View of Investigations…………………………………………………...26

UNRAVELING GENETICALLY ENCODED PATHWAYS LEADING TO BIOACTIVE METABOLITES IN GROUP V CYANOBACTERIA

by BRITTNEY MICHALLE BUNN

Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy

Dissertation Advisor: Dr. Rajesh Viswanathan

Department of Chemistry CASE WESTERN RESERVE UNIVERSITY January, 2016

CASE WESTERN RESERVE UNIVERSITY

SCHOOL OF GRADUATE STUDIES

We hereby approve the dissertation of

BRITTNEY MICHALLE BUNN

candidate for the degree of Doctor of Philosophy*.

Committee Chair

Robert Salomon, PhD

Committee Member

Anthony Pearson, PhD

Committee Member

Michael Zagorski, PhD

Committee Member

John Mieyal, PhD

Date of Defense

August 31, 2015

*We also certify that written approval has been obtained

for any proprietary material contained therein.

This thesis is dedicated to my parents, Glenn and Michalle Bunn. I am forever grateful for your boundless love and unwavering support and encouragement.

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Table of contents

Chapter 1: General Introduction…………………………………………………………..1

1.1 Introduction to Cyanobacteria…………………………………………………2

1.2 Cyanobacteria as a Source of Bioactive Natural Products……………………...3

1.3 Cyanobacterial Natural Product Biosyntheses…………………………….…...6

1.4 Group V Cyanobacteria’s Hapalindole-type Alkaloid Family of Natural

Products…………………………………………………………………..10

1.5 Hapalindole-type Alkaloid Biosynthesis……………………………….…….19

1.6 Synthetic Biology Approach to Bioactive Natural Products……………….…23

1.6.1 Introduction to Synthetic Biology as a Tool for Biosynthetic

Investigations…………………………………………………….23

1.6.2 Introduction to Synthetic Biology as a “Green” Synthesis

Methodology Toward Novel Natural Product Analogs…………..25

1.7 Overview of Investigations…………………………………………………...26

1.7.1 Heterologous Expression and in vitro Reconstitution of Isonitrile

Synthase (WelI1) and Fe(II)-α-Ketoglutarate Dependent Oxygenase

(WelI3)…………………………………………………………...26

1.7.2 Substrate Tolerance of Isonitrile Synthase and Fe(II)-α-Ketoglutarate

Dependent Dioxygenase affords Unnatural Variants of

Cyanobacterial Hapalindole Pathway Intermediate………………27

1.8 References…………………………………………………………………....28

Chapter 2: Heterologous Expression and in vitro Reconstitution of Isonitrile Synthase

