 25 30 2-50 Retention time (minutes)

D O SR O SR β‐scission + Co(II) O SR Light O HO

NH2 ethene NH2 H 2-51 2-52 H C O CH2 e-, H+ NH2 Co(III)

2-50 CN O SR NaCN Co(III) +

NH2 CNCbl 2-21

Figure 3.7 CysS-catalyzed reaction with brominated analog 2-49. (A) LCMS analysis of the CysS reaction with brominated analog. EICs of the adduct 2-50 [M + 2H]2+ (805.84 ± 0.02), red trace represents full reaction; green, black, blue, pink and black trace represent the full reaction without SAM, substrate, CysS, OHCbl and reducing system, respectively. (B) Mass spectrum of the adduct 2-50 in CysS catalyzed reaction (Exact mass: 805.8464, Accuracy: 9.4 ppm). (C) Structure of the Cbl substrate crosslinking species 2-50 (D) Different products generated from the crosslinking species under light condition or with NaCN. 56 In t e n s . x104 4

3 - CysS reaction - Model reaction 2

0 0 5 10 15 20 25 30 35 Time [min] 040616OHCbl NaBH4 BNAC_1-B,1_01_26911.d: EIC 805.8453±0.02 +All MS BrNAC OHCbl No SAM_1-E,2_01_26896.d: EIC 805.8453±0.02 +All MS EIC of 805.84

Figure 3.8 LC-MS detection of the CysS-catalyzed reaction with brominated analog 2-49 and the model reaction. EICs of the substrate cobalamin crosslinking adduct 2-50 [M + 2H]2+ (805.84 ± 0.02).

In t e n s . In t e n s . +MS, 24.1-24.2min #(1431-1435) x104 805.8434 +MS, 24.2-24.3min #(1438-1442) x104 805.8431

2.0 806.3440 806.3437 805.8434 805.8431 3 1.5 Accuracy: 0.4 ppm

2 806.8444 806.8450 1.0 Model reaction CysS reaction

1 807.3479 0.5 807.3443

807.8489 816.8313 807.8424 0 0.0 806 808 810 812 814 816 818 m/z 806 808 810 812 814 816 m/z MS MS B In t e n s . In t e n s . +MS2(806.3682), 24.1-24.3min #(1435-1443) +MS2(806.3248), 24.0-24.1min #(1428-1432) 4 x104 Model reaction x10 665.2943 665.2925 1.5 CysS reaction 0.8

0.6 1.0

359.0989 0.4 359.1007 0.5 147.0903 0.2 147.0907 971.4816 971.4693 486.7388 486.2370 0.0 0.0 200 400 600 800 1000 200 1200 400 1400 m/z 600 800 1000 1200 1400 m/z MS-MS MS-MS

Figure 3.9 LC-MS detection of the CysS-catalyzed reaction with brominated analog 2-49 and the model reaction. (A) MS spectrum at 24 min. (B) Mass fragments of 805.84.

To further confirm the formation of the carbon cobalt covalent bond, the enzymatic reaction was exposed to the light (Figure 3.7D). After light treatment, the

57 amount of the substrate Cbl crosslinking species 2-50 decreased while the intensity of several peaks was increased (Figure 3.10).

Intens . [mAU] x105 2.5 - Full reaction in dark - Full reaction under light 2.0

1.5

1.0

0.5

0.0 0 5 10 15 20 25 30 35 Time [min] Full BNAC light 4h_1-C,2_01_26591.d: UV Chromatogram, 325 nm Full BNAC dark _1-C,1_01_26590.d: UV Chromatogram, 325 nm UV of 325 nm

Figure 3.10 UV spectrum of CysS catalyzed reaction with (blue) and without (red) exposure to light.

By co-elution experiment with the synthetic standard, the peak at 21 minute corresponds to aminophenol compound 2-52 and the peak at 25 minute corresponds to the ethyl compound 2-21(Figure 3.11). Since carbon cobalt is considered weak, under the light condition, the carbon cobalt bond can go through homolytic cleavage to form ethyl radical 2-51, which either have a beta scission to form aminophenol compound 2-

52 or abstract a hydrogen atom to form the ethyl compound 2-21(Figure 3.7D). In order to test whether the radical abstract a hydrogen atom from the solvent, the reaction was performed in D2O. Compared with the H2O buffer, M+1 peak of 2-21 has increased from

19% to 65% of the M peak (Figure 3.12).

In t e n s . 5 x10 A 0.8 - CysS catalyzed reaction 0.6 - Co-elution with sythetic ONAc

0.4

0.2

0.0 0 5 10 15 20 25 30 35 Time [min] 031516 Full8 5 dilute f orONAC_1-D,5_01_26637.d: EIC 255.0798±0.05 +All MS EIC of 255.08 031516 Full8 5dilute coeluONAC_1-D,5_01_26639.d: EIC 255.0798±0.05 +All MS

Intens . 1+ BrNAC OHCbl No SAM_1-A,4_01_26873.d: +MS, 21.5-21.8min #1279-1295 x104 255.0781 2.0 B M+H+: 255.0781 Accuracy: 6.6 ppm

1.5 1+ 277.0605

1.0 M+H+: 255.0798

0.5

269.0425 0.0 240 250 260 270 280 m/z MS at 21 min

Figure 3.11 LC-MS detection of 2-52 in the CysS reaction using brominated analog 2-49 as substrate. (A) EICs of the aminophenol compound 2-52 [M + H]+ (255.08 ± 0.02). (B) Mass spectrum of the aminophenol compound 2-52.

In t e n s . 5 x10 A 3 - Full reaction - EthylNAC standard 2 - Coelution

0 0 5 10 15 20 25 30 35 Time [min] 87NACethyl 100uM 20ul_1-D,1_01_26633.d: EIC 283.1111±0.02 +All MS Full BNAC dark _1-C,1_01_26590.d: EIC 283.1111±0.02 +All MS 031516 Full8 coeluteethyl 50uM_1-D,3_01_26635.d: EIC 283.1111±0.02 +All MS EIC of 283.11

In t e n s . +MS, 25.5-25.9min #(1517-1541) x105 283.1108 2.0 B M+H+: 283.1108 H2O buffer

1.5 Accuracy: 3.0 ppm 281.0953 M+H+: 283.1111 1.0

0.5 284.1126

282.0982 285.1087 283:284 = 1: 0.19 0.0 275 280 285 290 295 m/z MS at 25 min

In t e n s . +MS, 25.7-25.7min #(1527-1531) x105 C 281.0959 283.1093 1.5

1.0 284.1158 283:284 = 1: 0.65 0.5 D2O buffer 282.0978 285.1146 286.1161 0.0 280 282 284 286 288 290 292 294 m/z MS at 25 min

Figure 3.12 LC-MS detection of the ethyl compounds in CysS reaction using brominated analog 2-49 as substrate. (A) EICs of the ethyl compound 2-21 [M + H]+ (283.11 ± 0.02). (B) Mass spectrum of the ethyl compound 2-21 in H2O buffer. (C) Mass spectrum of the ethyl compound in D2O buffer

The reaction mixture was also derivatized by adding cyanide (Figure 3.7D).

Complete consumption of the crosslinking species 2-50 was detected with the formation of the aminophenol compound 2-52 and cyanide cobalamin (CN-Cbl) by HPLC (Figure

3.13).

In t e n s . [mAU] Green : CysS catalyzed reaction with brominated substrate 2000 Red : after adding NaCN to the reaction 1500 Cbl adduct

1000

500 CNCbl

0 5 10 15 20 25 30 35 Time [min] No SAM_1-B,2_01_26585.d: UV Chromatogram, 325 nm UV of 325 nm031516 No SAM NaCN 2h_1-D,4_01_26636.d: UV Chromatogram, 325 nm

In t e n s . [mAU] x106 1.0 B - Derivatization of the reaction

0.8 - CNCbl standard

0.6

0.4

0.2

0.0 0 5 10 15 20 25 30 35 Time [min] 031516 No SAM NaCN 2h_1-D,4_01_26636.d: UV Chromatogram, 325 nm CNCb l 2 5 0 u M 1 5 u l_ 1 - D, 6 _ 0 1 _ 2 6 6 4 1 . d : UV Ch r o ma t o g r a m, 3 2 5 n m UV of 325 nm

Figure 3.13 HPLC spectrum of derivatization of CysS catalyzed reaction using brominated analog 2-49 as substrate by cyanide

In methionine synthase, Cbl(I) is the active species for the methyl transfer. Since we are not able to detect Cbl(I) by UV during the CysS reaction, it implies that Cbl(I) was transient and strongly coupled with the methyl transfer, which has also been observed for methionine synthase. However, we are able to design an electrophile Br-

NAC 2-49 to trap the nucleophile Cbl(I) species in the CysS active site. In CysS, Cbl(I) is also responsible for the regeneration of MeCbl using SAM as a methyl donor.

Here, Cbl(I) is methylated by Br-NAC 2-49 instead of SAM in the native reaction. This suggests that Br-NAC 2-49 is a better substrate for Cbl methylation than

SAM, and the ethyl group of 2-49 is closer to cobalt than the methyl group of SAM, or it blocks SAM to methylate Cbl in a certain amount of time. Since a large amount of 5′-dA

was not detected when Br-NAC 2-49 was used as a substrate for CysS, it indicated that radical chemistry was not favored unless the MeCbl was formed. When Cbl substrate crosslinking species was incubated with SAM, CysS and the reducing system, an increased amount of 5′-dA was detected (not as much as the native substrate). This indicated that radical chemistry was not initiated until the Cbl gets methylated, and the substrate is present. However, the binding affinity of the Cbl substrate crosslinking species probably is not as good as the native substrate. That’s why following radical mediated methylation step was not observed for the brominated substrate analog. The

Cbl substrate crosslinking species closely mimics the conformation when the methyl substrate is methylated by MeCbl to form ethyl group and suggests that the substrate is situated between MeCbl and SAM (Figure 3.14).

O OH 1+ NH2 S Fe S Fe S Co(III) OH Fe S O S Fe O OH A

H2N COSR A: adenine

Figure 3.14 Proposed orientation of adduct and cofactors in the CysS active sites

3.2.3. Identification of physiological cobalamin cofactor for CysS

As MeCbl can be regenerated in enzyme active through Cbl(I) species, we are wondering whether MeCbl function as an enzyme cofactor or a substrate for CysS.

CD3Cbl was synthesized and incubated with CysS, SAM, MV, NADPH. Unexpectedly, a majority of the methyl group was deuterium labeled even under single turnover condition (Figure 3.15). Furthermore, when CD3SAM was used with an excess of

CH3Cbl, 76% of isopropyl product was deuterium labeled (When mixed MeCbl and

OHCbl were used with CD3SAM, the percentage of deuterium incorporation will increase with the increased amount of OHCbl). It was found that OHCbl can be reduced to Cbl(II) more readily by MV, NADPH than MeCbl. These results indicate that CysS prefer the non-methylated Cbl than the methylated Cbl. CysS can perform sequential methylations with a catalytic amount of Cbl, which suggests that MeCbl is regenerated in situ in the enzyme active site. A SN2 mechanism requires in-line geometry of the methyl group donor- SAM and the methyl group acceptor-Cbl. Br-NAC 2-49 analog can also have a similar methyl transfer reaction, which suggests that the substrate and SAM probably share one binding site in at least one of the conformation. Thus, it’s very likely that SAM first methylates Cbl and then the substrate enters into the enzyme active site followed by the hydrogen abstraction, methylation. Then, the methylated substrate left the active site or have a substantial conformational change to allow the methyl group of the SAM align on the top of cobalamin for re-methylation for the next catalytic cycle.

A O SR O SR O SR

CysS, SAM, CD3Cbl + Reducing system H D O O O H D NH2 NH2 H NH2 D 2-24a 2-24b 2-24e

1 : 0.21

O SR O SR O SR

CysS, CD3SAM, CH3Cbl + Reducing system H D O O O H D NH2 NH2 H NH2 D 2-24a 2-24b 2-24e 0.26 : 1 Intens . Intens . Full CD3Cbl_1-B,2_01_24326.d: +MS, 29.1-29.6min #1728-1762 Full MeCbl CD3SAM_1-B,1_01_24976.d: +MS, 27.3-27.6min #1632-1646 x104 1.0 1+ B 456.2155 6000 C 459.2348 0.8

0.6 4000

0.4 1+ 2000 456.2150 457.2188 460.2396 0.2 1+ 459.2342 2+ 457.2191 458.2164 455.3509 461.2353 462.3447 460.2369 458.2211 0.0 0 452 454 456 458 460 462 455464 m/z456 457 458 459 460 461 462 463 m/z

Figure 3.15 CysS catalyzed methylation with (A) CD3Cbl, SAM (top) or CD3 SAM, CH3Cbl (bottom) in the presence of reducing system. (B) MS spectrum of the isopropyl product when CD3Cbl, SAM was used. (C) MS spectrum of the isopropyl product when CD3 SAM, CH3Cbl was used.

3.2.4. Measurement of the competitive and intrinsic isotope effect

The vast majority of radical SAM enzymes are known for generation of 5′dA•,

which can abstract a hydrogen atom from the substrate. Isotope effects have been used as

a powerful tool to interrogate transition states of carbon hydrogen bond cleavage. CD3-

pantethinyl substrate 2-53 was synthesized and mixed with its protium substrate 2-23 in

1:1 ratio in order to measure the intermolecular isotope effect. The enzymatic reaction was quenched at different time point (10 mins, 30 mins, 1h, 2.5h, 4h, 5.5h) and then analyzed by LC-MS/MS. The ratio of the methylated protium products 2-24a and the methylated deuterium product 2-54 was measured and the competitive isotope effect is

17 ± 0.3.

Usually, the kinetic isotope effect can be masked by slower chemical steps which are not isotope-sensitive and by slow product release. In order to obtain the intrinsic isotope effect for hydrogen abstraction, CD2H-pantetheinyl substrates 2-55 were synthesized. Here, we assumed that the three hydrogens on the methyl group could rotate freely in the enzyme active site and substitutions with deuterium will not have a significant effect. The KIE for CD2H-pantetheinyl substrates 2-55 was 18 ± 0.6 measured based on the products ratio (Figure 3.16). The large intermolecular isotope effect indicates that the C-H bond breaking happens before or at the first irreversible step in the CysS enzymatic reaction. The small difference between intrinsic isotope effect and the intermolecular isotope effect also suggests that there is no slow step that precedes the hydrogen abstraction step. Furthermore, all isotope effects are larger than the semiclassical limit predicted by transition state theory. Such large kinetic isotope effects are often interpreted as an indication of quantum mechanical tunneling of the hydrogen atom. This kind of substantial isotope effects has also been reported for other radical

SAM enzymes in the literature, like lipoyl synthase, biotin synthase (Figure 3.17).62-63 In the lipoyl cofactor biosynthesis, lipoyl synthase (LipA) catalyzes the insertion of two

sulfur atoms at C6 and C8 of an (N6-octanoyl)-lysyl residue on a lipoyl carrier protein

(LCP) in the final step of lipoyl cofactor biosynthesis. When 8,8,8-trideuterooctanoyl peptide was used as a substrate for LipA, a large isotope effect was observed with only minute quantities of the lipoyl product which the C8 hydrogen is abstracted64. Because of this significant isotopic discrimination, Booker’s group was able to trap a covalent cross-link intermediate between the peptide and LipA before the second hydrogen abstraction from C865.

O SR O SR

O SR MeCbl D D P1 kH O C D O C D Me D NH2 CH3 NH2 O C D 2-56 2-54 H Ad NH 2 O SR O SR 2-55 k CH2 D MeCbl Ad D D H P2 O C O C H NH Me 2 NH2 CH2D 2-57 2-58 Ad O O R: S N N OH H H OH

Figure 3.16 The strategy of intrinsic isotope effect measurement

A O O LipA N CH2 H N H H H S S N6-(octanoyl)lysine N6-(lipoyl)lysine

B O O O NH O NH H BioB H HN OH HN OH H H S

dethiobiotin biotin

Figure 3.17 (A) LipA in the biosynthesis of lipoic acid; (B) The conversion of dethiobiotin to biotin catalyzed by biotin synthase (BioB).

3.2.5. Efforts to obtain a crystal structure of CysS

Currently, no crystal structure is published for the B12 dependent radical SAM methyltransferase. The crystallography studies of CysS is collaborated with Dr. Drennan lab. From the size exclusion column analysis of purified CysS, it’s found that when

CysS is overexpressed and purified in E. coli B12, CysS can easily aggregate to form dimers, trimers until hexamers in solution. In order to solve the heterogeneous problem, different constructs were made for CysS, including cleavable His tag site on both C- terminal and N-terminal of the protein, a SUMO tagged and a C-terminal truncated version of CysS. We also overexpressed and made the peptide carrier protein CysG1 and native substrate of CysS – small molecule modified CysG1 to co-express or incubate with CysS in order to get homogeneous protein for crystallography studies. The best condition was obtained when CysS was co-expressed with a cobalamin uptake and

transport system in E. coli, developed by Dr. Booker.24 The cobalamin content and monomer species of CysS were increased. The crystallography studies are still in progress.

3.3. Conclusion

Based on the above result, we can build a detailed model of how CysS uses Cbl and radical SAM machinery to sequential methylate a methyl group to form ethyl, isopropyl, sec-butyl, and t- butyl groups (Figure 3.18). One molecule of SAM first binds to the [4Fe–4S] cluster in a conformation that can methylate Cbl. After the Cbl(I) is methylated by SAM via the SN2 mechanism, S-adenosyl homocysteine(SAH) is released. A second molecule of SAM binds to [4Fe–4S] cluster, because of the steric hindrance of MeCbl, the binding mode of the second molecule of SAM will be different from the first one, which favors the reductive cleavage and also opens up the active site for the binding of the substrate. Once the substrate enters into the active site, [4Fe–4S] cluster will cleave SAM to generate 5′-dA •, which abstracts a hydrogen atom from the methyl group of the substrate 2-23. The resulting radical 2-25 then undergoes a radical substitution with methylcobalamin to give the ethyl ether 2-24a. Regeneration of MeCbl from Cbl(II) can be achieved by reduction to Cbl(I) by the [4Fe−4S]+1 cluster followed by SAM-mediated methylation. Repetition of this sequence results in the successive formation of the isopropyl, t-butyl, and sec-butyl ethers of 2-23.