(WelI1) and Fe(II)-α-Ketoglutarate Dependent Oxygenase (WelI3)………………….…41

2.1 Introduction……………………………………….………………………….42

2.2 Results and Discussion………………………….……………………………44

2.2.1 Identification of the Putative Hapalindole-type Alkaloid Biosynthetic

Gene Cluster……………………………………………………...44

2.2.2 Bioinformatic Analysis of WelI1/3 & Homology to IsnA/B and

PvcA/B…………………………………………………………...45

2.2.3 In vitro Reconstitution and Unambiguous Characterization of WelI1

and WelI3………………………………………………………...52

2.3 Conclusion……………………………………….…………………………...58

2.4 Experimental Section………………………………………………………...61

2.4.1 Cyanobacterial Culturing…………………………………………..61

2.4.2 Genomic DNA Extraction………………………………………….61

2.4.3 Whole Genome Sequencing and Bioinformatics………….……….62

2.4.3.1 Homology Based Model Generation……………………..63

2.4.3.2 Nucleotide Accession Numbers…………………………..63

2.4.4 Gene Cloning for Heterologous Expression……………………...... 63

2.4.5 Heterologous Expression of WelI1 and WelI3……………………..63

2.4.6 Enzymatic Assay with Cell Lysates Containing WelI1 and WelI3 for

GC-MS and LC-MS……………………………………………...64

2.4.7 GC-MS Analysis…………………………………………….…..…65

2.4.8 LC-MS Analysis……………………………………………………65

2.4.9 Enzymatic Assay with Cell Lysates Containing WelI1 and WelI3 for

HPLC…………………………………………………………….66

2.4.10 HPLC Analyses……………..……………………….…….……...66

2.4.11 Synthesis and Spectroscopic Analysis of Indole-isonitrile…….…67

2.4.11.1 Synthesis of Cis and Trans Isomers of Indole-

isonitrile………………………………………….68

2.4.11.1.1 Synthesis of 3-Indolecarbaldehyde (Precursor for

Indole-isonitrile Synthesis………………………..68

2.4.11.1.2 Synthesis of Indole-isonitrile (3-(2-

Isocyanovinyl)indole)……………………………68

2.4.11.2 Spectroscopic Analysis of Indole-isonitrile…………….69

2.4.11.2.1 Cis Indole-isonitrile 1H and 13C NMR Data...... 68

2.4.11.2.2 Trans Indole-isonitrile 1H and 13C NMR Data...69

2.5 References…………….……………………………………………………...70

Chapter 3: Substrate Tolerance of Isonitrile Synthase and Fe(II)-α-Ketoglutarate

Dependent Dioxygenase affords Unnatural Variants of Cyanobacterial Hapalindole

Pathway Intermediate…………………………………………………………………….73

3.1 Introduction…………………………….…………………………………….74

3.1.1 Assessing the Substrate Promiscuity of WelI1 and WelI3………….74

3.1.2 Tri-Catalytic Biosynthetic Methodology to Access Novel Natural

Product Analogs………………………………………………….77

3.2 Results and Discussion………………………………………………………78

3.2.1 Heterologous Expression and Purification of WelI1 and WelI3……78

3.2.2 Heterologous Expression, Purification, and Enzymatic Assay of

TmTrpB1…………………………………………………………79

3.2.3 Assessment of Substrate Promiscuity of WelI1 and WelI3………...81

3.2.4 Homology-based Model Building and in silico Docking……….…85

3.3 Conclusion……………………………………………………………...... ….90

3.4 Experimental Section………………………………………………………...91

3.4.1 WelI1 and WelI3…………………………………………………...91

3.4.1.1 Vector Selection…………………………………….……91

3.4.1.2 Transformation…………………………………….……..91

3.4.1.3 Culturing and Induction……………………………….….91

3.4.1.4 Lysis………………………………………………….…..92

3.4.1.5 Purification……………………………………………….92

3.4.2 Enzymatic Assay with Purified WelI1 and WelI3……………….….93

3.4.2.1 General Procedure………………………………….…….93

VII

3.4.2.2 HPLC Analysis of Enzymatic Assay Extracts……………94

3.4.2.3 LC-HRMSMS Analysis………………………………….95

3.4.3 TmTrpB1…………………………………………………….……..96

3.4.3.1 Culturing and Induction………………………………….96

3.4.3.2 Lysis……………………………………….……………..96

3.4.3.3 Purification…………………………………….…………97

3.4.3.4 Enzymatic Assay with Purified TmTrpB1……….……….97

3.5 References………………………………………………….………………...99

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Chapter 4: Thesis Summary and Future Directions……………………………………..102

4.1 Thesis Summary……………………………………………………….……102

4.1.1 Part I: Heterologous Expression and in vitro Reconstitution of

Isonitrile Synthase (WelI1) and Fe(II)-α-Ketoglutarate Dependent

Oxygenase (WelI3)………………………………...…………...102

4.1.2 Part II: Substrate Tolerance of Isonitrile Synthase and Fe(II)-α-

Ketoglutarate Dependent Dioxygenase Affords Unnatural Variants

of Cyanobacterial Hapalindole Pathway Intermediate……….…104

4.2 Future Directions………………………………………….………………...106

4.3 References……………………….………………………………………….108

Appendix 1 Bioinformatic Analysis of WelI1 and WelI3……………………………….111

Appendix 1.1 Illustration of Gene Clusters…….……….……………………...111

Appendix 1.2 Translation of welI1 and welI3 using ExPaSy’s Translate Tool.....112

Appendix 1.2.1 Translation of welI1……………………………….…...112

Appendix 1.2.2 Translation of welI3…………………………….……...112

Appendix 1.3 BLASTP Results for WelI1 and WelI3……….………………....113

Appendix 2 Cis and Trans Indole-Isonitrile Synthetic Standard Characterization……..115

Appendix 2.1 NMR Spectra……………………………………………………115

Appendix 2.2 HRMS Spectra………………………………………….………..117

Appendix 3 Analysis of WelI1/3 Enzymatic Assays with E. coli Cell Lysates….…...…118

Appendix 3.1 Analysis of WelI1/3 Enzymatic Assay by LC-MS……………....118

Appendix 4 GC-MS Spectra of Synthesized Cis and Trans Indole-Isonitrile Standards119