A B C

O 1+ O O SAH 1+ OH S Fe OH 1+ OH S Fe S Fe Fe S NH2 NH2 Fe S NH2 Fe S Co(I) Fe S (III)Co S (III)Co CH S Fe S 3H3C S Fe S OH S Fe OH OH S Fe S Fe O O SAM O OH OH OH A A A SAM SAH SAM

D E F

NH 1+ O NH 2+ NH 2+ S Fe O S Fe O S Fe O O O Fe S Fe S Fe S S Fe S S Fe S S Fe S (III)Co OH (III)Co OH (III)Co OH S Fe S Fe S Fe O OH O OH O OH O O 5'dA O A A A 5'dA H N COSR 2 H2N COSR H2N COSR

2-23 2-23 2-25

O O G A R: S N N OH H H OH A: adenine NH 2+ O 1+ O S Fe Met, 5dA OH O S Fe Fe S NH Fe S S Fe S 2 O Co(II) OH Co(I) S Fe S S Fe OH S Fe OH H2N COSR O O 5'dA O A SAM, 2e- OH A 2-24b H2N COSR SAM 2-24a

Figure 3.18 Proposed model for CysS catalyzed sequential methylation.

Based on the isotope labeling cofactors experiment and Br-NAC 2-49 probe, we propose that Cbl is first reduced to Cbl(I) and then get methylated by SAM (Figure

3.18A-B). No 5′-dA was detected when Cbl is absent in the reaction. The result of an increased amount of 5′-dA was detected when Cbl substrate crosslinking species was used as the substrate for CysS indicates that radical chemistry was not initiated until the

Cbl get methylated, and the substrate is present. The formation of Cbl substrate crosslinking species 2-50 also suggests that the substrate is sandwiched between the Cbl cofactor and the radical SAM machinery (Figure 3. 18D-G), which is different from 69

GenK. For GenK, hydrogen abstraction is stereospecific and the methylation occurs with retention of configuration at C6′ of the substrate, which indicates MeCbl donor likely sits adjacent to the radical SAM machinery in the GenK active site. For Fom3, GenD1, and MoeK5, the methylations occur with inversion of configuration at the carbon center.

These suggest that MeCbl and the radical SAM machinery are probably situated on the opposite sides of the center to be methylated once bound in the active site. Here, the Cbl substrate crosslinking species generated in the CysS active site instead of MeCbl further reassembles the geometry of substrates, cofactors in the later cases.

Based on our experimental results and OxsB crystal structure, SAM is proposed to have one binding site but two conformations for methyl transfer and radical initiation in CysS active sites (Figure 3. 18C-D). A radical clock probe is used to trap the substrate radical (Figure 3. 18F) and estimate the radical substitution rate of a methyl group from cobalamin for the first time. From the large competitive isotope effect, we know that

CysS is a distributive rather than a processive enzyme, with the sequential reaction allowing free dissociation of the ethyl and isopropyl products.

To conclude, we are able to develop different substrate analogs and isotope labeling compounds to trap important intermediates and build a model for the sequential methylation of CysS. It is known that the C-Co in MeCbl is considered weak (BDE:

129.7kJ/mol). Thus, nature uses MeCbl as a catalyst to perform radical substitution for unusual methylation on sp3-hybridized carbon on substrate radical. Here, CysS is able to take advantage of these two complicated pieces of machinery- radical SAM and cobalamin cofactors to perform sequential methylations to form t-butyl group by radical

chemistry. The detail mechanistic study of CysS will help to unravel other methyltransferases in this large B12 radical SAM superfamily.

3.4. Experimental methods

3.4.1. Quantification of enzymatic products in the CysS catalyzed cyclopropyl substrate analog reaction

A typical enzymatic reaction was performed in 100 mM phosphate buffer, pH 7.5 containing 68 µM of CysS with 0.5 mM MeCbl, 0.5 mM SAM, 0.5 mM substrate with reducing systems (0.8 mM methyl viologen, 65 mM NADPH). The reaction was incubated at room temperature in an anaerobic chamber containing 95% nitrogen and

5% hydrogen. For LC-MS analysis the enzyme was removed by ultrafiltration using a

10kDa cut-off filter (VWR). The enzymatic assay was split into equal amounts. One of them was used to quantified the methylated cyclopropyl product, the other half was used to quantify the ring opening derived product aminophenol compound by adding TFA

(final concentration 0.1%). Calibration curves were constructed by measuring the peak intensity of synthetic standard 2-46 and 2-43 with known concentration using LC-MS.

Then the calibration curves were used to measure the concentration of 2-46 and 2-43 in the enzymatic reaction. The ratio of the methylted products and the ring opening products is 12.0. If we used the literature reported value (2 × 107 s−1) to estimate the value of the ring opening rate constant of the cyclopropyl substrate (kring) in CysS reaction, we can calculate the rate constant of the methyl group transfer from MeCbl to

8 −1 substrate radical (kme) as 2.4 × 10 s .

350000 10000000 y = 420907x + 8836.1 300000 y = 2,039,503.60x R² = 0.996 8000000 250000 R² = 0.996 200000 O SR 6000000 O SR 150000 4000000 peak areapeak 100000 OH peak area NH2 50000 2000000 O NH2 0 0 0 0.2 0.4 0.6 0.8 0 1 2 3 4 5 Concentration/ µM Concentration/ µM

Figure 3.19 calibration curves for the ring opening products and the methylated product

3.4.2. Model reaction for generating the Cbl crosslinking species 2-50

The preparation of Cbl(I) species is followed the protocol in the literature. A solution of hydroxocobalamin (50 mg) and cobalt nitrate (1 mg) in water (10 ml) is deoxygenated with a stream of argon. After 10 min a deoxygenated solution of sodium borohydride (15 mg) in water (1 ml) is added to the red cobalamin solution, which immediately turns brown and then blue-green, signifying that the reduction is complete.

The reaction was also monitored by UV in anaerobic environment.

3.4.3. Generation and derivatization of the Cbl crosslinking species in CysS catalyzed reaction

90 µM of CysS was incubated with 1 mM MeCbl, 1 mM SAM, 1.2 mM Br-NAC

2-49 with reducing systems (1.2 mM methyl viologen, 3.5 mM NADPH) at room temperature in an anaerobic chamber in dark for 12-15 hours.

3.4.4. Measurement of the competitive isotope effect of CysS

80 µM of CysS was incubated with 1 mM MeCbl, 1 mM SAM, 1 mM of

87pantCH3 2-23 and 87pantCD3 2-53 with reducing systems (1 mM methyl viologen, 3 72

mM NADPH) at room temperature in an anaerobic chamber. The reaction was quenched at different time point (10 mins, 30 mins, 1h, 2.5h, 4h, 5.5h) by ultrafiltration using a

10kDa cut-off filter. The reaction mixtures were analyzed by LC-MS/MS and the ratio of the methylated protium products 2-24a and the methylated deuterium 2-54 product was measured. Here, 10 min time point was used to obtain the competitive isotope effect, because after 10mins the dimethylated products can be detected by LC-MS/MS.

3.4.5. Measurement of the intrinsic isotope effect of CysS

20 µM of CysS was incubated with 270 µM MeCbl, 540 µM SAM, 540 µM of

87pantCD2H with reducing systems (1 mM methyl viologen, 3 mM NADPH) at room temperature in an anaerobic chamber. The reaction was quenched at different time point

(30 mins, 1h, 2h, 3h, 4h) by ultrafiltration using a 10kDa cut-off filter. The reaction mixtures were analyzed by LC-MS/MS and the ratio of the CDHMe products P1 and

CD2Me products P2 was measured. Under this condition, the dimethylated products were not formed in 4 hours. After the natural abundance correction of stable isotopes, the intrinsic isotope effect is 18 ± 0.6 using below equation:

[] r = = [ ]

3.4.6. Synthesis of (R)-S-pantetheinyl-4-amino-3-(cyclopropylmethoxy)- benzothioate

O O O O O OH HO 1. Pd/C, H2

DIAD, PPh 2. HCl OH 3 O O NO2 NO2 NH2

O O O S OH N N H H 1. Pantethine DTT NaHCO3 OH

2.EDC, DMAP, DCM O NH2

O O

O NO2 methyl 3-(cyclopropylmethoxy)-4-nitrobenzoate (2-61)

Diisopropyl azodicarboxylate (DIAD, 1.5 mmol) was added to the THF solution of triphenylphosphine (PPh3, 2.0 mmol) at 0° C. The reaction mixture was stirred for half an hour. Then to it cyclopropylmethanol (1.0 mmol) and methyl 3-hydroxy-4- nitrobenzoate (1.0 mmol) were added one by one. It was then stirred at rt. in dark for overnight. Then it was concentrated and purified by column chromatography (Si-gel,

1 25% ethyl acetate-hexane) to afforded 2-61. H NMR (400 MHz, CDCl3) δ 7.77 (d, J =

8.3 Hz, 1H), 7.69 (m, 1H), 7.67 – 7.64 (m, 1H), 4.02 (d, J = 6.8 Hz, 2H), 3.93 (s, 3H),

13 1.28 (m, 1H), 0.64 (m, 2H), 0.38 (m, 2H). C NMR (101 MHz, CDCl3) δ 165.26,

134.66, 132.10, 128.60, 125.13, 121.32, 116.09, 74.62, 52.76, 9.85, 3.30. 74

O OH

O NH 2 4-amino-3-(cyclopropylmethoxy)benzoic acid (2-62)

Using 10% Pd/C as catalyst, 50 mL of methyl methyl 3-(cyclopropylmethoxy)-4- nitrobenzoate 2-62 (1.0 mmol) in methanol was pumped through the H-Cube®. The pressure of the system was set to full hydrogen mode, and the temperature to 35 °C. The flow rate was 0.8 mL/min. The fraction was analyzed using TLC, which showed complete conversion to the product, and the solvent was reduced to dryness, affording the 4-aminobenzoic acids. The resulting methyl 4-amino-benzoate (1.0 mmol) was suspended in 5M HCl (15 ml) and the mixture was heated at 60 °C. The reaction was monitored by TLC till completed. The reaction mixture was concentrated under vacuum and purified by column chromatography (silica gel, 40:1 CH2Cl2: MeOH) to give 2-62.

1 H NMR (400 MHz, CDCl3) δ 7.60 (dd, J = 8.2, 1.5 Hz, 1H), 7.44 (d, J = 1.4 Hz, 1H),

6.66 (d, J = 8.2 Hz, 1H), 3.88 (d, J = 6.9 Hz, 2H), 1.30 – 1.25 (m, 1H), 0.62 (m, 2H),

13 0.34 (m, 2H). C NMR (101 MHz, CDCl3) δ 171.96, 145.42, 142.27, 125.06, 118.32,

113.08, 104.99, 77.33, 53.39, 10.27, 3.18.

O O O S OH N N H H OH

O NH 2

(R)-S-pantetheinyl-4-amino-3-(cyclopropylmethoxy)-benzothioate (2-41)

D-pantethine (0.2 mmol) was reduced to D-pantetheine by addition of DTT (0.2 mmol) and stirring for 1 h at room temperature. Water was removed by vacuum to leave a clear, viscous liquid. To a stirred solution of 4-amino-3-(cyclopropylmethoxy)benzoic acid 2-41 (0.4 mmol) in 3 mL of dichloromethane was added 1-Ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDC, 0.4 mmol), followed by 4- dimethylaminopyridine (DMAP, 0.05 mmol) at room temperature. The resulting mixture was added to the pantetheine solution. The reaction was stirred at room temperature for overenight. All solvent was removed by vacuum and was purified by column

1 chromatography (silica gel, 40:1 CH2Cl2: MeOH) to give 2-41. H NMR (400 MHz,

MeOD) δ 8.19 (s, 1H), 7.94 (s, 1H), 7.50 (dd, J = 8.3, 1.9 Hz, 1H), 7.35 (d, J = 1.8 Hz,

1H), 6.70 (d, J = 8.3 Hz, 1H), 3.94 – 3.87 (m, 3H), 3.44 (m, 6H), 3.16 (t, J = 6.6 Hz, 2H),

2.43 (t, J = 6.6 Hz, 2H), 1.31 (m, 1H), 0.93 (s, 6H), 0.70 – 0.58 (m, 2H), 0.44 – 0.35 (m,

2H). 13C NMR (101 MHz, MeOH) δ 189.61, 174.70, 172.60, 145.29, 144.14, 125.32,

122.54, 112.20, 109.78, 76.00, 73.08, 69.03, 39.25, 38.96, 35.10, 34.98, 27.53, 19.91,

19.56, 9.79, 2.21.

3.4.7. Synthesis of (R)-S-pantetheinyl-4-amino-3-(1-cyclopropylethoxy)- benzothioate

O OH O OH O OH HO Pd/C, H2

F O O LiHMDS NO NH NO2 2 2

O O O S OH N N 1. Pantethine DTT NaHCO H H 3 OH 2.EDC, DMAP, DCM O NH2

O OH

O NO2

3-(1-cyclopropylethoxy)-4-nitrobenzoic acid (2-63)

3-fluoro-4-nitrobenzoic acid (1.0 mmol) and 1-cyclopropylethan-1-ol (1.1 mmol) are added to lithium bis(trimethylsilyl)amide (LiHMDS) (1M in THF, 1.1 ml) at 0 °C.

KHMDS. 3.5 mL of LiHMDS solution was then added dropwise, resulting in a color change from light yellow to orange. This solution is stirred from 0 °C to room temperature over 2 h and then diluted with CH2Cl2 and washed once with saturated aqueous NH4Cl. The aqueous layer is back-extracted once with CH2Cl2. The organic layers are combined, dried over MgSO4, concentrated, and purified by column

1 chromatography (silica gel, 20:1 CH2Cl2: MeOH) to give 2-63. H NMR (400 MHz, 77

MeOD) δ 7.87 – 7.75 (m, 2H), 7.70 (m, 1H), 4.20 (dt, J = 12.5, 6.2 Hz, 1H), 1.44 (d, J =

6.1 Hz, 3H), 1.14 (m, 1H), 0.57 (m, 2H), 0.36 (m, 2H). 13C NMR (101 MHz, MeOD) δ

165.73, 149.90, 143.68, 134.46, 123.69, 120.82, 117.28, 79.95, 17.92, 15.42, 2.21, 0.24.

O OH

O NH2

4-amino-3-(1-cyclopropylethoxy)benzoic acid (2-64)

Compound 2-64 was prepared by the above procedure for methyl 4-amino-3-

(cyclopropylmethoxy)benzoic acid 2-63. 1H NMR (400 MHz, MeOD) δ 7.55 – 7.42 (m,

2H), 6.95 (m, 1H), 3.93 – 3.81 (m, 1H), 1.39 (dd, J = 6.1, 2.0 Hz, 3H), 1.14 (dt, J = 8.0,

5.0 Hz, 1H), 0.58 – 0.48 (m, 2H), 0.41 – 0.24 (m, 2H). 13C NMR (101 MHz, MeOD) δ

146.30, 144.19, 124.46, 123.80, 115.67, 113.07, 112.48, 79.38, 18.74, 16.41, 2.94, 0.88.

O O O S OH N N H H OH

O NH2

(R)-S-pantetheinyl-4-amino-3-(1-cyclopropylethoxy)-benzothioate (2-43)

Compound 2-43 was prepared by the above procedure for (R)-S-pantetheinyl-4- amino-3-(cyclopropylmethoxy)-benzothioate 2-41. 1H NMR (400 MHz, MeOD) δ 7.49

(dd, J = 8.3, 1.9 Hz, 1H), 7.39 (d, J = 1.8 Hz, 1H), 6.71 (d, J = 8.3 Hz, 1H), 3.92 – 3.84

(m, 2H), 3.55 – 3.38 (m, 6H), 3.16 (t, J = 6.6 Hz, 2H), 2.43 (t, J = 6.6 Hz, 2H), 1.41 (d, J

= 6.1 Hz, 3H), 1.21 – 1.11 (m, 1H), 0.93 (s, 6H), 0.56 (d, J = 8.2 Hz, 2H), 0.43 – 0.28 78

(m, 2H). 13C NMR (101 MHz, MeOD) δ 189.57, 174.62, 172.51, 145.28, 144.17,

125.27, 122.65, 112.81, 112.59, 79.41, 75.99, 69.02, 39.11, 38.96, 35.04, 34.96, 27.51,

19.90, 19.55, 18.70, 16.36, 3.01, 0.86.

3.4.8. Synthesis of S-(2-acetamidoethyl) 4-amino-3-(2-bromoethoxy)-benzothioate

O O O O O OH HO Br 1. Pd/C, H2

DIAD, PPh3 Br 2. HCl Br OH O O NO 2 NO2 NH2

O O S N H EDC, HOBT, TEA, SNAC Br O NH2

O O

Br O NO2 methyl 3-(2-bromoethoxy)-4-nitrobenzoate (2-65)

Compound 2-65 was prepared by the above procedure for methyl 3-

(cyclopropylmethoxy)-4-nitrobenzoate using 2-bromoethan-1-ol. 1H NMR (400 MHz,

MeOD) δ 7.87 – 7.80 (m, 2H), 7.73 (m, 1H), 4.57 (t, J = 11.4 Hz, 2H), 3.94 (s, 3H), 3.75

(t, J = 11.4 Hz, 2H). 13C NMR (101 MHz, MeOD) δ 172.95, 166.49, 152.06, 136.03,

126.11, 123.26, 117.12, 71.24, 53.27, 29.45.

O OH

Br O NH2

4-amino-3-(2-bromoethoxy)benzoic acid (2-66)

Compound 2-66 was prepared by the above procedure for methyl 4-amino-3-

(cyclopropylmethoxy)benzoic acid. 1H NMR (400 MHz, MeOD) δ 7.76 (m, 2H), 7.52

(m, 1H), 4.3 (t, J = 11.4 Hz, 2H), 3.81 (t, J = 11.4 Hz, 2H).13C NMR (101 MHz, MeOD)

δ 168.03, 152.58, 134.00, 125.04, 124.31, 124.13, 115.11, 53.04, 29.63.

O O S N H

Br O NH2

S-(2-acetamidoethyl) 4-amino-3-(2-bromoethoxy)-benzothioate (2-49)

To a solution of triethylamine (TEA, 1.0 mmol) in dichloromethane (3 mL) was added 4-amino-3-(2-bromoethoxy)benzoic acid 2-66 (0.5 mmol), (3- dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC, 0.5 mmol), 1- hydroxybenzotriazole (0.5 mmol) and N-acetylcysteamine (SNAC, 0.5mmol). The reaction mixture was stirred overnight under argon. The organic layer was washed with saturated NaHCO3 solution, 0.1 N HCl solution and brine. It was then dried over anhydrous sodium sulfate and concentrated under vacuum. Purification of the crude mixture by flash chromatography (silica gel, 1:1 to 2:1 of hexanes:EtOAc) afforded 2-

49. 1H NMR (400 MHz, MeOD) δ 7.50 (dd, J = 8.3, 1.9 Hz, 1H), 7.37 (d, J = 1.9 Hz, 80

1H), 6.70 (d, J = 8.3 Hz, 1H), 4.34 (t, J = 11.4 Hz, 2H), 3.74 (t, J = 11.4 Hz, 2H), 3.38 (t,

J = 6.7 Hz, 2H), 3.13 (t, J = 6.7 Hz, 2H), 1.92 (s, 3H). 13C NMR (101 MHz, MeOD) δ

190.83, 173.45, 145.84, 145.75, 126.68, 124.63, 113.91, 111.95, 70.15, 40.54, 30.62,

28.94, 22.51.

3.4.9. Synthesis of (R)-S-pantetheinyl-4-amino-3-(methoxy- d3)-benzothioate

O OH O OH O OH

CD3OH, LiHMDS Pd/C, H2

CD3 CD3 F O O NO NH NO2 2 2

O O O S OH N N H H 1. DCC, THF, NHS OH

2. Pantetheine, THF CD O 3 NH2

O OH

CD O 3 NO2

3-(methoxy-d3)-4-nitrobenzoic acid (2-67)

Compound 2-67 was prepared by the above procedure for methyl 4-amino-3-

(cyclopropylmethoxy)benzoic acid. 1H NMR (400 MHz, MeOD) δ 7.91 – 7.77 (m, 2H),

7.70 (dd, J = 8.3, 1.5 Hz, 1H). 13C NMR (101 MHz, MeOD) δ 167.68, 153.46, 144.08,

136.99, 125.93, 122.67, 115.80, 73.72.

O OH

CD O 3 NH2

4-amino-3-(methoxy- d3) benzoic acid (2-68)

Compound 2-68 was prepared by the above procedure for methyl 4-amino-3-

(cyclopropylmethoxy)benzoic acid. 1H NMR (400 MHz, MeOD) δ 7.49 – 7.37 (m, 2H),

6.68 (d, J = 8.2 Hz, 1H). 13C NMR (101 MHz, MeOD) δ 170.65, 147.41, 144.05, 125.58,

119.55, 113.85, 112.29.

O O O S OH N N H H OH

CD O 3 NH2

(R)-S-pantetheinyl-4-amino-3-(methoxy- d3)-benzothioate (2-53)

To a stirred solution of 4-amino-3-(methoxy- d3) benzoic acid 2-68 (0.5 mmol) in

10 mL of dry THF was added N-hydroxysuccinimide (NHS) (0.5 mmol), followed by N,

N'-Dicyclohexylcarbodiimide (DCC) (0.5 mmol) at room temperature. The resulting mixture was stirred overnight at room temperature. The white precipitate was filtered and then washed with EtOAc. The filtrate was concentrated under reduced pressure. The residue was triturated with CH2Cl2 to give the NHS ether of 2-68. Pantethine (0.075 mmol), sodium bicarbonate (1.9 mmol), and 1,4-dithiothreitol (DTT) (0.08 mmol) were dissolved in 3 mL of water, and left to stand for 10 minutes to allow reduction of the pantethine to pantetheine to take place. The NHS ester (0.13 mmol) was dissolved in 6 82

mL of tetrahydrofuran (THF) and added to the 3 mL water mixture. The reaction mixture was stirred overnight and then the solvent was evaporated under reduced pressure. The crude product was then dissolved in water and the insoluble residue was removed by filtration. The resulting solution was lyophilized and purified by flash chromatography

(silica gel, 20:1 to 10:1 CH2Cl2: MeOH). The fraction was further purified by Prep

HPLC using a SPLC-18DB column (10 x 250 mm, 5 µm, Supelco) at a flow rate of 2 mL/min on an Agilent 100 HPLC system with a quaternary pump and an automatic injector. 1H NMR (400 MHz, MeOD) δ 7.48 (dd, J = 8.3, 1.9 Hz, 1H), 7.35 (d, J = 1.9

Hz, 1H), 6.66 (d, J = 8.3 Hz, 1H), 3.88 (s, 1H), 3.59 – 3.33 (m, 6H), 3.14 (t, J = 6.7 Hz,

2H), 2.41 (t, J = 6.6 Hz, 2H), 0.91 (d, J = 1.4 Hz, 6H). 13C NMR (101 MHz, MeOD) δ

191.00, 176.04, 173.92, 147.47, 145.35, 126.70, 123.97, 113.41, 109.57, 77.40, 70.43,

40.54, 40.37, 36.45, 36.38, 28.91, 21.30, 20.95.

3.4.10. Synthesis of (R)-S-pantetheinyl-4-amino-3-(methoxy- d2)-benzothioate

O OH O OH O OH

CHD2OH LiHMDS Pd, H2

CD2H CD2H F O O NO NH NO2 2 2

O O O S OH N N H H 1. Pantethine DTT NaHCO3 OH

2. EDC, DMAP, DCM CD H O 2 NH 2

O OH

CD H O 2 NO 2

3-(methoxy-d2)-4-nitrobenzoic acid (2-69)

Compound 2-69 was prepared by the above procedure for methyl 4-amino-3-

(cyclopropylmethoxy)benzoic acid. 1H NMR (400 MHz, MeOD) δ 7.86 – 7.79 (m, 2H),

7.70 (dd, J = 8.3, 1.4 Hz, 1H), 3.96 (s, 1H).

O OH

CD H O 2 NH2

4-amino-3-(methoxy-d2) benzoic acid (2-70)

Compound 2-70 was prepared by the above procedure for methyl 4-amino-3-

(cyclopropylmethoxy)benzoic acid. 1H NMR (400 MHz, MeOD) δ 7.50 – 7.35 (m, 2H),

6.68 (d, J = 8.2 Hz, 1H), 3.82 (s, 1H). 13C NMR (101 MHz, MeOD) δ 170.90, 147.44,

143.97, 125.57, 119.88, 113.89, 112.33, 55.69, 55.47, 55.25.

O O O S OH N N H H OH

CD H O 2 NH2

(R)-S-pantetheinyl-4-amino-3-(methoxy-d2)-benzothioate (2-55)

Compound 2-55 was prepared by the above procedure for (R)-S-pantetheinyl-4- amino-3-(cyclopropylmethoxy)-benzothioate using 4-amino-3-(methoxy- d2) benzoic acid. 1H NMR (400 MHz, MeOD) δ 7.51 (dd, J = 8.3, 1.9 Hz, 1H), 7.38 (d, J = 1.9 Hz,

1H), 6.69 (d, J = 8.3 Hz, 1H), 3.91 (s, 1H), 3.88 – 3.84 (m, 1H), 3.57 – 3.38 (m, 6H),

3.17 (t, J = 6.7 Hz, 2H), 2.44 (t, J = 6.7 Hz, 2H), 0.94 (d, J = 1.4 Hz, 6H). 13C NMR (101

MHz, MeOD) δ 189.61, 174.63, 172.51, 146.06, 143.93, 125.29, 122.57, 112.01, 108.17,

75.99, 69.02, 54.36, 54.14, 53.92, 39.13, 38.96, 35.04, 34.97, 27.51, 19.91, 19.54.

3.4.11. Synthesis of S-(2-acetamidoethyl) 4-amino-3-hydroxybenzothioate (2-52)

O O OH O S N H EDC, HOBT, TEA, SNAC

OH OH NH 2 NH2

Compound 2-52 was prepared by the above procedure for S-(2-acetamidoethyl)

4-amino-3-(2-bromoethoxy)-benzothioate using 4-amino-3-hydroxybenzoic acid. 1H

NMR (400 MHz, MeOD) δ 7.72 – 7.63 (m, 1H), 7.63 – 7.48 (m, 2H), 3.46 (t, J = 6.7 Hz,

2H), 2.81 (t, J = 6.7 Hz, 2H), 1.93 (s, 3H).

3.4.12. Synthesis of (R)-S-pantetheinyl-4-amino-3-hydroxybenzothioate (2-46)

O O O OH O S OH N N H H 1. Pantethine DTT NaHCO3 OH

2. EDC, DMAP, DCM OH OH NH NH2 2

Compound 2-46 was prepared by the above procedure for (R)-S-pantetheinyl-4- amino-3-(cyclopropylmethoxy)-benzothioate using 4-amino-3-hydroxybenzoic acid. 1H

NMR (400 MHz, MeOD) δ 7.40 (dd, J = 8.3, 2.0 Hz, 1H), 7.31 (d, J = 2.0 Hz, 1H), 6.67

(d, J = 8.3 Hz, 1H), 3.91 (s, 1H), 3.51 – 3.38 (m, 6H), 3.14 (t, J = 6.7 Hz, 2H), 2.43 (t, J

= 6.7 Hz, 2H), 0.94 (s, 7H). 13C NMR (101 MHz, MeOD) δ 189.67, 174.63, 172.51,

143.42, 143.02, 125.71, 121.23, 112.69, 112.22, 75.98, 69.01, 39.14, 38.95, 35.02,

34.97, 27.41, 19.90, 19.52.

4. TRAPPING RADICAL INTERMEDIATES IN AN ANEROBIC HEME

DEGRADATION PATHWAY

4.1. Introduction

Iron is known to play a crucial role in the living system. Although iron is the fourth most common element in the Earth's crust, it is not readily bioavailable at neutral pH due to oxidation to the insoluble ferric oxide. For the soluble reduced Fe2+, it can catalyze the generation of toxic free radicals through Fenton chemistry. These reactive oxygen species (ROS) can react with membrane lipids, proteins, and nucleic acids, which cause irreversible cell damage.66 Thus, the free iron concentration has to remain very low. Mammals employ heme, iron-binding proteins (transferrin, lactoferrin) to reduce the levels of free extracellular iron to around 10−18 M, which is orders of magnitude below the 10−6 M required for bacterial growth.67 The majority of iron within the host, approximately 95%, is sequestered in the form of heme, primarily as hemoglobin.68 However, free heme is considered toxic to the cells.69

In order to compete for the host’s iron during infection, pathogens have developed sophisticated mechanisms to utilize heme as a source of iron.67 Bacteria employ surface proteins to seize heme from hemoproteins and then shuttle heme across the outer-membrane/cell wall onto specific transporters for import across the cytoplasmic membrane.70 Inside the bacterial cell, heme is incorporated directly into proteins or it is further degraded to release the iron.71 Oxidative degradation by heme oxygenases (HOs) is the most common mechanism used to liberate iron from heme.

In most of the cases, heme oxygenase catalyzes oxidative cleavage of heme.72

Heme oxygenase requires three molecules of oxygen and seven electrons to regiospecifically cleave heme at the a-meso-carbon to yield biliverdin IXa via a-meso- hydroxyheme and verdoheme.73-75

However, it’s known that pathogens can colonize the nonsterile region of the lower intestines, which is an anaerobic environment. For example, Vibrio cholerae and

Escherichia coli O157:H7 are hemolytic enteric pathogens, and a bona fide heme oxygenase has not been characterized for them. Both organisms can use heme as an iron source and contain a heme uptake operon that is up-regulated during iron duress.76-77

The heme uptake operon includes a heme uptake and transport machinery, and three additional genes, chuw, chux, and chuy (Figure 4.1). ChuW is assigned as a radical

SAM enzyme and has high sequence similarity with coproporphyrinogen III oxidase

(HemN), which is a characteristic feature of Class C radical SAM methyltransferases.

Class C RSMTs are proposed to use two molecules of SAM to methylate sp2-hybridized carbon centers.19

Radical SAM Reductase enzyme

Figure 4.1 The heme uptake operon in Escherichia coli O157:H7

In 2016, Dr. Lanzilotta demonstrates that ChuW is able to catalyze a radical- mediated reaction to break the protoporphyrin ring of heme to release iron and form a tetrapyrrole product “anaerobilin” anaerobically (Figure 4.2).78 5′-dA and SAH were produced at 1:1 stoichiometry in the presence of heme, ChuW and flavodoxin,

NADPH/flavodoxin reductase system. When [13C-methyl]- SAM was used in the assay, the isolated product exhibited an enrichment of 13C. This suggests that ChuW also catalyzes the transfer of a methyl group from the SAM moiety to the product.

Furthermore, the anaerobilin can be used as a substrate by anaerobilin reductase ChuY in the same operon. Nevertheless, the deuteroheme-derived tetrapyrrole product deuteroanaerobilin (DAB) is not stable under the light condition. Exposure of DAB to sunlight for 5 min resulted in a significant change in the UV-visible spectrum. The light sensitivity of anaerobilin and DAB has prohibited the accumulation of product for NMR characterization.

CH2

N N ChuW, SAM NH N Fe Reducing system N N NH HN

HOOC COOH HOOC COOH Heme Anaerobilin 4-1 4-2

Figure 4.2 ChuW-catalyzed degradation of heme anaerobically

Here, we try to design substrate analogs to trap the possible racial intermediates generated on the tetrapyrrole ring during the ChuW catalyzed reaction in order to study the mechanism.

4.2. Result and discussion

4.2.1. Reconstitution of ChuW catalyzed heme degradation

The chuW gene was obtained from Dr. Lanzilotta and was cloned into a pTHT vector. When heme was used as a substrate for ChuW with E. coli flavodoxin

(EcFldA)/ferredoxin (flavodoxin): NADP+ oxidoreductase (EcFpr)/ NADPH system as the reducing system, a new species (455nm, 800nm) was formed similarly as the reported anaerobilin with heme degraded over time based on the UV spectrum (Figure

4.3). The proposed anaerobilin was not stable and degraded under light conditions for several minutes.

0 min: — 420 10 mins: — 30 mins: — 455 50 mins: — 80 mins: — 120 mins: — overnight: —

560 800

Figure 4.3 UV spectrum of the time course of the ChuW catalyzed heme degradation.

LC-MS analysis of ChuW degradation of heme reaction mixtures showed a large amount formation of 5′-dA and SAH in the reaction (Figure 4.4).

Intens . 5 x10 NH2 2.5 N N 2.0 N N - Full reaction 1.5 O - No heme 1.0 - No ChuW OH OH 5’dA 0.5

0.0 0 5 10 15 20 25 30 35 Time [min] 050617No Heme2_1-B,2_01_1233.d: EIC 252.1091±0.02 +All MS 050617 ChuWheme1_1-B,1_01_1232.d: EIC 252.1091±0.02 +All MS, Smoothed (4.64,1,GA) 050617No ChuW3_1-B,3_01_1234.d: EIC 252.1091±0.02 +All MS EIC of 252.1091 Intens . x104

6 NH2 N N - Full reaction 4 N N - No heme HOOC S O - No ChuW 2 NH2 OH OH SAH

0 0 5 10 15 20 25 30 35 Time [min] 050617No Heme2_1-B,2_01_1233.d: EIC 385.1289±0.2 +All MS, Smoothed (2.51,1,GA) 050617 ChuWheme1_1-B,1_01_1232.d: EIC 385.1289±0.2 +All MS, Smoothed (2.32,1,GA) 050617No ChuW3_1-B,3_01_1234.d: EIC 385.1289±0.2 +All MS, Smoothed (2.38,1,GA) EIC of 385.1289

Figure 4.4 LC-MS analysis of ChuW catalyzed heme degradation. Upper panel: EICs of 5′-dA [M+H]+ (252.11±0.02). Lower panel: EICs of SAH [M+H]+ (385.12±0.02).

HPLC analysis of ChuW degradation of heme reaction mixtures also showed formation of a new speak under 455 nm (Figure 4.5).

N N Fe3+ ChuW, SAM N N Blue: Full FldA, FldR, NADPH Red: No ChuW

O HO O OH

UV of 455 nm

Figure 4.5 HPLC analysis of ChuW catalyzed heme degradation.

LC-MS analysis of ChuW degradation of heme reaction mixtures showed the new peak of 455 nm had a mass of 577.28, which corresponds with the mass of the ring opening product anaerobilin (Figure 4.6).

Intens . x105 - Full reaction 0.8 - No ChuW N N ChuW, SAM 0.6 Fe Reducing system N N 0.4

0.2 HOOC COOH

0.0 0 5 10 15 20 25 30 35 Time [min] 051117ChuWhemeh2o_2_1-B,2_01_1355.d: EIC 577.2809±0.2 +All MS 051117NoChuWhemeh2o_4_1-B,1_01_1354.d: EIC 577.2809±0.2 +All MS EIC of 577.2809

Intens . 051117ChuWhemeh2o_2_1-B,2_01_1355.d: +MS, 14.7-15.0min #1737-1773 4 x10 577.2849

5 577.2849 4 Accuracy: 6.9 ppm NH HN

3 N N

HOOC COOH 1 Exact Mass: 577.2809 643.2787 627.2827 784.7211851.9532 758.5782764.6673805.9240813.0767828.9185828.3019854.9024873.2531898.5424906.8425967.63961001.1100 1106.1702 0 500 750 1000 1250 1500 1750 2000 2250 2500 2750 m/z

Figure 4.6 LC-MS analysis of ChuW catalyzed heme degradation. Upper panel: EICs of anaerobilin [M+H]+ (577.28±0.02). Lower panel: mass spectrum of anaerobilin at 15 min.

4.2.2. Design of cyclopropyl heme analog as a radical trap for ChuW catalyzed reaction

Since there is one mass unit of 5′-dA increased from 11% to 60% when [d3- methyl]-SAM was used in the chuW reaction, it’s proposed that 5′-dA generated from the first molecular of SAM abstracts a hydrogen atom from the second molecular of

SAM. The resulting SAM radical can attack the unsaturated bond on the porphyrin ring of heme. This resulting radical species can be delocalized on the porphyrin ring to initiate the ring cleavage and the methylation. Since the ring opening of the cyclopropylcarbinyl radical is a well-recognized fast radical reaction,79 the displacement of the vinyl group from the heme with a cyclopropyl group could be used to trap the radicals generated on the protoporphyrin ring.

The functionalization of vinyl group of tetrapyrrolic ring has been explored extensively for new photosensitizer libraries.80 One of the useful functionalizations is a reaction with 1,3-dipolar compounds via a concerted cis-cycloaddition mechanism using diazomethane. The resulting 1-pyrazolines can be photolyzed or pyrolyzed to give cyclopropanes.81 Cyclopropyl heme analog 4-6 is synthesized using the literature route as shown in Figure 4.7.