Appendix 5 NMR Spectra of 2-Methyl-L-Tryptophan………………..………………...123

Appendix 6 Analysis of WelI1/3 Enzymatic Assay Results with L-Tryptophan and

Tryptophan Analogs……………..……………………………………………...125

Appendix 6.1 HPLC Analysis…………………………………………………..125

Appendix 6.2 LC-MS and MSMS Analysis of WelI1/3 Enzymatic Assay Results

with L-Tryptophan and Tryptophan Analogs…………………………...126

Appendix 6.2.1 Assay with L-Tryptophan…………………………..…….……126

Appendix 6.2.2 Assay with 2-Methyl-L-Tryptophan…………………..…………….…128

Appendix 6.2.3 Assay with 1-Methyl-L-Tryptophan……………………..…….………130

Appendix 6.2.4 Assay with 4-Fluoro-DL-Tryptophan……………………..…………...132

Appendix 6.2.5 Assay with 5-Methyl-DL-Tryptophan……...………………………….134

Appendix 6.2.6 Assay with 6-Methyl-DL-Tryptophan……………………….…..…….136

Appendix 6.2.7 Assay with 5-Methoxy-L-Tryptophan……………………...………….138

Appendix 6.2.8 Assay with 5-Hydroxy-L-Tryptophan………………………….……...140

Appendix 7 WelI3 Homology Modeling, Docking and Multiple Alignments…………..142

Appendix 7.1 Method for Generation of Homology Model for WelI3………………....142

Appendix 8 References to Appendices………………………………………………….143

Bibliography……………………………………………………………………………144

List of Tables

Table 1.1 Hapalindole analogs that have been isolated from group V cyanobacteria…...11

Table 1.2 Fischerindole analogs isolated from group V cyanobacteria………………….14

Table 1.3 Ambiguine analogs isolated from group V cyanobacteria……………………15

Table 1.4 Welwitindolinone analogs isolated from group V cyanobacteria…………….17

Table 3.1 List of LC-MS retention times, molecular formulas, percent yields, and MS2

fragmentation patterns for products (3.3a-h)……………………………………..84

Table 3.2 Total volume, protein volume, and protein concentration data for assays with purified WelI1 and WelI3………………………………………………………..93

Table A1.1 BLASTP results for WelI1………………………………………………...113 Table A1.2 BLASTP results for WelI3………………………………………………...114

List of Figures

Figure 1.1 Chemical structures of well-known cyanobacterial toxic natural products…...4

Figure 1.2 Chemical structures of cyanobacterial natural products with therapeutic

potential due to their interesting bioactivities. Hapalosin (1.4) has shown anti-

MDR properties and dolastatin 10 (1.5) has shown to be cytotoxic………..……...6

Figure 1.3 Graphical representation of available cyanobacterial genomes……………….9

Figure 1.4 Structures of the first isolated hapalindole-type alkaloids, hapalindole A (1.6)

and hapalindole B (1.7) isolated from Hapalosiphon fontinalis………………….10

Figure 1.5 The common tetracyclic core of the hapalindoles includes an indole ring, an

isonitrile or isothiocyanate moiety, and a cyclized isoprenoid unit……………….12

Figure 1.6 A) Representative structures of the fischerindole sub-family within the

hapalindole-type natural products. B) General structures for the tetracyclic

hapalindoles and fischerindoles highlighting the difference in ring

fusion………………………………………………………………………….….13

Figure 1.7 Structures of a few members of the ambiguine sub-family of natural products;

unique structural characteristics are highlighted, including the presence of a tert-

prenyl unit, a cyclized tert-prenyl unit, and a nitrile moiety………………………15

Figure 1.8 Structures of some of the structurally intriguing and biologically active s

welwitindolinones………………………………………………………………..16

Figure 1.9 Structures of xanthocillin, diisocyanoadociane, and hazimicin. Proposed

sources of the isonitrile nitrogen and carbon are highlighted………………....…..19

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Figure 1.10 Summary of biosynthetic investigations and hypotheses towards hapalindole

biosynthesis. L-tryptophan and L-glycine are putative substrates for formation of

indole-isonitrile, which is proposed to combine with geranyl diphosphate (pathway

A) or a derivative thereof, (Z)-3,7-dimethyl-1,3,6-octatriene through a chloronium-

ion induced condensation (pathway B)………………………………………...21

Figure 1.11 Relationship of the hapalindole-type alkaloids; the family is thought to share

a common set of genes that biosynthesize the hapalindole structural scaffold which

is then modified in higher order pathways, leading to the more complex

hapalindole-type alkaloids……………………………………………………….22

Figure 2.1 Synthetic biology approach utilized to unambiguously characterize

biocatalytic activity of enzymes responsible for hapalindole-type alkaloid

biosynthesis………………………………………………………………………43

Figure 2.2 Illustration of the wel gene cluster from Westiella intricata UH strain HT-29-

1. This investigation focuses on the isonitrile synthase genes welI1 and welI3…...45

Figure 2.3 A) Clustal Omega alignment of WelI1, IsnA, and PvcA. Clustal Omega key:

*(asterisk): fully conserved residue, : (colon): groups with highly similar properties,

. (period) groups with weak similarity. Conserved domains within the three

sequences are highlighted. B) Homology based 3-D model of WelI1 with the

potential active site highlighted…………………………………………………..48

Figure 2.4 A) Clustal Omega alignment of WelI3, IsnB, and PvcB. Clustal Omega key:

*(asterisk): fully conserved residue, : (colon): groups with highly similar properties,

. (period) groups with weak similarity. The amino acids comprising the metal

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binding motif common to α-ketoglutarate dependent oxygenases are indicated. B)

Homology based 3-D model of WelI3. The Fe (II)-binding motif is

highlighted……………………………………………………………………….50

Figure 2.5 Biosynthetic hypothesis towards cis indole-isonitrile (2.3b) utilizing

biocatalysts WelI1 and WelI3, substrates L-tryptophan (2.1) and ribose-5-

phosphate (2.7), and cofactor/cosubstrate ammonium iron (II) sulfate and α-

ketoglutarate (2.8)………………………………………………………………..51

Figure 2.6 Vector map for plasmids hosting welI1 and welI3; the plasmids contain

kanamycin resistance and are under control of the T7 promoter………………….52

Figure 2.7 SDS-PAGE analysis of E. coli BL21(DE3) cell lysates hosting WelI1 and

WelI3…………………………………………………………………………….53

Figure 2.8 A) GC of assay extract indicating the production of both the cis (2.3b) and

trans (2.3a) isomers of the indole-isonitrile. B) MS of the peak at Rt 6.42 min from

the GC; notice the parent peak with a mass of 168.06. C) MS of the peak at Rt

6.70 min from the GC; notice the parent peak with a mass of 168.06. ……………55

Figure 2.9 HPLC was analyzed at 310 nm with a UV detector. X-axis – retention time in

minutes (min). Y-axis - intensity in arbitrary units. Presented as a stacked Y-plot

and is drawn to relative intensity units. Peaks show only relative intensities and are

not normalized for concentration of metabolites. 1) Synthesized cis indole-isonitrile

only (2.3b) (Rt = 8.8 min). 2) Synthesized trans (2.3a) indole-isonitrile only (Rt =

13.1 min). 3) Co-injection of synthetic standards of cis (2.3b) and trans indole-

isonitrile (2.3a). 4) Control for enzyme assay where cell lysates of E. coli

BL21(DE3) were subjected to assay conditions without WelI1 and WelI3. 5) WelI1

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and WelI3 enzyme assay after 16 h incubation at 25°C. 6) Control sample (4) spiked

with cis indole-isonitrile (2.3b) after 3 h incubation. 7) Control sample (4) spiked

with cis indole-isonitrile (2.3b) after 16 h incubation. 8) Co-injection of cis (2.3b)

and trans (2.3a) indole-isonitrile with enzyme assay mixture……………………57

Figure 2.10 Depiction of various C10-C11 stereochemistries among the hapalindole-type

alkaloids………………………………………………………………………….60

Figure 3.1 A) Existing synthetic methodology for production of hapalindole biosynthesis

intermediate, indole isonitrile (3.3); B) This work employs sequential biocatalysis

of TmTrpB1 and WelI1/3 in order to produce indole-isonitrile (cis and trans-3.3),

which could be carried through the reminder of the biosynthetic pathway to produce

novel hapalindole-type alkaloids…………………………………………………76

Figure 3.2 Reaction catalyzed by TmTrpB1…………………………………………….77

Figure 3.3 A) SDS-PAGE analysis of fractions from the Ni-NTA purification of 6x-His

tagged WelI1. B) SDS-PAGE analysis of fractions from the Ni-NTA purification

of 6x-His tagged WelI3. …………………….……………………………………78

Figure 3.4 SDS-PAGE analysis of purified WelI1 and WelI3…………………………...79

Figure 3.5 SDS-PAGE analysis of purified TmTrpB1…………………………………..80

Figure 3.6 Sequential biocatalysis involving three enzymatic steps utilized in this

Investigation……………………………………………………………………...81

Figure 3.7 List of substrate analogs (3.8a-h) incorporated into the Wel-I1-3 catalysis,

resulting in isonitriles 3.3 a-h. HRMS results and cis:trans analysis are also

displayed…………………………………………………………………………82

Figure 3.8 Pairwise Clustal alignment between biochemically characterized PaPvcB

and WelI3………………………………………………………………………86 Figure 3.9 A) Image of WelI3 docked with L-tryptophan isonitrile (3.10). WelI3’s active

site components are shown as sticks, the docked substrate is shown in magenta, and

the Fe(II) is shown in orange. PaPvcB’s hydrophobic pocket residues (Met114-

Tyr115-Leu-116) are shown for comparison to WelI3’s smaller triad Ala114-

Phe115-Ala116. B) Side view of WelI3 docked with L-tryptophan isonitrile (3.10).