N N

NH N N NH N N CH(OMe)3 CH2N2 NH N HN N HN N N HN EtOH, H2SO4

HOOC COOH MeOOC COOMe MeOOC COOMe 4-1a 4-3 4-4 reflux o-dichlorobenzene

N N Acetic acid NH N Fe2+ N N N HN FeSO4, HCl

HOOC COOH MeOOC COOMe 4-6 4-5

Figure 4.7 Synthetic route of cyclopropyl heme analog

4.2.3. Cyclopropyl heme analog as a substrate for ChuW

When cyclopropyl heme 4-6 was used as a substrate for ChuW with EcFldA/

EcFpr/ NADPH system as the reducing system, a new species (455nm, 780nm) was formed, which is blue shifted when compared with the anaerobilin based on the UV spectrum (Figure 4.8).

0min 5min 1.4 10min 15min 1.2 20min 25min

1 30min 40min 455 0.8 420 75min 110min

Absorbance (AU) 0.6 780 0.4

0.2

0 270 370 470 570 670 770 Wavelength/ nm

Figure 4.8 UV spectrum of the time course of the ChuW catalyzed cyclopropyl heme degradation.

LCMS analysis of ChuW degradation of cyclopropyl heme 4-6 reaction mixtures showed a large amount formation of 5′-dA in the reaction, which suggests that ChuW takes cyclopropyl heme as a substrate (Figure 4.9).

NH2 N N - Full reaction N N - No ChuW O

OH OH 5’dA

EIC of 252.1091

Figure 4.9 LC-MS analysis of ChuW catalyzed cyclopropyl heme 4-6 degradation. EICs of 5′-dA [M+H]+ (252.11±0.02).

HPLC analysis of ChuW degradation of cyclopropyl heme 4-6 reaction mixtures also showed the formation of new species which have an absorbance around 500nm. The high wavelength products are not stable under air and light condition. The 500nm products decrease over time (Figure 4.10).

Blue: Full Red: No ChuW

Blue: 0 min Red: 40 mins Green: 80 mins Pink: 120 mins Grey: 160 mins Purple: 200 mins

UV of 500 nm

Figure 4.10 HPLC analysis of ChuW catalyzed cyclopropyl heme degradation under UV of 500 nm.

LCMS analysis of new compounds with high wavelength showed a new peak of

500 nm had a mass of 605.31, which corresponds with the mass of the methylated ring opening product (Figure 4.11).

ChuW, SAM N N - Full reaction Fe Reducing system N N - No ChuW

HOOC COOH 4-6

EIC of 605.3122

Intens . 041317UV1ChuWP256P4_30min_1-E,2_01_1093.d: +MS, 23.7-24.1min #2789-2834 4 x10 605.3133 4-7 4 605.3133 N HN 3 Accuracy: 1.8 ppm N N

2 606.3155

HOOC COOH 1 Exact Mass: 605.3122

607.3184 619.2978 621.3277 609.3393 622.3292 0 595 600 605 610 615 620 625 630 m/z

Figure 4.11 LC-MS analysis of ChuW catalyzed cyclopropyl heme degradation. Upper panel: EICs of [M+H]+ (605.31±0.02). Lower panel: mass spectrum of degradation product at 15 min.

13 When CD3-SAM or CH3-SAM was used in the ChuW catalyzed cyclopropyl heme degradation reaction, there were two mass units and one mass unit increase in the

605.31 peak respectively (Figure 4.12). This suggests that the methyl group is transferred to the new product from SAM. However, this compound is also not stable under the light condition. The high wavelength peak will disappear when it’s exposed to light.

Intens . 0109 ChuW P256P4 TiCi_1_SAMCD3_1-B,2_01_4941.d: +MS, 13.2-13.6min #1553-1605 x105 607.3290 1.5 CD3-SAM D D 607.3290 Accuracy: 6.9 ppm N HN 1.0 N N 606.3213

608.3305 0.5 605.3151 611.3578 HOOC COOH 609.3377 Exact Mass: 607.3248 612.3598 621.3347 610.3471 622.3366 613.3608 620.3209 0.0 600.0 602.5 605.0 607.5 610.0 612.5 615.0 617.5 620.0 622.5m/z

Intens . 0109 ChuW P256P4 TiCi_2_SAM13C_1-B,1_01_4942.d: +MS, 13.2-13.6min #1552-1607 x105 13 13 606.3198 1.0 CH3-SAM CH2 606.3198 Accuracy: 6.9 ppm 0.8 N HN

0.6 N N

607.3221 0.4 610.3494

HOOC COOH 0.2 605.3134 611.3504 621.3330 608.3247 622.3272 Exact Mass: 606.3156 609.3396 612.3513 620.3279 0.0 600.0 602.5 605.0 607.5 610.0 612.5 615.0 617.5 620.0 622.5m/z

Figure 4.12 Mass spectrum of ChuW catalyzed cyclopropyl heme degradation product at 13 15 min with (A) CD3-SAM or (B) CH3-SAM.

4.2.4. Detection of protoporphyrin ring opening product in ChuW catalyzed cyclopropyl heme 4-6 degradation

Under the UV wavelength of 390 nm, there was a new peak formation around 34 min in HPLC chromatography, which has a similar UV spectrum as the protoporphyrin

(Figure 4.13). Because of the highly conjugated p-electron systems of the tetrapyrrole ring, the porphyrins have characteristic UV-visible spectra that consist of two distinct region regions: in the near ultraviolet and the visible region.82 The porphyrins display extreme intense bands, the so-called Soret or B-bands in the 380–500 nm range with molar extinction coefficients of 105 M-1 cm-1. Moreover, at longer wavelengths, in the

500–750 nm range, the porphyrins spectra contain a set of weaker, but still considerably intense Q bands with molar extinction coefficients of 104 M-1 cm-1. As for the new peak

generated in the ChuW catalyzed cyclopropyl heme 4-6 degradation, it has a large absorbance around 390 nm in the Soret bands region, and some weaker absorbance in the 500, 530, 570 nm in the Q-bands region (Figure 4.13). This suggests that this new peak should still maintain the intact porphyrin ring. This product is considered stable under the light condition for several hours (Figure 4.14).

N N - Full reaction Fe - No ChuW N N

HOOC COOH

HPLC (UV of 390nm)

NH N

N HN

HOOC COOH

UV spectrum of heme

UV spectrum of 34 min

Figure 4.13 HPLC analysis of ChuW catalyzed cyclopropyl heme degradation. Upper panel: HPLC UV trace of 390 nm. Lower panel left: UV spectrum of heme; Lower panel right: UV spectrum of product at 34 min.

100

Blue: Under light 0 h Red: Under light 2 h Green: Under light 4 h

HPLC (UV of 400nm)

Figure 4.14 HPLC analysis of stability of products in ChuW catalyzed cyclopropyl heme degradation over time under 400 nm

LC-MS analysis of the new compound showed it had a mass of 591.3011, which corresponds with the mass of cyclopropyl heme analog without the iron (Figure 4.15B).

When CD3-SAM was used in the ChuW catalyzed reaction, 60% of the product have one mass unit increased (Figure 4.15C). To further explore whether the cyclopropyl side chain is open, the porphyrin product was purified from HPLC and was hydrogenated in a short amount of time. A new peak with a mass of 593.31 was formed (Figure 4.15D).

This suggests that there is a double bound on the side chain of the porphyrin ring and probably is the cyclopropyl ring opening product.

101

A Red: Full reaction Blue: No ChuW N N ChuW, SAM Fe Reducing system N N

HOOC COOH

EIC of 591.30 Intens . B 0718ChuW P256p4_1-B,1_01_2201.d: +MS, 18.3-18.8min #2156-2205 Intensx104 . 0718ChuW P256p4_1-B,1_01_2201.d: +MS, 18.3-18.8min #2156-2205 x1054 591.3011 5 4-9 591.3011 591.3011 591.3011 Accuracy: 7.6 ppm 4 Accuracy: 7.6 ppm NH2 N 4 NH N N HN 3 3 N H2N

2 2 HOOC COOH HOOC COOHExact Mass: 591.2966 1 619.3016 1 Exact Mass: 591.2966 619.3016 1181.5902 0 1181.5902 0 500 750 1000 1250 1500 1750 2000 2250 2500 2750 m/z 500 750 1000 1250 1500 1750 2000 2250 2500 2750 m/z C Intens . 081017ChuW_2 p256p4CD3SAM50_1-D,3_01_2744.d: +MS, 19.2-19.8min #2265-2338 x105 592.3078 592.3078

1.5 Accuracy: 8.2 ppm N ChuW, CD3-SAM N 591.3015 Fe Reducing system N N 1.0

593.3095

0.5 HOOC COOH 584.4733 Exact Mass: 592.3029 594.3105 0.0 584 586 588 590 592 594 596 598 m/z

Intens . 100917_0927ChuWP256p4_1 H2_4h_1-B,3_01_3790.d: +MS, 13.9-14.1min #1636-1657 D 593.3116 4000 593.3116 NH N Pd/C NH N Accuracy: 7.4 ppm N HN 3000 H2 N HN

594.3146 2000 603.2972 HOOC COOH HOOC COOH Exact Mass: 593.3122 1000 595.3025 596.2772 597.2931 598.2813 586.2794 588.3729 591.2977 592.2926 599.2865 600.2773 601.2855 587.2722 589.2728 590.4005 602.2882 593.7858 594.7980 595.7917 596.9558 597.7839 598.7811 0 586 588 590 592 594 596 598 600 602 604 m/z

Figure 4.15 LC-MS analysis of ChuW catalyzed cyclopropyl heme degradation. (A) EICs of [M+H]+ (591.30±0.02). (B) Mass spectrum of the porphyrin degradation product at 19 min. (C) Mass spectrum of the porphyrin degradation product when the reaction used CD3-SAM as cofactor. (D) Mass spectrum of the porphyrin degradation product after hydrogenation reaction.

102

4.2.5. Detection of SAM adducts in ChuW catalyzed cyclopropyl heme degradation

LC-MS analysis of the unstable high wavelength products showed a compound

had a mass corresponding to the crosslinking species 4-10 between cyclopropyl heme

derived product and SAM, which were absent in the control (Figure 4.16). Another

compound also had a mass corresponding to the crosslinking species 4-11 between

cyclopropyl heme derived product and methylthioadenosine (Figure 4.17). It is known

that SAM can degrade to L-homoserine lactone (HSL) and 5-methylthioadenosine

(MTA) under high and low pH condition.83 After purification and acid treatment of the

reaction mixture, SAM crosslinking species 4-10 can be degraded to MTA crosslinking

species 4-11.

Intens . x104

1.0

0.8

0.6 - Full reaction 0.4 - No ChuW

0.2

0.0 5 10 15 20 25 30 Time [min] 0718No ChuW P256p4_1-B,2_01_2200.d: EIC 502.2284±0.02 +All MS EIC of 502.2284 0718ChuW P256p4_1-B,1_01_2201.d: EIC 502.2284±0.02 +All MS

Intens . Intens . 0718ChuW P256p4_1-B,1_01_2201.d: +MS, 12.2-12.4min #1432-1453 0718ChuW P256p4_1-B,1_01_2201.d: +MS, 12.1-12.5min #1427-1471 4000 502.2298 2+ 4000 502.2298 Exact mass [M + 2H] :502.2284 4-10 NH Accuracy: 2.8 ppm HOOC 2 HO OH 3000 502.7327 N N O N 3000 S NH 2000 N 2

503.2327

1000 N N 2000 503.7350 501.7282 0 500 501 502 503 504 505 506 N HN m/z Exact Mass [M + H]1+ : 1003.4495 1000 635.3240 1003.4503 Accuracy: 0.8 ppm 754.3669 HOOC COOH 902.3981 1125.2160 1285.8227 1500.2770 1799.9458 0 500 750 1000 1250 1500 1750 2000 2250 2500 2750 m/z

103

- Full reaction - No ChuW

EIC of 902. 4018 Intens . 0718ChuW P256p4_1-B,1_01_2201.d: +MS, 13.5-13.5min #1588-1591 HO OH N N 902.4036 4-11 O N 5000 S NH 902.4036 N 2 Exact Mass: 902.4018 Accuracy: 1.3 ppm 4000 N N 3000 N HN 2000

1000 HOOC COOH 877.2840 918.3804 934.3944 957.1390 869.5517 884.7571 890.0639 894.7973 910.1733 924.4050 941.0453 944.7548 949.3901 962.5444 968.6263 0 870 880 890 900 910 920 930 940 950 960 970 m/z

Figure 4.17 LC-MS analysis of ChuW catalyzed cyclopropyl heme degradation. Upper panel: EICs of [M+H]+ (902.40±0.02). Lower panel: mass spectrum of the degradation porphyrin cross-linking species 4-11 at 13.5 min.

When CD3-SAM was used in the reaction, the majority of the crosslinking species have 2-4 mass unit increased for the +1-charged compound (Figure 4.18, 4.19).

13 Similarly, when CH3-SAM was used in the reaction, the majority of the crosslinking species have 2 mass unit increased for SAM adduct 4-10 (Figure 4.18, 4.19). MS/MS fragmentations of the crosslinking species also match with two methylene groups from

SAM get incorporated into the product (Figure 4.20).

104

Intens . COOH 0109 ChuW P256P4 TiCi_1_SAMCD3_1-B,2_01_4941.d: +MS, 11.1-11.2min #1308-1320 x104 503.7384 504.2412 CD -SAM NH 504.2412 3 HOOC 2 N D C 1.0 N 2 S Accuracy: 0.5 ppm 503.2366 CD2 OH 0.8 NH N HOOC 504.7441 O OH Exact Mass: 0.6 N 505.2468 N 504.2409 0.4 N

N 502.7324 0.2 H N 505.7460 2 502.2318 497.2369 497.7372 498.2260 499.2321 499.7406 500.2374 501.2310 506.2618 507.2683 508.2865 510.2380 510.7256 0.0 498 500 502 504 506 508 510 m/z Intens . COOH 0109 ChuW P256P4 TiCi_2_SAM13C_1-B,1_01_4942.d: +MS, 11.1-11.3min #1308-1328 x104 503.2353 13 503.2353 NH CH3-SAM HOOC 2 2.5 N 13 Accuracy: 7.5 ppm N H2 C S

13 OH 2.0 CH2 NH HOOC N O Exact Mass: 503.7364 1.5 OH N 503.2317 1.0 N N 502.7335 504.2367 0.5 N H2N 504.7366 499.2235 502.2306 505.2427 510.2272 510.7287 0.0 498 500 502 504 506 508 510 m/z

Figure 4.18 Mass spectrum of the porphyrin cross-linking species (503.22) when ChuW 13 used (upper panel) CD3-SAM, (lower panel) CH3-SAM as cofactor.

COOH CD3-SAM Intens . NH HOOC 2 0109 ChuW P256P4 TiCi_1_SAMCD3_1-B,2_01_4941.d: +MS, 11.1-11.3min #1304-1332 N N D C 2 S 1006.4654 1007.4709 CD2 OH 1005.4602 1007.4709 NH N 3000 HOOC O Accuracy: 3.7 ppm OH Exact Mass: N

2000 N N 1007.4746

N H2N 980.4291 1000 976.4768 1026.4052 1043.4130 982.4361 990.4270 999.4672 1014.6741 1020.4329 985.4486 993.2767 1034.5486 1038.3839 1050.0065

0 980 990 1000 1010 1020 1030 1040 1050 m/z

13 COOH Intens . CH3-SAM 0109 ChuW P256P4 TiCi_2_SAM13C_1-B,1_01_4942.d: +MS, 11.1-11.3min #1300-1332 1005.4575 NH 8000 HOOC 2 1005.4575 N N 13 H2 C S Accuracy: 3.7 ppm 13CH OH NH 2 6000 HOOC N O OH Exact Mass: N 4000 N 1005.4562 N

2000 N H N 1025.3935 982.4260 2 1020.4042 1043.4210 977.8807 987.4346 990.4152 996.4384 1012.3825 1017.4048 1032.4252 1051.4992 0 980 990 1000 1010 1020 1030 1040 1050 m/z

Figure 4.19 Mass spectrum of the porphyrin cross-linking species 4-10 (1003.45) when 13 ChuW used (upper panel) CD3-SAM, (lower panel) CH3-SAM as the cofactor.

105

Intens . CH3-SAM 1105 ChuW_P271P4_1-C,2_01_4182.d: +MS2(502.2295), 25.1eV, 11.0-11.0min #1288-1294 COOH 8000 619 619.3275 619 HOOC NH2 N N S 6000 OH N HOOC N 606 O 4000 OH N N N 606 2000 N 606.3178 H N 2 591.2944 575.3390 0 540 560 580 600 620 640 660 m/z

COOH CD3-SAM 623 NH Intens . HOOC 2 0109 ChuW P256P4 TiCi_1_SAMCD3_1-B,2_01_4941.d: +MS2(504.2402), 35.1eV, 11.1min #1299 N N D2C S 1500 621.3405 CD2 OH 608 NH 608.3295 HOOC N 608 O OH 623 1000 N

592.3131 N N

N 500 H2N

0 590 600 610 620 630 640 650 660 670 m/z Intens . 0109 ChuW P256P4 TiCi_2_SAM13C_1-B,1_01_4942.d: +MS2(503.2331), 35.1eV, 10.6-10.7min #1251-1257 13CH -SAM COOH 1250 3 621 621 621.3335 NH HOOC 2 N N 13 1000 H2 C S

13 OH CH2 NH N 750 HOOC 607 O OH 609.3393 500 N 607 N N 592.3070 250 548.3048 N H2N 0 540 560 580 600 620 640 660 m/z

Figure 4.20 Mass fragmentation of the porphyrin cross-linking species (502.23) when 13 ChuW used (upper panel) CH3-SAM, (middle panel) CD3-SAM, (lower panel) CH3- SAM as cofactor.

106

Intens . 0109 ChuW P256P4 TiCi_1_SAMCD3_1-B,2_01_4941.d: +MS, 11.7-11.8min #1379-1391 x104 HO OH CD -SAM N N 906.4279 3 905.4217 6 S O N NH2 906.4297 D C N 2 904.4166 CD2 Accuracy: 6.9 ppm 907.4319 4 N N

N HN

2 908.4348 903.4071 902.4016 HOOC COOH 909.4385 Exact Mass: 906.4269 910.4439 0 Intens . 895.0 897.5 OH 900.0 902.5 905.0 907.5 910.0 0109 ChuW P256P4 TiCi_2_SAM13C_1-B,1_01_4942.d: +MS, 11.7-12.0min #1380-1408912.5 915.0 m/z x104 HO 13 N N 904.4130 CH3-SAM S O N 904.4130 6 NH2 13 N H2 C 13 CH2 905.4166 Accuracy: 5.0 ppm

4 N N 906.4227 N HN 903.4069 2 907.4235 HOOC COOH 908.4265 Exact Mass: 904.4085 902.3982 909.4249 0 895.0 897.5 900.0 902.5 905.0 907.5 910.0 912.5 915.0 m/z

Figure 4.21 Mass spectrum of the degradation porphyrin cross-linking species 4-11 13 (902.40) when the ChuW used (upper panel) CD3-SAM, (lower panel) CH3-SAM as cofactor.

When CD3-SAM was used as the cofactor and titanium citrate was used as the reducing agent in the ChuW catalyzed reaction, the [A+1] peak for 5'-dA increased from

13% to 98% of [A], the [A+2] peak for 5'-dA increased from 1.8% to 31% of [A]

(Figure 4.23). It’s been shown in the literature that 5'-dA radical can abstract a hydrogen atom from the second molecule of SAM (Figure 4.22). Thus, it’s expected that there will be 1 mass unit increased for the 5'-dA when CD3-SAM was used. However, the increase of [A+2] peak indicated that the ring opening radical probably could abstract a hydrogen atom from 5'-dA, which then can abstract a hydrogen atom again from CD3-SAM.

107

NH HOOC 2 HO OH N N O N [4Fe-4S]1+ HOOC S [4Fe-4S]2+ NH2 H3C N NH2 SAM2 NH2 OH N N HOOC HO H N H2N N 2 N O S N O N CH N 3 N N S O N N NH2 OH N HO OH HO CH2 N H SAM1 5'dA H N 2 N O SAM2

N N HO OH 5'dA

Figure 4.22 The general scheme of SAM radical generated in the Class C radical SAM enzyme.

Intens . 0111ChuW p256P4_1 SAMCD3 flite_1-C,1_01_4968.d: +MS, 18.3-18.6min #1085-1105 x104 1+ CD3-SAM 252.1103 1+ 8 253.1155

4 1+ 254.1208 2 1+ 255.1235 0 250 252 254 256 258 260 m/z

Intens . 0111ChuW p256P4_2 SAM13C flite_1-C,2_01_4969.d: +MS, 18.2-18.5min #1081-1097 x105 13 1+ CH3-SAM 252.1110 NH3 1.5 N N

N N 1.0 O HO

0.5 HO 1+ 253.1121 Exact Mass: 252.1091

0.0 250 252 254 256 258 260 m/z

Figure 4.23 Mass spectrum of 5'-dA when ChuW used (upper panel) CD3-SAM, (lower 13 panel) CH3-SAM as the cofactor.

4.2.6. ChuW catalyzed cyclopropyl protoporphyrin degradation

Since protoporphyrin can also be a substrate for ChuW, cyclopropyl protoporphyrin 4-12 was synthesized and was tested whether it could also be a substrate

108

for ChuW. LC-MS analysis of ChuW degradation of cyclopropyl protoporphyrin reaction mixtures showed a large amount formation of 5′-dA in the reaction, which suggests that ChuW takes cyclopropyl protoporphyrin as a substrate (Figure 4.24).

Intens . x106 1.5 Blue: No sub N NH ChuW, SAM Red: Full Rxn 1.0 N HN 5’dA FldA, FldR, NADPH

0.5

O HO O OH 4-12 0.0 0 5 10 15 20 25 30 35 Time [min] 1105 ChuW NoP271P4_1-C,3_01_4136.d: EIC 252.1091±0.02 +All MS 1105 Full ChuW P271P4_1-C,1_01_4137.d: EIC 252.1091±0.02 +All MS EIC of 252.1091

Figure 4.24 LC-MS analysis of ChuW catalyzed cyclopropyl protoporphyrin degradation. EICs of 5'-dA [M+H]+ (252.10±0.02).

HPLC analysis of ChuW degradation of cyclopropyl protoporphyrin reaction mixtures also showed the formation of new species which have absorbance around 455 nm (Figure 4.25). The high wavelength products are not stable under air and light condition. The 500nm products will decrease over time, which is similar to the case when cyclopropyl heme 4-6 is used as a substrate for ChuW.

109

Blue: Full NH N ChuW, SAM Red: No ChuW N HN FldA, FldR, NADPH

O HO O OH

UV of 455 nm

Figure 4.25 HPLC analysis of ChuW catalyzed cyclopropyl protoporphyrin 4-12 degradation under 455 nm UV.

Similarly, as the cyclopropyl heme substrate, LC-MS analysis of high wavelength compounds showed a mass of 605.31, which corresponds with the mass of the ring opening product 4-7 (Figure 4.26). What’s more, a new peak with a mass of

607.32 was also detected, which corresponds with the further reduced form of the ring opening product (Figure 4.27).

110

NH N ChuW, SAM Black: No sub N HN FldA, FldR, NADPH Red: Full Rxn

O HO O OH

EIC of 605.3122

Intens . 1105 ChuW_P271P4_1-C,2_01_4182.d: +MS, 12.7-13.4min #1501-1577 5 x10 4-7 605.3176 605.3176 2.5 Accuracy: 8.9 ppm

2.0 N HN

1.5 N N 606.3185

1.0

COOH 0.5 HOOC 607.3198 Exact Mass: 605.3122 619.3279 609.3416 603.2964 608.3225 610.3445 0.0 595.0 597.5 600.0 602.5 605.0 607.5 610.0 612.5 615.0 617.5 620.0 m/z

Figure 4.26 LC-MS analysis of ChuW catalyzed cyclopropyl protoporphyrin degradation. Upper panel: EICs of the ring cleavage product [M+H]+ (605.31±0.02). Lower panel: mass spectrum of the ring cleavage product 4-7 at 13 min.

Intens . x106

1.0

NH N Black: No sub 0.8 ChuW, SAM HN Red: Full Rxn 0.6 N FldA, FldR, NADPH

0.4

0.2 O HO O OH

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 Time [min] 1105 NoChuW_P271P4_1-C,1_01_4179.d: EIC 607.3281±0.02 +All MS EIC of 607.3279 1105 ChuW_P271P4_1-C,2_01_4182.d: EIC 607.3277±0.02 +All MS

Intens . 4-13 1105 ChuW_P271P4_1-C,2_01_4182.d: +MS, 12.4-12.4min #1457-1461 x105 607.3330 607.3330 3 N HN Accuracy: 8.4 ppm

NH HN 2 608.3338

621.3407 1 HOOC COOH 653.3251 Exact Mass: 607.3279 639.3187 635.3213 669.3267 0 560 580 600 620 640 660 680 m/z

Figure 4.27 LC-MS analysis of ChuW catalyzed cyclopropyl protoporphyrin degradation. Upper panel: EICs of the reduced form of ring cleavage product [M+H]+

111

(607.33±0.02). Lower panel: mass spectrum of the reduced form of ring cleavage product 4-13 at 12 min.

Mass corresponding with the crosslinking species between cyclopropyl ring product and SAM was also detected (Figure 4.28). When CD3-SAM was used in the reaction, the majority of the crosslinking species 2-4 mass units increased for the SAM adduct 4-10 (Figure 4.29). When D4-SAM was used in the reaction, the crosslinking species also have 4 mass units increased for the SAM adduct 4-10 (Figure 4.29). The degradation and reduced form of the crosslinking species was also detected (Figure

4.30).

Intens .

8000

N - Full reaction NH ChuW, SAM 6000 - No ChuW N HN FldA, FldR, NADPH 4000

O HO O 2000 OH

0 0 5 10 15 20 25 30 35 Time [min] 1105 NoChuW_P271P4_1-C,1_01_4179.d: EIC 502.2284±0.02 +All MS EIC of 502.2284 1105 ChuW_P271P4_1-C,2_01_4182.d: EIC 502.2284±0.02 +All MS

NH2 OH Intens . HOOC HO N 0214_1ChuW P271P4 TiCi CH3SAM_1-B,3_01_5459.d: +MS, 11.0-11.3min #1296-1332 Figurex104 4.28 LC-MS analysis ofN ChuW catalyzed cyclopropyl protoporphyrin 3 S O N 502.2306 NH2 + degradation. EICs of the porphyrinN cross-linking species [M+H]502.2307 (502.23±0.02). Accuracy: 4.5 ppm 2 N N 502.7325

N HN

1 503.2337 HOOC COOH 503.7334 510.2426 509.2375 510.7441 511.2418 Exact mass: 502.2284 504.2380 506.2655 507.2658 509.7349 0 500 502 504 506 508 510 m/z

Intens . 0214ChuW P271P4 TiCi CD3SAMm_1-B,2_01_5418.d: +MS, 11.1-11.3min #1302-1330 504.2408 NH2 8000 HOOC HO OH 504.2408 N N

CD3-SAM S O N Accuracy: 0.5 ppm NH2 N D2C CD 6000 2 504.7438

N N

4000 N HN 112 503.7380 Exact Mass: 503.2357 504.2409 505.2448 HOOC COOH 2000 505.7488 502.7318 508.3027 511.2481 502.2300 506.2507 507.2654 500.2530 501.2449 509.2678 510.2647510.7416 0 500 502 504 506 508 510 m/z

Intens . 0214_1ChuW P271P4 TiCi CH3SAM_1-B,3_01_5459.d: +MS, 11.0-11.3min #1296-1332 x104 NH HOOC 2 HO OH 502.2306 3 N N S O N 502.2307 NH2 N Accuracy: 4.5 ppm

2 N N 502.7325

N HN

1 503.2337 HOOC COOH 4-10 503.7334 510.2426 509.2375 510.7441 511.2418 Exact mass: 502.2284 504.2380 506.2655 507.2658 509.7349 0 500 502 504 506 508 510 m/z

Intens . 0214ChuW P271P4 TiCi CD3SAMm_1-B,2_01_5418.d: +MS, 11.1-11.3min #1302-1330 COOH CD3-SAM 504.2408 8000 504.2408 NH HOOC 2 N N D2C S Accuracy: 0.5 ppm CD 6000 2 OH 504.7438 NH HOOC N O OH 4000 N 503.7380 Exact Mass: 503.2357 N N 505.2448 504.2409 2000 N 505.7488 H2N 502.7318 508.3027 511.2481 502.2300 506.2507 507.2654 500.2530 501.2449 509.2678 510.2647510.7416 0 500 502 504 506 508 510 m/z

NH2 OH Intens . HOOC HO 1123 ChuW p271p4_1 D4SAM L_1-B,2_01_4286.d: +MS, 11.0-11.2min #1287-1319 N 1500 D N D4-SAM D 504.2399 S O N D NH2 4Da increase D N 504.2399 1250 504.7419 Accuracy: 2.1 ppm 1000 N N D4-SAM

750 N HN

505.2387 500 Exact mass: 504.2409 513.0477 COOH HOOC 505.7451 508.3121 509.2543 250 506.2425 507.2277 511.2389 512.0511 514.0599 497.2321 499.2100 500.7318 502.2176 510.2331 495.2446 496.2110 498.2309 503.2239 508.7210

0 496 498 500 502 504 506 508 510 512 514 m/z

Figure 4.29 Mass spectrum of the porphyrin cross-linking species (502.23) when the ChuW used (upper panel) CH3-SAM, (middle panel) CD3-SAM, (lower panel) D4-SAM as cofactor.

113

Intens . x104

1.25

NH N - Full reaction 1.00 ChuW, SAM N HN - No ChuW 0.75 FldA, FldR, NADPH

0.50

O HO O 0.25 OH

0.00 0 5 10 15 20 25 30 35 Time [min] 1105 NoChuW_P271P4_1-C,1_01_4179.d: EIC 902.4018±0.02 +All MS 1105 ChuW_P271P4_1-C,2_01_4182.d: EIC 902.4015±0.02 +All MS EIC of 902.4018 4-14 Intens . 4-11 1105 ChuW_P271P4_1-C,2_01_4182.d: +MS, 11.4-11.8min #1345-1389 x104 OH HO HO OH 904.4150 IntensN . 1123 ChuW p271p4_1 D4SAM L_1-B,2_01_4286.d: +MS, 11.2-11.8min #1311-1384 N N N 908.4405 1.5 904.4150 S O N2500 O N NH2 S NH 908.4405 N N 2 Accuracy: 2.6 ppm 2000 Accuracy: 2.2 ppm

N N 1.0 1500 N HN 909.4433 905.4188 N HN 902.4009 NH HN D4-SAM 1000 903.4024 910.4446 913.3657 901.3237 903.3384 906.4226 919.3368 905.3483 907.4309 0.5 500 904.3493 911.3345 915.3593 917.3307 920.3355 897.3110 898.3871 899.3177 902.3434 912.8617 916.3514 922.3612 923.8155 HOOC COOH 900.3286 905.8872 918.2850 921.3075 HOOC COOH 906.4202 Exact Mass: 902.4018 0 Exact Mass: 904.4174 900 907.4164 905 910 915 920 m/z 893.8210 918.3836 0.0 890 895 900 905 910 915 920 925 930 m/z

Intens . 0214ChuW P271P4 TiCi CD3SAMm_1-B,2_01_5418.d: +MS, 11.7-12.1min #1369-1421 4 x10 CD3-SAM 908.4415

0.8 906.4282 906.4282 HOOC D C N N 2 S Accuracy: 6.9 ppm CD2 OH 907.4336 909.4458 0.6 NH HOOC N O OH 0.4 N N N Exact Mass: 910.4478 N 905.4198 0.2 H2N 906.4269 911.4517 924.4347 899.3651 903.4104 912.4435 915.3814 917.4483 920.4331 0.0 895 900 905 910 915 920 925 m/z

Figure 4.30 LC-MS analysis of ChuW catalyzed cyclopropyl protoporphyrin degradation. Upper panel: EICs of the degradation product of porphyrin cross-linking species [M+H]+ (902.40±0.02). Middle panel: mass spectrum of the degradation product of porphyrin cross-linking species at 11.5 min; Zoon in graph: mass spectrum of the degradation product of porphyrin cross-linking species at 11.5 min when D4-SAM was used as cofactor; Lower panel: mass spectrum of the degradation product of porphyrin cross-linking species at 11.5 min when CD3-SAM was used as cofactor.

When a strong reducing reagent, such as titanium citrate, was used in the ChuW catalyzed reaction, a crosslinking species 4-15 was detected with just one methylene group from SAM got incorporated (Figure 4.31). Based on the UV spectrum, the 114

crosslinking species most likely contains the ring opening form of the porphyrin ring.

When CD3-SAM was used in the reaction, the majority of the crosslinking species have

2 mass units increased, which suggested that there was one methylene group from the methyl group of SAM (Figure 4.32).

Intens . x104

6 NH N ChuW, SAM - Full reaction N HN 4 - No ChuW TiCi

2 O HO O OH 0 0 5 10 15 20 25 30 35 Time [min] 1105 NoChuW_P271P4_1-C,1_01_4179.d: EIC 888.3861±0.02 +All MS EIC of 888.38 1129 ChuW P256P4 TiCi_1_1-B,1_01_4324.d: EIC 888.3861±0.02 +All MS, Smoothed (0.94,1,GA) 200 300 400 500 600 700 800 900 Wavelength [nm] Intens . 201 UV, 11.8-12.1min #3490-3569 [mAU] 441

200 497 100 258 717 0 Intens . 1129 ChuW P256P4 TiCi_1_1-B,1_01_4324.d: +MS, 11.8-12.1min #1384-1416 5 Intensx10 . 1129 ChuW P256P4 TiCi_1_1-B,1_01_4324.d: +MS, 11.1-11.5min #1296-1344 OH x104 HO 886.3749 1.0 888.3902 N N 0.5 902.4038 S O N 924.3300 0.04 NH2 888.3902 N 860 870 Accuracy: 4.6 880 ppm 890 900 910 920 m/z N 3 N

N H2N 904.4170

2 918.4028 HOOC COOH 1 Exact Mass: 888.3861 902.3962 934.4021 886.3728 4-15 916.4110 942.3816 950.3966 966.4080 983.0267 0 880 900 920 940 960 980 m/z

Figure 4.31 LC-MS analysis of ChuW catalyzed cyclopropyl protoporphyrin degradation using titanium citrate as reducing agent. Upper panel: EICs of the cross-linking species [M+H]+ (888.38±0.02). Middle panel: UV spectrum of the cross-linking species at 11 min; Lower panel: mass spectrum of the cross-linking species at 11 min.

115

OH Intens . HO 0214ChuW P271P4 TiCi CD3SAMm_1-B,2_01_5418.d: +MS, 11.5-11.6min #1350-1365 N N 890.3977 890.3977 S O N CD3-SAM NH2 D2C N Accuracy: 2.8 ppm 3000 N N 891.4027

N H2N 2000

889.3906 875.3050 892.4034 HOOC COOH 888.3874 1000 874.3023 Exact Mass: 890.3987 876.3313 893.4125 904.4143 907.3899 884.3005 886.4612 908.4167 877.8664 879.3371 881.4262 896.9389 898.2063 900.4625 903.3868 0 875 880 885 890 895 900 905 m/z

Figure 4.32 Mass spectrum of the cross-linking species in ChuW catalyzed cyclopropyl protoporphyrin degradation using titanium citrate as reducing agent.

4.2.7. Proposed mechanisms for the ring opening product of ChuW catalyzed cyclopropyl substrates reaction

Based on the result that 5′-dA radical abstracts a hydrogen atom from a methyl group of SAM and the methylene group from SAM is transferred to the ring opening tetrapyrrole product in the ChuW catalyzed reaction, several mechanisms can be proposed for the formation of protoporphyrin ring opening product 4-9. Here, two mechanisms are shown in Figure 4.33 and Figure 4.34. After 5′-dA radical abstracts a hydrogen atom from a methyl group of SAM, the SAM radical attacks the unsaturated bond of the 4-6 to form 4-21. In pathway a, deprotonation and tautomerization of 4-21 lead SAM left as a ylide, and the resulting radical delocalize on the porphyrin and induces the ring opening of the cyclopropyl group on porphyrin A ring to give 4-23, which can be quenched by abstracting a hydrogen atom from SAM (Figure 4.33). This can account for the deuterium incorporation of the porphyrin product when CD3-SAM was used. For pathway b, the early step is the same as the pathway a, which forms the intermediate 4-21. The resulting radical induces the ring opening of the cyclopropyl 116

group on porphyrin B ring. The radical on 4-21 attacks back on the unsaturated bond of pyrrole to form a five-member ring on porphyrin B ring. Then 4-28b goes through a beta-scission to give the 4-29. Both 4-9 and 4-29 have the same mass and can be reduced under hydrogenation. NMR characterization of the enzymatic product will help to distinguish which ring is open. Since porphyrin with 18 π electron core is considered more stable than anaerobilin under light condition, it’s possible to purify compound using solid phase extraction followed by HPLC in a short amount of time. However, since the yield of enzymatic product is low and the purification steps under dark are tedious, there is not enough compound for NMR characterization yet. More compounds have to be purified in the future in order to obtain an NMR spectrum. Reference compounds 4-9 and 4-29 may also be made from irradiation of cyclopropyl protoporphyrin 4-12.