C) Top view of WelI3 docked with L-tryptophan isonitrile (3.10)……………….87

Figure 4.1 Biosynthetic route to the first stable intermediate en route to the hapalindole-

type alkaloids, indole isonitrile (4.5), catalyzed by WelI1 and WelI3 from the wel

cluster of W. intricata (HT-29-1). WelI1 utilizes L-tryptophan (4.1) and ribose-5-

phosphate (4.2) to yield an unstable intermediate (4.3). This intermediate (4.3) is

then oxidatively decarboxylated by WelI3, an oxygenase that requires Fe(II) and α-

ketoglutarate…………………………………………………………………….102

Figure 4.2 This work employs sequential biocatalysis of TmTrpB1, WelI1, and WelI3 in

order to generate analogs of indole isonitrile. All three biocatalysts were

heterologously expressed utilizing T7 inducible plasmids and purified to

homogeneity for enzymatic assays. TmTrpB1 catalyzes the formation of

tryptophan analogs (4.1), which can be utilized by WelI1 to generate an isonitrile-

containing intermediate (4.3) that is oxidatively decarboxylated by WelI3 to yield

analogs of indole isonitrile (4.3)………………………………………………...105

Figure A1.1 Illustration of the hapalindole (hpi), ambiguine (amb) and welwitindolinone

(wel) biosynthetic gene clusters. A) hpi gene cluster from Fischerella sp. ATCC

43239.1 B) hpi gene cluster from Fischerella sp. PCC 9339 (JGI IMG/ER:

2516653082). C) amb gene cluster from Fischerella ambigua UTEX 1903.2 D)

XVI

amb gene cluster from Fischerella ambigua UTEX 1903.1 E) wel gene cluster from

Hapalosiphon welwitschii UTEX B1830.3 F) wel gene cluster from Hapalosiphon

welwitschii UH strain IC-52-3.1 G) wel gene cluster from Westiella intricata UH

strain HT-29-1.1 H) wel gene cluster from Fischerella sp. PCC 9431 (JGI IMG/ER:

2512875027). I) wel gene cluster from Fischerella muscicola SAG 1427-1 (JGI

IMG/ER: 2548876995)…………………………………………………………111

Figure A2.1 1H NMR for cis indole-isonitrile………………………………………….115

Figure A2.2 13C NMR for cis indole-isonitrile…………………………………………115

Figure A2.3 1H NMR for trans indole-isonitrile………………………………………..116

Figure A2.4 13C NMR for trans indole-isonitrile……………………………………….116

Figure A2.5 HRMS for a sample containing a mixture of cis and trans isomers……….117

Figure A3.1 LC-MS of WelI1/3 enzymatic assay with cell lysates…………………..118

Figure A4.1 GC trace for trans indole-isonitrile…………………………………….…119

Figure A4.2 MS spectrum of peak with Rt of 7.91 in Figure A4.1……………………..120

Figure A4.3 GC trace for cis indole-isonitrile………………...………………………..121

Figure A4.4 MS spectrum of peak with Rt of 7.51 in Figure A4.3……………………...122

Figure A5.1 1H NMR spectrum of 2-methyl-L-tryptophan…………………………….123

Figure A5.2 13C NMR spectrum of 2-methyl-L-tryptophan…………………………...124

Figure A6.1 HPLC traces for all 8 enzymatic assay extracts……….………………….125

Figure A6.2 A) Analysis of ethyl acetate extract of WelI1-I3 enzymatic assay with L-

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tryptophan. Biosynthetic reaction involving WelI1-I3 leading to cis indole

isonitrile. B) EIC for reaction mixture and HRMS data for peak at 3.83 min. C)