As for the formation of SAM crosslinking species 4-10, 4-11, the early steps are the same as the formation of porphyrin product. However, after the SAM radical attacks the unsaturated bond of the porphyrin ring, instead of SAM leaving as ylide, the radical goes through β‐scission to cleave the porphyrin ring. The pyrrole racial 4-26 gets trapped by the ring opening of side chain cyclopropyl group. After tautomerization,

SAM leaves as SAH. The resulting ring opening radical 4-27 can abstract a hydrogen atom from 5′-dA, which can again abstract a hydrogen atom from SAM. Then the SAM radical can attack the methylene group of the ring opening product 4-7, which further gets quenched by obtaining an electron and a proton to give the crosslinking species 4-

10. Under current purification condition, 4-10 can be degraded to 4-11. When

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cyclopropyl protoporphyrin 4-12 was used as a substrate, and titanium citrate was used as a reducing agent, the intermediate SAM adduct 4-15 can be detected from the reduction of 4-26.

SAM1

SAM2 NH2 HO OH NH HOOC 5’dA HOOC 2 HO OH N N N N S O N O N NH2 5’dA S NH H2C N N 2 N N SAM2 H Fe2+ N Path a N N NH NH N

N N N N SAM

O HO O OH HOOC COOH HOOC COOH 4-6 4-21 4-22

H H

e-, H+ N NH N NH NH N NH N N N N N N N N HN

COOH HOOC COOH HOOC HOOC COOH HOOC COOH 4-23 4-22 4-24 4-9

Figure 4.33 Proposed mechanism 1 for how cyclopropyl heme substrate analog 4-6 trap radical intermediate by formation of porphyrin ring opening product in the ChuW catalyzed reaction.

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SAM1 SAM2 NH2 NH 5’dA HOOC HO OH HOOC 2 HO OH N N N N 5’dA S O N O N NH2 S NH N N 2 N N SAM2 Fe2+ N Path b N N NH NH N

N N N HN

O HO O OH HOOC COOH HOOC COOH 4-6 4-21 4-28a

NH2 OH HOOC HO NH2 HO OH N HOOC N N N S O N NH2 S O N N NH2 N H+, e- N NH NH N NH N

N HN N HN N HN SAM

COOH HOOC HOOC COOH HOOC COOH 4-28b 4-29 4-29

Figure 4.34 Proposed mechanism 2 for how cyclopropyl heme substrate analog 4-6 trap radical intermediate by formation of porphyrin ring opening product in the ChuW catalyzed reaction.

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SAM1

SAM2 NH HOOC 2 HO OH NH 5'dA N N HOOC 2 HO OH N N S O N NH2 5'dA N S O N NH2 N N N 2+ SAM2 Fe NH N N N N N N HN N HN

O HO O OH HOOC COOH HOOC COOH 4-6 4-21 4-22

NH HOOC 2 HO OH NH N N HOOC 2 HO OH N O N N S NH 2 O N N S NH N 2 N N N N N N N HN N HN SAH N N

HOOC COOH HOOC COOH HOOC COOH 4-25 4-26 4-27

NH SAM 5’-dA HOOC 2 HO OH N N 5’-dA 5’-dA SAM O N S NH N 2

N N N N N N N N N N N N

HOOC COOH HOOC COOH HOOC COOH 4-27 4-7 4-7

NH HOOC 2 HO OH HO OH N N N N S O N S O N NH NH2 e- H+ N 2 N degradation

N N N N

HOOC COOH HOOC COOH 4-10 4-11

Figure 4.35 Proposed mechanism for the formation of the crosslinking species in ChuW catalyzed cyclopropyl heme degradation. 120

4.2.8. Proposed mechanisms for the ring opening product of ChuW catalyzed heme degradation

Based on the previous results, cyclopropyl substrate analog studies and the proposed mechanism for NosN37-38, TbtI42, C10P41, a similar mechanism can be proposed for the heme degradation by ChuW (Figure 4.36).

SAM1 SAM2

5’dA R1 R1 S R2 S R2 5’dA N- N SAM2 N- N Fe2+ N N Fe2+ N N- Fe2+ N- N N N-

HOOC COOH COOH HOOC HOOC COOH 4-1 4-30 4-31

R1 R1 R1 S R2 S R2 S R2

N N N N NH N e- H+ N HN N HN N HN

HOOC COOH HOOC COOH HOOC COOH 4-32 4-34 4-35

R1 S R2 NH2

R1: COOH NH N NH N - + 2e 2H NH2 N NH HN N N N N R2: N O OH HOOC COOH HOOC COOH 4-36 4-37 OH

Figure 4.36 Proposed mechanism of ChuW catalyzed heme degradation.

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4.3. Conclusion

Cyclopropyl heme analog was designed as a radical probe for ChuW catalyzed heme degradation. ChuW can take cyclopropyl heme analog as a substrate and several ring opening products were detected by LC-MS/MS, HPLC. A protoporphyrin ring opening product was further characterized by hydrogenation and is considered more stable for NMR characterization. However, there is no NMR spectrum currently available for this porphyrin compound, because the low yield of the enzymatic product and the potential light and air sensitivity of the compound.

Similar as anaerobilin, porphyrin cleavage products are photolabile, including

SAM anaerobilin adducts. The adducts were further confirmed by isotope labeling SAM studies. The cyclopropyl ring opening products and the SAM adducts suggested that a radical is generated on the porphyrin ring of heme by the attack of a SAM radical species. Based on the result, a mechanism was deduced for heme degradation by ChuW.

4.4. Experimental methods

4.4.1. Synthesis of cyclopropyl heme analog

The overall scheme is shown in Figure 4.5. Each detailed step is shown as below:

Synthesis of the protoporphyrin-IX dimethyl ester (4-3)

The protoporphyrin-IX (100 mg) was added to a stirred solution of the trimethyl orthoformate (10 ml) and the methanol (10 ml). Concentrated sulfuric acid (1 ml) was then added slowly, with cooling to room temperature, and the solution was stirred at room temperature for 45 minutes. The mixture was then diluted with water, the pH was adjusted to 4.5 by the addition of dilute sodium hydroxide and the mixture was extracted

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with dichloromethane. The organic extracts were washed with water and the solvent was removed under reduced pressure. The resulting solid was purified by column chromatography (silica gel, 20:1 CH2Cl2: MeOH) to give protoporphyrin-IX dimethyl ester. 1H NMR (400 MHz, Chloroform-d) δ 10.09 (d, J = 4.2 Hz, 2H), 9.97 (s, 2H), 8.24

(m, 2H), 6.36 (d, J = 17.8 Hz, 2H), 6.19 (d, J = 11.5 Hz, 2H), 4.39 (t, J = 7.8 Hz, 4H),

3.82 – 3.53 (m, 18H), 3.28 (t, J = 7.8 Hz, 4H).

Synthesis of 3,8-di- [3' (R, S) -(1' -pyrazolinyl)]-deuteroporphyrin IX dimethyl ester

(4-4)

All reactions were carried out in the dark. A protective shield was used during diazomethane cycloaddition. The starting protoporphyrin-IX dimethyl ester (100 mg) was dissolved in dichloromethane (100 ml) and an ethereal solution of diazomethane (50 ml) was added to the reaction mixture. The resulting solution was sealed with a rubber septum and kept at room temperature for 48 – 96 h. The solvent was degassed by bubbling with nitrogen and the resulting solution evaporated. The resulting solid was purified by column chromatography (silica gel, 100:1 - 10:1 CH2Cl2: acetone) to give

3,8-di- [3' (R,S)-(1' -pyrazolinyl)]-deuteroporphyrin IX dimethyl ester. 1H NMR (400

MHz, Chloroform-d) δ 10.05 (d, J = 21.4 Hz, 2H), 9.78 (dd, J = 34.3, 6.0 Hz, 2H), 7.03 –

6.83 (m, 2H), 5.64 – 5.49 (m, 2H), 4.94 – 4.75 (m, 2H), 4.39 (td, J = 7.8, 3.2 Hz, 4H),

3.74 – 3.50 (m, 18H), 3.29 (td, J = 7.8, 2.7 Hz, 4H), 2.94 – 2.78 (m, 2H), 2.32 – 2.16 (m,

2H).

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Synthesis of 3,8-dicyclopropyldeuteroporphyrin IX dimethyl ester (4-5)

The 8-di- [3' (R, S) -(1' -pyrazolinyl)]-deuteroporphyrin IX dimethyl ester (31 mg) were dissolved in o-dichlorobenzene (20 ml) and heated at reflux for 45 min. The reaction solution was diluted with hexane (200 ml), and the formed residue was filtered and purified by column chromatography (silica gel, 100:1 CH2Cl2: acetone) to give the

3,8-dicyclopropyldeuteroporphyrin IX dimethyl ester. 1H NMR (400 MHz, Chloroform- d) δ 10.41 (d, J = 8.6 Hz, 2H), 10.03 (d, J = 10.0 Hz, 2H), 4.43 (m, 4H), 3.80 – 3.55 (m,

18H), 3.32 (m, 4H), 3.14 – 3.01 (m, 2H), 1.70 (m, 4H), 1.54 – 1.44 (m, 4H).

Synthesis of Ferric 3,8-dicyclopropyldeuteroporphyrin IX (4-6)

Synthesis of ferric 3,8-dicyclopropyldeuteroporphyrin IX was followed a protocol to synthesize ferric meso-methylmesoheme regioisomers.84 The 3,8- dicyclopropyldeuteroporphyrin IX dimethyl ester was hydrolyzed in 25% (v/v) HCl at

25 °C in the dark for 8 h. The hydrolysis reaction was worked up by neutralizing to pH

2-4, extracting with CH2Cl2, washing the organic phase with water before drying it with anhydrous Na2SO4, and removing the solvent under vacuum. The porphyrins were used without further purification for the subsequent reaction in which the iron was inserted by the ferrous sulfate method. An argon-purged, saturated aqueous Fe2SO4 solution (1mL) was added to a stirred, argon-purged solution of the porphyrins in 1 mL of pyridine and

20 mL of acetic acid. The mixture was warmed for 10 min under a stream of argon. The stirred reaction mixture was then exposed to the air while it cooled to promote autooxidative formation of the ferric state before it was combined with 25 mL of brine and extracted with diethyl ether. The organic layer was washed with 25% HCl to remove

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unreacted porphyrin, washed with water, and dried over Na2SO4. Removal of the solvent under vacuum provided the ferric 3,8-dicyclopropyldeuteroporphyrin IX, which were

+ purified by reversed-phase HPLC. HRMS m/z 644.2113, calculated for FeC36H36N4O4

644.2080. lmax (CH2C12) 420, 540, 640 nm.

4.4.2. Overexpression and purification of ChuW

chuW was cloned into pTHT vector (modified from pET28b with TEV cleavage site). ChuW was over expressed in E. coli strain BL21 DE3 and was co-expressed with a plasmid encoding the suf operon. A 15 mL overnight culture was grown in LB medium in the presence of 40 µg/mL kanamycin and 30 µg/mL chloramphenicol. This was then added to 1.5 L of LB medium containing 40 µg/mL kanamycin and 30 µg/mL chloramphenicol. The cultures were incubated at 37°C with shaking (180 rpm) until the

OD600 reached 0.45. The culture was then incubated at 4°C without shaking for 1 hour.

Expression was induced by the addition of 100 µM IPTG followed by ferrous ammonium sulfate (120 mg) and cysteine (120 mg) and the culture was grown at 15 °C with shaking (110 rpm) overnight. The cells were harvested by centrifugation and stored in liquid nitrogen until further use. All steps for protein purification were carried out in an anaerobic chamber (COY laboratories). Cell pellets (~12 g) were thawed and resuspended in 30 mL lysis buffer (100 mM Tris-HCl, pH 7.5) in the presence of lysozyme (0.2 mg/mL) and benzonase (100 units). This mixture was cooled in an ice- bath with continuous stirring for 1-2 hrs. The suspension was then sonicated to complete cell lysis for 5 × 120 s using a Misonix Sonicator XL-2000 (setting = 9). Cell debris was removed by centrifugation, and the lysate was loaded onto a Histrap column pre-

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equilibrated with lysis buffer. The column was washed with 5-10 column volumes of wash buffer (100 mM Tris-HCl, 30 mM Imidazole, pH 7.5). The CysS protein was then eluted using elution buffer (100 mM Tris-HCl, 300 mM NaCl, 250 mM imidazole, pH

7.5). The brown-colored fractions, containing the desired protein, were pooled and buffer exchanged to final buffer (100 mM Tris-HCl, pH 7.5 at 25 °C, 30% glycerol) using Econo-Pac 10DG size exclusion chromatography. The purified CysS was divided into aliquots in eppendorf tubes, frozen in liquid nitrogen, and stored at −80 °C, submerged in liquid nitrogen, until future use. Each tube has a pin hole in the cap to allow for gas release during thawing. Protein concentration was measured by the absorbance at 280 nm (A280) when the protein was exposed to air or the [4Fe-4S]2+ cluster was reduced to [4Fe-4S]1+ by dithionite (prevent the interference from absorbance from [4Fe-4S]2+) with an extinction coefficient calculated using the

ProtParam tool of the ExPASy proteomics server.

4.4.3. In vitro activity assay with ChuW:

For the UV-Visible spectroscopy of monitoring ChuW activity, ChuW (5 µM) was incubated with 20 µM substrate and EcFld (5 µM) /EcFpr (2 µM), NADPH (200

µM) reducing system anaerobically in dark. Assays were initiated by the addition of

SAM (250 µM) and UV spectra were recorded every 10-20 minutes.

For the HPLC and LCMS analysis of ChuW activity, 150 µM ChuW was incubated with 300 µM substrate, 1 mM SAM, 1 mM NADPH, 100 µM EcFld, and 50

µM EcFpr in pH 7.5, 100 mM phosphate buffer anaerobically for 2.5 h at room temperature and in the dark. The assay mixture was applied to the SPE column pre-

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equilibrated with 1 eq of methanol, 1 eq of H2O, and 1 eq of assay buffer. Once the assay mixture ran through the column, it was washed with 1eq of buffer,2 eq of water,1eq of

1%TFA, and 3eq of water, before elution in 100% methanol in dark. The elution solution was further lyophilized before analyzed by HPLC and LCMS.

4.4.4. HPLC parameters for the analysis of the ChuW reaction mixture

HPLC analysis was performed on a ZORBAX Eclipse XDB-C18 column (15cm

4.6mm, 5µm particles, Agilent Technologies, flow rate1⁄41mL/min). Typical injection volumes were in the range of 10–80µL. Data were processed using ChemStation ver.

B.04.01 SP1 (Agilent Technologies).

LC conditions: A- 0.1 % formic acid in H2O, B- 0.1 % formic acid in acetonitrile

LC method: 0 min – 100% A, 2 min – 100% A, 4 min – 80% A 20% B, 27 min –

100% B, 29 min – 100% B, 30 min – 100% A, 40 min – 100% A.

4.4.5. LC-MS parameters for the analysis of the ChuW reaction mixture

LC-ESI-TOF-MS on small molecules was performed using an Agilent 1260

(15 cm x 4.6 mm, 5 µm particles, Agilent).

LC conditions: A- 5 mM Ammonium acetate buffer, pH 6.6, B- 75 % Methanol and 25 % Water.

LC method: 0 min – 100% A, 2 min – 100% A, 4 min – 80% A 20% B, 27 min –

100% B, 29 min – 100% B, 30 min – 100% A, 40 min – 100% A.

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5. TRAPPING A FOUR-CARBON FRAGMENT IN THIC CATALYZED

REARRANGEMENT IN THIAMINE BIOSYNTHESIS

5.1. Introduction

Thiamine pyrophosphate (vitamin B1) is a crucial cofactor that is present in all living systems, in which it helps to catalyze essential biochemical reactions. Thiamine pyrophosphate is synthesized in the cytosol and is required in the cytosol for the activity of transketolase and in the mitochondria for the activities of pyruvate-, oxoglutarate-, branched chain keto acid dehydrogenases and thiamin acts as a stabilizer of an acyl carbanion biosynthon. The deficiency of thiamine in the diet will lead to the peripheral nervous system disease beriberi in human.

In all organisms, the thiazole and pyrimidine moieties of thiamin monophosphate

(ThMP) are generated in separate branches of the pathway and then joined by a coupling enzyme. The mechanisms of most enzymes in thiamin biosynthesis pathway are well understood.85-86 One of the exceptions is ThiC, a pyrimidine synthase in the thiamin biosynthetic pathways of bacteria and plants.

ThiC catalyzes the complex rearrangement of aminoimidazole ribonucleotide

(AIR) 5-1 to 4- amino-5-hydroxymethyl-2-methylpyrimidine phosphate (HMP-P) 5-2

(Figure 5.1). The origin of all the atoms of the product and the fate of most of the atoms of the substrate have been determined using isotope labeling studies.4, 15, 87-88

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NH H 2 H H N H 2- H O ThiC N OPO3 N H H O 2- solv H CO O3PO H H N Hsolv NH2 H OH HO OH Hsolv AIR 5-1 HMP-P 5-2

NH2 H H H H HO + O N O N S N NH H N O 2 NH2 HO HO OH N N OH N N SAM 5’dA

Figure 5.1 ThiC catalyzed rearrangement from AIR to HMP-P

From the experimental data, we know that the proS hydrogen at C5′ is first abstracted by 5′-dA radical and then later replaced by a hydrogen atom from 5′-dA with retention of stereochemistry. The resulting 5′-dA radical has the second abstraction of a hydrogen atom from C4′ of AIR. C2′ of AIR becomes the methyl group C8 of HMP-P and C4′, C5′ remain in the HMP-P. C1′ of AIR is released as formate and C3′ is lost as carbon monoxide. N5 of AIR is retained as the N4 of the HMP-P. C3′ hydrogen of AIR is transferred to C8 of HMP-P while C2′ hydrogen remains in the C8 of HMP-P. One hydrogen on C8 and the hydrogen on C6 of HMP-P are from the solvent.

Based on the current data, a mechanism for ThiC catalyzed rearrangement can be proposed (Figure 5.2). The 5′-dA radical first abstracts a hydrogen atom from C5′ of

AIR. The resulting radical goes through a beta scission and then a reduction by the amino imidazole to give 5-5. The radical on the amino imidazole induces the cleavage of the N-glycosyl bond. Then the amino imidazole is reoriented in the active site to add on the enol phosphate to form 5-8, which abstracts a hydrogen atom from 5′-dA. The 5′-dA

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radical further abstracts a hydrogen atom from C4′. Cyclization and hydrogen atom transfer of the resulting radical give 5-13. Then, C1′ leaves as formate to form the radical stabilized carbanion 5-14. After protonation, the C3′ radical goes through a ribonucleotide reductase type of dehydration to form a-keto radical 5-16. Similar as the

Dowd–Beckwith ring-expansion reaction, another cyclization gives bicyclic intermediate

5-17 followed by two beta scission and oxidation to give acylium ion 5-19. CO elimination and tautomerization give the final product HMP-P 5-2.

A ThiC paralog, BzaF, can catalyze the conversion of AIR to hydroxybenzimidazole – one form of the axial ligands of vitamin B12. Labeling studies have established that the early steps in the ThiC and BzaF-catalyzed reactions are same.

EPR studies on BzaF using AIR isotopologues show a transient radical with a structure that is consistent with intermediate 5-5.89 Since desamino-AIR is not a substrate for

ThiC, the amino group may play a role for in activating the substrate for the electron transfer shown in the conversion of 5-4 to 5-5 in Figure 5.2.

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N N N N

H2N N H2N N H2N N H2N N PO O PO O PO HO PO HO H H H H H Ad H Ad H H H H H Ad H Ad H OH OH OH OH OH OH OH OH H H H 5-5 5-1 5-3 5-4

H H N N N H N H N N N H2N N N NH NH H PO O PO HO PO HO PO HO H H OH H OH H OH Ad H H H H H Ad H H Ad H OH OH OH OH Ad OH OH OH H H 5-9 5-6 5-7 H 5-8

H N H N N N 2 N 2 N H N H H H2N 2 PO PO N N N OH NH OH PO HO PO HO H H OH H OH H OH H H H H OH HO H Ad H Ad OH OH OH OH OH OH OH 5-10 5-11 Ad 5-12 5-13

H2N H N H2N H H N H H N H N H2N N 2 N 2 N H O PO H H H H NH PO PO PO PO OH N N N N H H HO H H H HO H O O O 5-14 OH 5-15 5-16 H 5-17 OH H 5-18 H

H2N H2N H2N H2N H H H PO NH H PO NH H PO N H PO N H O O H N N NH N H C O H 5-19 H 5-10 H 5-21 H 5-2

Figure 5.2 Proposed mechanism for ThiC catalyzed AIR 5-1 to HMP-P 5-2 rearrangement.

Compared with the canonical CX3CX2C cluster-binding motif for radical SAM enzyme, ThiC adopts an unusual CX2CX4C motif for the iron-sulfur cluster binding.

Several crystal structures of ThiC with bound [4Fe-4S] cluster, small molecules (SAH, 131

5′-dA, methionine, AIR, imidazole ribonucleotide) are available. These structures not only reveal a novel second iron site involved in amino acid binding but also give an insight into possible protein residues in the active site that may involve in the catalysis.

The active D383N mutant suggests that the N-glycosyl bond cleavage is not strongly dependent on acid catalysis. Several other mutants (D383N, M248L, C474S and H417A) still needs further investigation.

Although the activity of ThiC was reconstituted in vitro more than ten years ago,87 we still don’t know most of the steps in the mechanism because of the low activity and narrow range of substrates tolerance. While we tried to identify the new products and metabolites in ThiC mutants, a four-carbon fragment from AIR was detected during

ThiC catalyzed reaction.

5.2. Result and discussion

5.2.1. Identification of bound metabolites co-purifying with ThiC

When wild-type ThiC (wtThiC), recombinantly over-expressed in E. coli in LB medium and purified in a glove box, several bound metabolites were detected by HPLC under UV of 250 nm after ThiC was heat denatured (Figure 5.3). By comparing with the

UV and the retention time of the standard, around 4 min peak is identified as SAM and around 17 min peak as 5′-dA. After the treatment of alkaline phosphatase from calf intestinal (CIP), the early retention time peaks were disappeared or decreased with the increase of 16 min peak and appearance of a new peak around 15 min. For the ThiC mutant C474S, besides the same peaks that were detected in the wtThiC, there was one

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more peak around 2 min, which disappeared under CIP treatment with the formation of a new peak around 14 min (Figure 5.4).

250 nm 2500 260 nm 280 nm

2000

1500 Blue: WT heat mAu Red: WT CIP treatment 1000 SAM 5’dA 500

0 0 5 10 15 20 T/min 280 nm 250 nm 260 nm 280 nm

Figure 5.3 HPLC analysis of bound metabolites in ThiC under 250 nm. UV spectrum of different bound metabolites metabolites.

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2500 250 nm 280 nm WT heat_CIP 2000 WT heat

1500 C474S heat_CIP

mAu C474S heat 1000

500

0 0 5 10 15 20 T/min

250 nm

280 nm

Figure 5.4 HPLC analysis of bound metabolites in the ThiC mutant C474S under 250 nm. UV spectrum of different bound metabolites metabolites.

Based on the UV similarity and co-elution studies of the bound metabolites with different nucleosides, the 17 min peak is identified as adenosine (Figure 5.5). The 15 min peak is identified as deoxyguanosine. The 14 min peak is identified as guanosine.

So, the earlier time point peaks on the HPLC could probably be the corresponding nucleotides.

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250 nm 260 nm

280 nm

Guanosine adenosine

Exact Mass: 268.1040 Blue: C474S bounded metabolites O NH3 Red: C474S bounded metabolites after CIP N HN N N Green: Adenosine N N H2N N N Pink: Guanosine O O HO HO OH OH HO HO Guanosine adenosine

Blue: Deoxyguanosine Red: C474S bounded metabolites after CIP

HN N N H2N N O OH HO deoxyguanosine

UV of 250 nm

Figure 5.5 Upper panel: UV spectrum of guanosine and adenosine. Middle panel: co- elution HPLC traces of bound metabolites in the ThiC mutant C474S and guanosine, adenosine under 250 nm. Lower panel: co-elution HPLC traces of bound metabolites in the ThiC mutant C474S and deoxyguanosine.

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5.2.2. Detection of a four-carbon fragment in ThiC reaction by LC-MS/MS

When Arabidopsis thaliana ThiC (AtThiC) was incubated with AIR, SAM, dithionite in an anaerobic condition, a new peak with a mass of 204.10 was detected by

LC-MS/MS after heat quenching (Figure 5.6). This peak only presents in the full reaction and is absent in all other controls. By using isotope labeling AIR substrates, it was found that four carbons from the ribose part of the AIR but not the nitrogen of the amino group and C1′ of AIR were incorporated in this new compound (Figure 5.7).

Intens . x104 Red: Full reaction 1.5 Orange: No ThiC Black: No AIR Blue: No SAM 1.0 Pink: No Dit Purple: No AIR ThiC_HMP

0.5

0.0 0 5 10 15 20 25 30 35 Time [min] 0619_2No sub_HMP_CIP_pos_1-C,2_01_2364.d: EIC 204.1000±0.02 +All MS 0619_1Full_CIP_pos_1-C,1_01_2363.d: EIC 204.1000±0.02 +All MS 0504_5_No Dit_CIP_pos_1-E,5_01_1489.d: EIC 204.1000±0.02 +All MS 0504_2_No e_CIP_pos_1-E,2_01_1486.d: EIC 204.1000±0.02 +All MS EIC of 204.10540504_3_No sub_CIP_pos_1-E,3_01_1487.d: EIC 204.1000±0.02 +All MS 0504_4_No SAM_CIP_pos_1-E,4_01_1488.d: EIC 204.1000±0.02 +All MS

Intens . 1+ 0619_1Full_CIP_pos_1-C,1_01_2363.d: +MS, 14.1-14.5min #837-863 204.1054

8000

6000

4000

2000 1+ 205.1091 1+ 206.1069 210.1162 212.8498 0 200 202 204 206 208 210 212 214 m/z

Figure 5.6 LC-MS analysis of ThiC catalyzed AIR to HMP rearrangement. Upper panel: EICs of 5′-dA [M+H]+ (204.10±0.02). Lower panel: mass spectrum of 204 compound at 14 min.

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A Intens . 13 0508_3_FullAIR1_CIP m_pos_1-C,1_01_1549.d: +MS, 14.2-14.4min #844-856 2ʹ- C-AIR 1+ 205.1070 6000 N 1Da increase NH2 NH2 O O 5000 P N NH ThiC, SAM O 2 N OPO 2- CIP N OH OH 3 HO 4000 Dithionite OH OH N N

3000 250.0935 2000 245.1270 1000 1+ 207.0979 261.0749 189.0477 201.1046 224.1289 228.5783 233.1328 242.1419 255.1494 259.1049 0 180 190 200 210 220 230 240 250 260 m/z B Intens . 0514_4FullAIR3_pos_1-D,4_01_1658.d: +MS, 14.3-14.5min #849-861 13 1+ 1ʹ,2ʹ,3ʹ,4ʹ,5ʹ- C5-AIR 208.1187 3000 N NH2 4Da increase NH2 O O P N NH ThiC, SAM O 2 N OPO 2- CIP N OH OH 3 2000 HO Dithionite OH OH N N

1000 212.8493 219.0615

1+ 212.0732 207.1088 209.1219 214.8493 224.1356 204.1102 221.0634 223.1221 226.1106 228.0928 0 195 200 205 210 215 220 225 m/z

Intens . C 0514_1FullAIR2_pos_1-D,1_01_1655.d: +MS, 14.2-14.3min #844-852 3000 2 205.1121 5ʹ,5ʹ- H2-AIR 2500 N 1Da increase D NH2 D NH2 D O O D 2000 P N NH ThiC, SAM O 2 N OPO 2- CIP N OH OH 3 HO Dithionite 212.0757 1500 OH OH N N

212.8508 1000 219.0620 206.1146 500 214.8472 196.1049 197.1037 207.1068 210.1147 211.0818 0 195 200 205 210 215 m/z

Intens . D 1+ 0627_1Full_15N_pos_1-B,1_01_2579.d: +MS, 13.8-14.3min #818-849 4000 1+ 15N-AIR 204.1044 N 15 15 NH2 204.1044NH2 3000 O O P N 15NH ThiC, SAM O 2 N OPO 2- CIP N OH OH 3 HO Dithionite OH OH N N 2000

1000 1+ 205.1074 1+ 206.1028 0 198 200 202 204 206 208 210 212 m/z

Figure 5.7 Isotope labeling pattern in 204.10 shunt product in ThiC catalyzed reaction. Mass spectrum of the in 204.10 compound when the substrate is (A) 2′-13C-AIR; (B) 13 2 15 13 2 1′,2′,3′,4′,5′- C5-AIR; (C) 5′,5′- H2-AIR; (D) N-AIR; (E) 1′- C-AIR; (F) 2′- H-AIR; (G) 4′-2H-AIR.

137

Intens . E 0805_1Full_pos_1-D,1_01_3115.d: +MS, 13.8-14.2min #823-843 x104 13 1+ 1ʹ- C-AIR 204.1048 N 2.5 NH2 NH2 O O P N NH ThiC, SAM O 2 N OPO 2- CIP N OH OH 3 2.0 HO Dithionite OH OH N N

1.5

1.0

231.0421 0.5 193.0702 207.0734 217.0997 187.0360 233.0428 0.0 180 190 200 210 220 230 m/z

Intens . F 1023_4FullAIR2D_pos_1-B,4_01_4435.d: +MS, 14.1-14.4min #838-854 2 205.1115 3000 2ʹ- H-AIR 1Da increase 2500 N 207.0723 O O N NH 2000 P O 2 ThiC, SAM OH HO D Dithionite 1500 OH OH

1000 206.1326 212.0928 218.1220 219.0638 213.0033

195.0563 201.0755 222.1118 500 208.0760 203.1026 204.1070 209.0736 215.0655 196.1122 197.0151 198.0056 200.0886 202.1092 216.0859 221.0534 199.0899 210.1664 211.0513 217.1075 0 195 200 205 210 215 220 m/z Intens . 0607_5Full 4DAIR4_pos_1-B,5_01_2227.d: +MS, 14.1-14.3min #838-851

G 2 205.1102 600 4ʹ- H-AIR 170.0906 N 1Da increase O O N P NH2 HO O 400 HO D OH OH

200 193.0651 203.1007 206.1124 228.0835 212.8485

0 190 195 200 205 210 215 220 225 m/z

138

5.2.3. Identification of the methionine as the trapping agent for the four-carbon fragment of the AIR

When CD3-SAM was used in the reaction, there was an increase of three mass

13 units in the mass of 204.10 peak. Similarly, when CH3-SAM was used as the cofactor, there was an increase of one mass in the mass of 204.10 peak (Figure 5.8). Considered the error range of the mass instrument we used, the error of the measured mass and the exact mass should be less than 10 ppm. Based on the reactants in ThiC reaction, one of the most possible chemical formula of 204.1048 contains a sulfur atom. These results lead us to think the sulfur as well as the methyl group of SAM could be incorporated into this shunt product of the ThiC reaction.

139

Intens . 1+ 0627_4Full_CD3_pos_1-B,4_01_2581.d: +MS, 13.8-14.4min #820-853 3000 207.1247 207.1247 CD3-SAM

2500

2000

1500 231.0390

212.8517 1000

204.1047 216.1722 500 214.8487 193.0589 228.1024 234.9141 0 180 190 200 210 220 230 240 m/z

Intens . 0611_1Full_13CSAM_pos_1-B,1_01_2278.d: +MS, 14.4-14.6min #853-865 x104 1+ 205.1090 1.0 13C-SAM 205.1090

0.8

0.6

0.4

204.1061 0.2 1+ 206.1120 207.0881

0.0 202 204 206 208 210 m/z

Figure 5.8 Isotope labeling pattern in 204.10 compound from different isotope labeling SAM. Mass spectrum of the in 204.10 compound when (Upper panel) CD3-SAM; 13 (Lower panel) CH3-SAM.

In order to explore whether the other parts of SAM were incorporated in this

13 204.10 peak, different labeling SAM was biosynthesized. When (adenosyl- C10)-SAM was used as the cofactor in ThiC reaction, the mass of byproduct peak remained the same, which suggested that the adenosyl part of SAM was not incorporated into this compound (Figure 5.9). However, when (methionine-D8)-SAM was used in the reaction, there was an increase of eight mass units in this compound (Figure 5.9).

140

Intens . 0826_1Full_pos_1-D,1_01_3457.d: +MS, 13.7-13.9min #817-825 1+ 8000 13 207.0717 SAM- C10 NH2 * N * N CH * 1+ 3 N N * 204.1044 6000 HOOC S * * O NH2 * * * * 4000 OH OH

2000 1+ 1+ 208.0747 209.0674 1+ 1+ 205.1031 1+ 213.0001 206.1070 210.0891 215.0719 0 202 204 206 208 210 212 214 m/z

Intens . 1001_1FullSAMD8_pos_1-E,1_01_3986.d: +MS, 14.5-14.6min #861-869 212.1536 2500 SAM-D8 213.0012 NH2 N 8Da increase 2000 D CD3 N D D HOOC S N N

1500 D O NH2 D OH OH 1000 Exact Mass: 407.1947 1+ 210.0772 214.9980 500 213.1551 214.0014 211.1460 214.1467 210.5612 211.5483 212.5017 213.5062 214.5308 215.4244215.6037 0 210 211 212 213 214 215 m/z

Figure 5.9 Isotope labeling pattern in 204.10 compound from different isotope labeling SAM. Mass spectrum of the in 204.10 compound when different isotope labeling SAM 13 was used in the ThiC catalyzed reaction. Upper panel: (adenosyl- C10)-SAM; Lower panel: (methionine-D8)-SAM.

To further explore whether the methionine in the 204.10 peak comes from SAM, fivefold excess of D8-methionine (compared with the concentration of SAM) was incubated in the ThiC reaction. LC-MS analysis of the reaction mixture revealed that more than 80% of the product was deuterium labeled (212.15) (Figure 5.10). This suggests that methionine part that gets incorporated into the peak does not have to come from the cleavage product of the SAM.

141

Intens . 1+ 1026_2FullD8Met_pos_1-F,3_01_4509.d: +MS, 14.1-14.6min #835-867 x104 212.1543 NH2 HO D D D D 212.1543 D 3 O D S Accuracy: 5.6 ppm D D

2 Exact Mass: 212.1555

1 1+ 204.1048 211.1483 213.1571 1+ 207.0738 214.1522 0 202 204 206 208 210 212 214 216 218 220 222 m/z

Figure 5.10 Isotope labeling pattern in 204.10 compound when excess of D8-methionine was added to the ThiC catalyzed reaction.

In order to probe whether the AIR fragments trapped by methionine happened in the enzyme active site, D-methionine, L-methionine, cysteine, L-homocysteine were also incubated with the ThiC reaction respectively. Only D/L-methionine showed the incorporation in the unknown compound, while there is no much difference between D- methionine or L-methionine.

5.2.4. Determination of possible reference compounds for the detected shunt product in ThiC catalyzed reaction

Based on the experimental data in section 5.2.2-5.2.4, the shunt product consists of four carbon atoms (2′,3′,4′,5′) from AIR substrate and methionine from SAM or buffer. It’s not known how the four-carbon fragment is connected to the methionine.

Based on the ThiC proposed mechanism, the crystal structure and the electrophilic property of the atoms in methionine, the most possible structure of the shunt product is the sulfonium compounds 5-21, where the four-carbon is linked to methionine through the sulfur atom (Figure 5.11). In one of the conformation in ThiC, the distance between sulfur atom of SAH and the 5′C of the AIR is 4.4 Å (Figure 5.12). Once the ribose ring

142

of AIR is broken, the distance could be smaller. Synthesis of compounds 5-21 are in progress.

NH NH NH2 2 HO 2 HO HO

O S O S O S

5-21a 5-21b 5-21c

Figure 5.11 Possible structures for the shunt product.

Figure 5.12 The distance between sulfur atom of SAH and the 5′C of the AIR in ThiC crystal structure (4S28)

143

Based on the analysis of MS/MS fragments of the shunt product, it is also possible that the four-carbon is linked to methionine through the nitrogen atom of the methionine (Figure 5.13, 5.15). Compounds 5-22a and 5-22b were synthesized.

However, from the retention time and MS/MS fragments of these compounds, neither of them match the enzymatic shunt product generated in the ThiC catalyzed reaction. It’s possible that 5-21 can have an arrangement to form 5-22, which gives the observed

MS/MS fragmentations.