MS/MS data for molecular ion identified in B………………….……………….126

Figure A6.3 MS/MS data for molecular ion identified in Figure A6. 2B with fragment ion

annotation for fingerprinting identity of cis indole isonitrile……………………127

Figure A6.4 A) Analysis of ethyl acetate extract of WelI1-I3 enzymatic assay with 2-

methyl-L-tryptophan. Biosynthetic reaction involving WelI1-I3 leading to 2-

methyl indole isonitrile. B) EIC for reaction mixture and HRMS data for peak at

5.17min. C. MS/MS data for molecular ion identified in B……………………..128

Figure A6.5 MS/MS data for molecular ion identified in Figure A6. 4B with fragment ion

annotation for fingerprinting identity of 2-methyl indole

isonitrile………………………………………………………………………...129

Figure A6.6 A) Analysis of ethyl acetate extract of WelI1-I3 enzymatic assay with 1-

methyl-L-tryptophan. Biosynthetic reaction involving WelI1-I3 leading to 1-

methyl indole isonitrile. B) EIC for reaction mixture and HRMS data for peak at

4.00 min. C) MS/MS data for molecular ion identified in B…………………….130

Figure A6.7 MS/MS data for molecular ion identified in Figure A6.6 B with fragment ion

annotation for fingerprinting identity of 1-methyl indole isonitrile……………..131

Figure A6.8 A) Analysis of ethyl acetate extract of WelI1-I3 enzymatic assay with 4-

fluoro-DL-tryptophan. Biosynthetic reaction involving WelI1-I3 leading to 4-

fluoro indole isonitrile. B) EIC for reaction mixture and HRMS data for peak at

5.23 min. C) MS/MS data for molecular ion identified in B…………………..132

Figure A6.9 MS/MS data for molecular ion identified in Figure A6. 8B with fragment ion

XVIII

annotation for fingerprinting identity of 4-fluoro indole isonitrile………………133

Figure A6.10 A) Analysis of ethyl acetate extract of WelI1-I3 enzymatic assay with 5-

methyl-DL-tryptophan. Biosynthetic reaction involving WelI1-I3 leading to 5-

methyl indole isonitrile. B) EIC for reaction mixture and HRMS data for peak at

4.45 min. C) MS/MS data for molecular ion identified in B…………………….134

Figure A6.11 MS/MS data for molecular ion identified in Figure A6. 10B with fragment

ion annotation for fingerprinting identity of 5-methyl indole isonitrile...... 135

Figure A6.12 A) Analysis of ethyl acetate extract of WelI1-I3 enzymatic assay with 6-

methyl-DL-tryptophan. Biosynthetic reaction involving WelI1-I3 leading to 6-

methyl indole isonitrile. B) EIC for reaction mixture and HRMS data for peak at

4.40 min. C) MS/MS data for molecular ion identified in B…………………..136

Figure A6.13 MS/MS data for molecular ion identified in Figure A6.12 with fragment ion

annotation for fingerprinting identity of 6-methyl indole isonitrile……………..137

Figure A6.14 A) Analysis of ethyl acetate extract of WelI1-I3 enzymatic assay with 5-

methoxy-L-tryptophan. Biosynthetic reaction involving WelI1-I3 leading to 5-

methoxy indole isonitrile. B) EIC for reaction mixture and HRMS data for peak at

3.95 min. C) MS/MS data for molecular ion identified in B…………………..138

Figure A6.15 MS/MS data for molecular ion identified in Figure A6. 14B with fragment

ion annotation for fingerprinting identity of 5-methoxy indole isonitrile………..139

Figure A6.16 A) Analysis of ethyl acetate extract of WelI1-I3 enzymatic assay with 5-

hydroxy-L-tryptophan. Biosynthetic reaction involving WelI1-I3 leading to 5-

hydroxy indole isonitrile. B) EIC for reaction mixture and HRMS data for peak at

4.31 min. C) MS/MS data for molecular ion identified in B…………………..140

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Figure A6.17 MS/MS data for molecular ion identified in Figure A6. 16B with fragment

ion annotation for fingerprinting identity of 5-hydroxy indole isonitrile………..141

List of Schemes

Scheme 2.1 A) IsnA catalyzes the reaction between L-tryptophan (2.1) and ribose-5-

phosphate, which yields an unstable intermediate (2.2). The intermediate is

oxidatively decarboxylated by Isn B, which requires Fe(II) and α-ketoglutarate to

yield trans indole isonitrile (2.3a). B) PvcA catalyzes the reaction between L-

tyrosine (2.4) and ribulose-5-phosphate, which yields an unstable intermediate

(2.5). The intermediate is oxidatively decarboxylated by Isn B, which requires

Fe(II) and α-ketoglutarate to yield an oxidized tyrosine-isonitrile (2.6)………….46

Scheme 3.1 A) Possible mechanism of WelI3 based on its analogy to PaPvcB. B) PaPvcB

mechanism……………………………………………………………………….89

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List of Abbreviations

Å Angstrom ATCC American Type Culture Collection aq Aqueous amb Ambiguine BGI Beijing Genome Institute BLAST Basic Local Alignment Search Tool BLASTP Basic Local Alignment Search Tool Protein C Celsius δ Delta, chemical shift in NMR DMF N,N-Dimethylformamide E. coli Escherichia Coli equiv. Equivalents eDNA Environmental DNA EIC Electrospray Ion Chromatography ESI Electrospray Ionization et al Et alia, “and others” ExPASy Expert Protein Analysis System FA Fischerella ambigua UTEX 1903 FS Fischerella sp. ATCC 43239 g Gram gDNA Genomic DNA GC-MS Gas Chromatography-Mass Spectrometry h Hour He Helium HMG-CoA 3-Hydroxy-3-Methylglutaryl-Coenzyme A hpi Hapalindole HPLC High-performance Liquid Chromatography