Intens . 1029_1Full Met_pos_1-F,1_01_4515.d: +MS2(204.1150), 35.0eV, 14.2-14.3min #841-848 NH HO 2 110.0975 2000 O S

5-21a N 1500 H Exact Mass: 204.1053 Exact Mass: 110.0964

S 1000 S 128.0693 133.0302 O HO N HO HO N H2 N 500 O H S 158.0980 O H Exact Mass: 133.0318 Exact Mass: 204.1053 Exact Mass: 158.0998 5-22a Exact Mass: 128.0706 136.0605 0 100 120 140 160 180 200 m/z

Intens . 1026_2FullD8Met_pos_1-F,3_01_4509.d: +MS2(212.1479), 35.0eV, 13.7-14.4min #815-856 D8-methionine 1+ 114.1221 2000 D D D D HO D S D N D D H O 1500 O D D D D HO D D D Exact Mass: 129.0769 D D D N D N 1+ S H H 129.0770 D D 1000 Exact Mass: 114.1215 Exact Mass: 141.0820 Exact Mass: 166.1500 1+ 141.0813 166.1521 1+ 500 151.0462

133.0338 212.1479 156.0908 189.0499 0 100 120 140 160 180 200 220 m/z

Figure 5.13 Mass fragmentation of the shunt product (204.1053) when ThiC catalyzed reaction with SAM, AIR, dithionite and methionine (upper panel), D8-methionine (lower panel).

144

S S

HO HO N N H H O O 5-22a 5-22b

Figure 5.14 Possible structures for the shunt product.

Intens . A 0508_3_FullAIR1_CIP m_pos_1-C,1_01_1549.d: +MS2(205.1139), 35.0eV, 14.0-14.1min #832-839 13 111.0985 600 2ʹ- C-AIR N NH2 NH2 O O N P NH2 ThiC, SAM 2- CIP 400 O N OPO N OH OH 3 HO Dithionite 136.0628 OH OH N N 129.0774

200

0 80 100 120 140 160 180 m/z

Intens . B 0720_1FullAIR3_CIP_pos_1-E,1_01_2797.d: +MS2(208.1221), 35.0eV, 14.0-14.1min #831-839

114.1078 13 1ʹ,2ʹ,3ʹ,4ʹ,5ʹ- C5-AIR 600 N NH2 NH2 O O P N NH ThiC, SAM O 2 N OPO 2- CIP N OH OH 3 HO Dithionite 400 OH OH N N

132.0841

200

0 110 120 130 140 150 160 170 180 190 200 210 m/z

Figure 5.15 Mass fragmentation of the shunt product (204.1053) in ThiC catalyzed 13 13 2 reaction when substrate is (A) 2′- C-AIR; (B) 1′,2′,3′,4′,5′- C5-AIR; (C) 4′- H-AIR.

145

Intens . 0701_1FullAIR54D_pos_1-C,1_01_2648.d: +MS2(205.1182), 35.0eV, 13.9-14.2min #824-846 111.1023 4ʹ-2H-AIR N 1500 O O N P NH2 HO O HO D 1000 129.0757 OH OH

159.1044

500 133.0309

98.9838

0 100 120 140 160 180 200 220 m/z

Figure 5.15 Mass fragmentation of the shunt product (204.1053) in ThiC catalyzed 13 13 2 reaction when substrate is (A) 2′- C-AIR; (B) 1′,2′,3′,4′,5′- C5-AIR; (C) 4′- H-AIR.

5.3. Conclusion

By using high resolution LC-MS, isotope labeling AIR, SAM and methionine, a four-carbon fragment and methionine adduct was detected. Since the adduct does not have a structure like compounds 5-22. It’s possible that the shunt product has structures like compounds 5-21. From the isotope labeling studies, we learn that this four-carbons fragment forms after 5′-dA abstracts a hydrogen atom from the C5′ of the AIR but before the second hydrogen abstraction from C4′ of the AIR. Also, it forms after the C1′ release as formate and before imidazole recombines with the sugar backbone or the imidazole gets hydrolyzed after recombines with the sugar backbone. If it’s not the latter case, it implies that C1′ leaves before the imidazole recombines during the ThiC catalyzed AIR rearrangement.

146

5.4. Experimental methods

5.4.1. Overexpression and purification of AtThiC

ΔN71-AtThiC in pET-28 plasmid was transformed into E. coli BL21(DE3) cells.

ThiC was over expressed in E. coli BL21 DE3 and was co-expressed with a plasmid encoding the suf operon. A 15 mL overnight culture was grown in LB medium in the presence of 40 µg/mL kanamycin and 30 µg/mL chloramphenicol. This was then added to 1.5 L of LB medium containing 40 µg/mL kanamycin and 30 µg/mL chloramphenicol. The cultures were incubated at 37°C with shaking (180 rpm) until the

OD600 reached 0.45. The culture was then incubated at 4°C without shaking for 1 hour.

Expression was induced by the addition of 100 µM IPTG followed by ferrous ammonium sulfate (120 mg) and cysteine (120 mg) and the culture was grown at 15°C with shaking (110 rpm) overnight. The cells were harvested by centrifugation and stored in liquid nitrogen until further use. All steps for protein purification were carried out in an anaerobic chamber (COY laboratories). Cell pellets (~12 g) were thawed and resuspended in 30 mL lysis buffer (100 mM Tris-HCl, pH 7.5) in the presence of lysozyme (0.2 mg/mL) and benzonase (100 units). This mixture was cooled in an ice- bath with continuous stirring for 1-2 hrs. The suspension was then sonicated to complete cell lysis for 5 × 120 s using a Misonix Sonicator XL-2000 (setting = 9). Cell debris was removed by centrifugation, and the lysate was loaded onto a Histrap column pre- equilibrated with lysis buffer. The column was washed with 5-10 column volumes of wash buffer (100 mM Tris-HCl, 30 mM Imidazole, pH 7.5). The CysS protein was then eluted using elution buffer (100 mM Tris-HCl, 300 mM NaCl, 250 mM imidazole, pH

147

ProtParam tool of the ExPASy proteomics server.

5.4.2. Enzymatic synthesis of AIR from AIRs:

A typical reaction mixture consisted of 25 µL 100 mM AIRs, 10 µL 400 mM

ATP, 5 µL 1 M MgCl2, 900 µL 25 mM KPi (pH 7.5) and 100 µL of the AIRs kinase stock.

The reaction mixture was allowed to incubate at room temperature for 1 hr and was lyophilized till dry. The residue was transferred into the glove box and resuspended in degassed water to a final volume of 100 µL (final AIR concentration of ~25 mM).

This AIR stock was directly used for ThiC reactions.

5.4.3. Enzymatic assay of ThiC reaction

A typical enzymatic reaction was performed in 100 mM phosphate buffer, pH 7.5 containing 250 µM ThiC, 1 Mm AIR, 1.2 mM SAM and 5 mM dithionite. The reaction was incubated at room temperature anaerobically for 2 hours. EcFld (5 µM) /EcFpr (2

µM), NADPH (200 µM) reducing system anaerobically in dark. For HPLC, LC-MS analysis, the enzymatic reaction was heat quenched at 90 °C for two minutes and then centrifuged and filtered through a 10kDa cut-off filter.

148

5.4.4. Alkaline phosphate treatment of ThiC reaction

Alkaline phosphatase 1µL (1000 units) was added to 100 µl to 200 µl of ThiC enzymatic reaction. The mixture was incubated at 37°C for 30 minutes and then quenched through 10kDa cut-off filter before it was analyzed by HPLC or LC-MS.

5.4.5. HPLC parameters for the analysis of the ThiC reaction mixture

HPLC analysis was performed on a ZORBAX Eclipse XDB-C18 column (15cm

4.6mm, 5µm particles, Agilent Technologies, flow rate1 mL/min). Typical injection volumes were in the range of 10–80µL. Data were processed using ChemStation ver.

B.04.01 SP1 (Agilent Technologies).

HPLC conditions: (Flow rate 1mL/min)

A. Water.

B. 100 mM potassium

acetate, pH 6.6. C. Methanol.

phosphate

buffer, pH

6.6/10 mM ammonium

HPLC method:

0min—100% B, 5min—100% B, 14min—7% A 70% B 23% C, 25min—25% A

0% B 75% C, 28min—25% A 0% B 75% C, 32min—100% B, 36min—100% B.

5.4.6. LC-MS parameters for the analysis of the ThiC reaction mixture

LC-ESI-TOF-MS on small molecules was performed using an Agilent 1260

HPLC system equipped with a binary pump and a 1200 series diode array detector

149

followed by a MicroToF-Q II mass spectrometer (Bruker Daltonics) using an ESI source in positive mode. The analysis was performed on a ZORBAX Eclipse XDB-C18 column

(15 cm x 4.6 mm, 5 µm particles, Agilent).

LC conditions: A- 5 mM Ammonium acetate buffer, pH 6.6, B- 75 % Methanol and 25 % Water.

LC method: 0 min – 100% A, 2 min – 100% A, 4 min – 80% A 20% B, 27 min –

100% B, 29 min – 100% B, 30 min – 100% A, 40 min – 100% A.

150 6. SUMMARY AND OUTLOOK

6.1. Summary

Here, we have explored unusual reactions catalyzed by radical SAM enzymes. In cystobactamid biosynthesis, CysS utilizes B12, SAM and [4Fe-4S] to perform sequential methylations on an sp3-hybridized carbon. In anaerobic heme degradation pathway,

ChuW not only methylates the porphyrin ring but also cleaves the ring to release the iron. In thiamin biosynthesis pathway, ThiC catalyzes the most complicated rearrangement from AIR to HMP-P. In the first two cases, classic probes (radical trap, isotope labeling compounds) were designed to trap the unstable intermediates to study the detailed mechanism or conformations in the enzyme active site. For the last case, because of the narrow substrates tolerance of the ThiC and the lack of chromophore in the substrate, it’s harder to use substrate analogs to study the mechanism. Here, we are able to trap a four-carbon fragmentation from AIR by methionine using unbiased LC-

MS/MS analysis and isotope labeling approaches. These give us a better insight into how nature uses complex protein architecture and inherent chemistry property of substrates to perform novel reactions.

6.2. Outlook

6.2.1. B12 dependent radical SAM methyltransferase-CysS

6.2.1.1. Crystallography

Currently, there is no crystal structure that is published for the B12 dependent radical SAM methyltransferase. However, there is a crystal structure for a B12-dependent

151 radical SAM enzyme, OxsB, which performs a ring contraction reaction to generate oxetanocin A. Although no substrate or product is bound to the crystal, cobalamin,

SAM, [4Fe-4S] cluster are found in the crystal, which gives us insight into how a B12- dependent radical SAM transferase could position the SAM and Cbl to perform both racial reaction and methyl transfer. Bridwell-Rabb was able to obtain two conformations of SAM binding to the unique Fe of the [4Fe-4S] cluster.22 In one of the conformation, the S atom of SAM is 3.0 Å to the unique Fe, which is in the range (3.4 Å) that is permitted for reductive cleavage of SAM while the methyl group of the SAM is 8.8 Å from Co of Cbl. In another conformation, the S atom of SAM is 6.5 Å to the unique Fe while the methyl group of the SAM is 5.8 Å from Co of Cbl, which favors the methyl transfer reaction to the Cbl. In this case, it seems SAM can both initiate the radical chemistry and perform the methyl transfer in the same active site. Nevertheless, OxsB is not a methyltransferase. It’s not clear why SAM adopt two conformations to perform the ring contraction and why Cbl is needed. CysS has a sequence similarity around 16% with OxsB and mostly align in the Cbl-binding domain and SAM radical domain.

Compared to the OxsB, CysS lacks the N-terminal domain of OxsB and has great variation in the C-terminal helix bundle of OxsB, which is proposed for the conformational change upon the binding of the substrate. We have made several constructs and substrate analogs for the crystallography of CysS and the study is still in progress. It will be interesting to see whether CysS adopts similar conformations for

SAM to achieve the unusual methylation with Cbl.

152 6.2.1.2. The evolution of radical SAM enzymes

Currently, the radical SAM superfamily contains 113,000 unique sequences that represent more than 65 distinct reactions across all domains of life. The catalytic activities of radical SAM enzymes are usually low compared to other highly efficient enzymes. SAM is not always used as a cofactor instead is consumed as a substrate for many reactions catalyzed by radical SAM enzymes. Both [4Fe-4S] cluster and SAM are not complicated cofactors in the living organism. Therefore, the radical SAM enzymes are considered as ancient enzymes instead of well evolved modern enzymes, especially for their capability to mediate radical reactions under anaerobic condition.

Adenosylcobalamin (AdoCbl)-dependent enzymes also catalyze unusual rearrangement or elimination reactions through radical chemistry. The catalytic cycle starts with the homolytic cleavage of the Co–C bond to generated the 5′-dA radical, which abstracts a hydrogen atom from a substrate. The substrate radical can undergo rearrangement and subsequent hydrogen exchange to yield the final product.90 Most of the radical SAM enzymes also use 5′-dA radicals to initiate the reaction. However, The

C–Co bond of AdoCbl is around 30 kcal/mol while the 5′-C-S bond of SAM is around

60 kcal/mol. Also, in order to cleave SAM, there must be a one-electron reduction step.

Considered SAM is not as a chemically elegant molecule as adenosylcobalamin, SAM is regarded as “a poor man’s adenosylcobalamin.91 However, in terms of the number of the enzymes and reaction diversity, the radical SAM enzymes far exceed of the AdoCbl- dependent enzymes. There are only 12 groups of AdoCbl-dependent enzymes have so far been identified, which include skeleton mutase, eliminase and aminomutase.

153

Furthermore, it takes nearly 30 enzymatic steps to biosynthesize cobalamin while SAM only requires one step by methionine adenosyltransferase using methionine and ATP.

From these aspects, it’s possible that AdoCbl-dependent enzymes evolve from radical

SAM enzymes.

Cobalamin-dependent radical SAM enzymes are a large class of enzymes that use both radical SAM machinery and cobalamin to catalyze unusual reactions, such as inactive carbon methylation, phosphorous atoms methylation, ring contraction. From the crystal structure of OxsB, we know that SAM bound [4Fe-4S] cluster and Cbl are just 4

Å apart, which indicates that it’s quite conceivable that same substrate can bind to a single position and react with two cofactors in a single active site. This active site combines both SAM radical core unit and Cbl-binding Rossmann domain. It’s proposed that OxsB can be considered as a radical SAM enzyme that has been adapted to bind

Cbl.92 It’s likely that cobalamin-dependent radical SAM enzyme is an ancient ancestry of the AdoCbl-dependent enzyme.

From the mechanistic studies of CysS, we learn that the enzyme prefers Cbl(II) to start with and 5′-dA radical can be generated by reductive cleavage of radical SAM enzyme. With a close vicinity of Cbl(II) and a 5′-dA radical, it’s interesting to speculate the possibility of generating the AdoCbl, which could be a random event for the first appearance of the sophisticated cofactor AdoCbl.

154

6.2.2. ChuW in anaerobic heme degradation pathway

ChuW has high sequence similarity with HemN. HemN is a radical SAM enzyme that catalyzes the oxidative decarboxylation of the propionate side chains of rings A and

B of coproporphyrinogen III to form protoporphyrinogen IX. It is shown that EPR can be used to detect a comparatively stable delocalized substrate radical intermediate.93

Here for ChuW, we are able to use a cyclopropyl heme analog to trap a radical intermediate on the porphyrin ring by forming stable porphyrin compound and SAM adducts. It’s conceivable that we can directly use EPR to detect the possible radical intermediate in the ChuW catalyzed cyclopropyl heme analog reaction.

ChuW is assigned as the class C radical SAM enzyme. NosN, another enzyme in this family, catalyzes both carbon transfer from SAM and formation of the ester linkage between Glu6 and the appended C4 carbon unit of 3,4-dimethylindolic acid (DMIA) motif in nosiheptide biosynthesis.38 It is shown that NosN can cleave an allyl analog of

SAM (allyl-SAM) efficiently and the resulting 5′-dA radical can be captured by the olefin moieties of allyl-SAM or 5′-allylthioadenosine (ATA).94 It’s possible that ChuW can also take allyl-SAM as a substrate and the resulting product may provide insight of whether 5′-dA radical abstracts a hydrogen atom from SAM or methylthioadenosine, and the orientation of two SAM molecules in the active site. It will also be useful to get a crystal structure because currently there is no crystal structure available for this kind of enzymes.

155

6.2.3. HMP-P synthase ThiC in thiamin pyrimidine biosynthesis

6.2.3.1. Formation of the four-carbon fragment with methionine

From the structure characterization of the four-carbon fragment, we know that all the oxygen is lost in this shunt product. It will be useful to know whether the reducing agent- dithionite used for the ThiC reaction is strong enough to reduce the leaking intermediate to form the four-carbon fragment or the two oxygen indeed is lost during the enzymatic reaction. If it’s the latter case, the oxygen of the by-product carbon monoxide in the ThiC catalyzed reaction should come from water instead of oxygen from the AIR. We can try to obtain the isotope pattern of oxygen of carbon monoxide in

ThiC reaction under D2O condition by GC-MS.

6.2.3.2. The function of the mononuclear metal center in ThiC

Compare to other classic radical SAM enzymes, ThiC not only has a different binding mode for SAM but also have an extra Fe/Zn binding site. It will be interesting to know whether Fe is the actual metal that function in ThiC and whether it has a redox role in catalyzing the reaction. Also, the ThiC crystal structure has shown the amino and carboxylate group of methionine are coordinated with the metal. The metal may also play a role in trapping the four-carbon fragment with methionine. Mutants study of the ligands (H417A, H481A) on the effect of the formation of the four-carbon fragment with methionine will give some hints about how the four-carbon fragment forms.

6.2.3.3. ThiC mutants’ studies

Besides H417A and H481A in Caulobacter crescentus ThiC (CcThiC), other mutants (C474S, Y277F, M248L in CcThiC, D383N in AtThiC) show the 5′-dA radical

156

abstracts a hydrogen atom from AIR, which suggests the radical chemistry is initiated.

Potential intermediate can be trapped by radical probes for these mutants.

6.2.3.4. Substrate analogs studies

In the proposed mechanism for ThiC catalyzed rearrangement, there is a dehydration reaction induced by the radical, which is similar to the case in ribonucleotide reductase (RNR) for the step 5-14 to 5-16 in Figure 5.2. There have been extensive substrate analogs studies for RNR. Similar strategy can also be applied to study the mechanism of ThiC.

157

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