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HW Hapalosiphon welwitschii UH Strain IC-52-3 Hz Hertz iPrOH Isopropanol IPTG Isopropyl β-D-1-Thiogalactopyranoside kan Kanamycin kb Kilobase kDa Kilodalton L Liter LB Luria Bertani LC-HRMS Liquid Chromatography-High Resolution Mass Spectrometry LC-MS Liquid Chromatography-Mass Spectrometry m Meter M Molar (M)+ Molecular Ion MDR Multi-Drug Resistance mg Milligram MHz Megahertz min Minute mL Milliliter mm Millimeter mM Millimolar MS/MS Tandem Mass Spectrometry m/z Mass-to-Charge Ratio µg Microgram µL Microliter µm Micrometer µM Micromolar µmol Micromol

XXIII ng Nanogram NCBI National Center for Biotechnology Information NCE New Chemical Entities

NHI Non-Heme Iron-dependent nm Nanometer NRP Nonribosomal Peptide NRPS Nonribosomal Peptide Synthase NTA Nitrilotriacetic Acid OD Optical Density PCR Polymerase Chain Reaction PHYRE Protein Homology/Analogy Recognition Engine pI Isoelectric Point PK Polyketide PKS Polyketide Synthase PLP Pyridoxal 5'-Phosphate ppm Parts per Million psi Pounds per Square Inch QTOF Quadrupole Time-of-Flight RBS Ribosomal Binding Site rpm Revolutions per minute

Rt Retention Time SAM S-Adenosyl-Methionine SDS-PAGE Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis S.O.C. media Super Optimal broth with Catabolite Repression SIB Swiss Institute of Bioinformatics TCEP Tris(2-CarboxyEthyl)Phosphine THF Tetrahydrofolate TLC Thin Layer Chromatography

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TM-score Template Modeling Score Tris Tris(hydroxymethyl)aminomethane UTEX University of Texas v/v Volume/Volume wel Welwitindolinone WI Westiella intricata UH strain HT-29-1

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Amino Acid Abbreviations and One Letter Codes

Ala A Alanine

Arg R Arginine

Asn N Asparagine

Asp D Aspartic acid

Cys C Cysteine

Glu E Glutamic Acid

Gln Q Glutamine

Gly G Glycine

His H Histidine

Ile I Isoleucine

Lys K Lysine

Met M Methionine

Phe F Phenylalanine

Pro P Proline

Ser S Serine

Thr T Threonine

Trp W Tryptophan

Tyr Y Tyrosine

Val V Valine

XXVI

Acknowledgements

I would first like to thank the Department of Chemistry at Case Western Reserve

University. I would like to thank them for their investment in my future and for all of the opportunities and support that they have provided.

I would like to extend a special thank you to my research advisor, Dr. Rajesh

Viswanathan. I am appreciative to have had a patient mentor that adapted to my learning style. He taught me how to be an independent thinker and scientist. I have learned much from my advisor, both scientifically and personally, and I am grateful for the understanding guidance he has provided.

I would also like to thank Kathryn Howard for all that she has taught me, both inside of the laboratory and out. I appreciate how she has helped me mature as a scientist and person, in general.

I would like to express my gratitude to my committee members, Drs. Robert

Salomon, Anthony Pearson, John Mieyal, and Michael Zagorski. I appreciate the advice they have provided throughout my graduate career and how they have challenged me to become a better scientist.

I appreciate Drs. Mary Barkley and Robert Salomon for the use of some of their laboratory equipment and space—without their support my research would not have been possible.

I would like to extend a special thank you to my fellow graduate students and to my friends, especially Katie Doud, Anton Kovalsky, Caitlin Meyer, and Julie Bochard. I appreciate their friendship, their support, and their advice.

XXVII

Lastly, but certainly not least, I would like to express my extreme gratitude to my family, especially my parents, Glenn and Michalle Bunn. Their abundant love, amazing support, encouragement, and bravery inspire me every day—and for that I am very thankful.

XXVIII

Unraveling Genetically Encoded Pathways Leading to Bioactive Metabolites in Group V Cyanobacteria

Abstract

BRITTNEY MICHALLE BUNN

The ability of cyanobacteria to inhabit almost every aquatic and terrestrial environment and their ubiquitous survival is accredited to their exceptionally diverse genetics. This in turn, has led to their successful combination of metabolic pathways, allowing the majority of cyanobacteria to encode a variety of unique secondary metabolites with powerful bioactivities.

A specific family of bioactive cyanobacterial natural products produced by Group

V cyanobacteria, the hapalindole-type alkaloids, offers great pharmaceutical potential.

Explorations on the biosynthesis of this family of isoprenoid-indole alkaloids was hindered by the lack of genomic data available until recently.

This thesis reports a synthetic biology approach utilized to characterize two enzymes involved in hapalindole-type alkaloid biosynthesis (Chapter 2). A putative biosynthetic gene cluster was identified to be involved in hapalindole-type alkaloid biosynthesis within the genome of a strain of Group V cyanobacteria. Two genes, welI1 and welI3, were hypothesized to encode for biocatalysts responsible for producing an intermediate en route to the hapalindole-type alkaloids. Bioinformatic analysis revealed homology of WelI1 and WelI3 to an isonitrile synthase and an oxidative decarboxylase, respectively. Within this investigation, the two enzymes putatively involved in isonitrile

XXIX biosynthesis were heterologously expressed in E. coli and the bacterial lysates were assayed for enzymatic activities. Reconstitution of these enzymatic steps led to the production of the first stable intermediate toward the hapalindole-type alkaloid biosynthetic pathway. Products from the assay were verified through GC-MS, LC-MS, and HPLC analyses with comparison to synthetically prepared authentic standards. The enzymatic steps catalyzed by WelI1 and WelI3 were found to produce both cis and trans isomers of indole isonitrile.

Chapter 3 describes an investigation probing the substrate promiscuity of WelI1 and WelI3. The two enzymes were reconstituted in vitro, and their substrate versatility was evaluated utilizing seven tryptophan derivatives, including one derivative biosynthesized by a third biocatalyst, the tryptophan synthase TmTrpB1. All seven tryptophan derivatives yielded corresponding cis and trans indole-isonitrile analogs. The results were characterized by HPLC for turnover and product identities were established by LC-HRMS and MS/MS. The relaxed substrate specificity of WelI3 was rationalized based on a 2.1 Å crystal structure of one of its homologs, PaPvcB.

XXX

Chapter 1: General Introduction

Portions of the discussion within this chapter are included in a manuscript under the following citation:

Brittney M. Bunn* and Rajesh Viswanathan.* “Taking Stock of Nostoc: Review of

Recent Metabolite and Biosynthetic Literature of this Cyanobacterial Genus with an

Outlook for Future Biocatalytic Discoveries.” In preparation.

*Indicates corresponding author.

This chapter introduces cyanobacteria (Section 1.1) and their natural products as a source of bioactive compounds (Section 1.2). The motivation behind studying cyanobacterial natural product biosyntheses is discussed, as well as how the techniques used to do so have changed with evolving biotechnology (Section 1.3). A specific family of cyanobacterial natural products, the hapalindole-type alkaloids, is discussed (Section

1.4) along with investigations on the biosynthesis of these compounds (Section 1.5).

Some of the synthetic biology techniques used to complete the work described within this thesis are introduced (Section 1.6) and a brief overview of the investigations described in Chapter 2 and Chapter 3 is provided (Section 1.7).

1.1 Introduction to Cyanobacteria

Cyanobacteria are one of the oldest life forms on Earth having flourished for approximately 3.5 billion years.1 Due to their evolutionary adaptation to a multitude of habitats (terrestrial, marine and freshwater niches, deserts, hot springs, rocks, etc.) cyanobacteria are considered one of the largest, most diverse, highly specialized groups of

Gram-negative, photosynthetic prokaryotes. They are important members of the environment ecologically, being the only prokaryotes capable of photosynthetically

2 converting solar energy and CO2 into organic matter. In addition, many cyanobacteria contribute to the supply of reduced nitrogen in the environment. In some cases, cyanobacteria thrive as symbionts with other organisms and supply them directly with reduced nitrogen.

Cyanobacteria are characterized into five subsections, I-V (Chlorococcales,

Pleurocapsales, Ocillatoriales, Nostocales, and Stigonematales, respectively) based on the complexity of the strain’s morphology.3 Groups I and II are unicellular cyanobacteria that divide by binary fission or budding (Group I) or multiple fission (Group II). Groups III-V are filamentous cyanobacteria that can be non-heterocystous and divide in one plane

(Group III), heterocystous and divide in one plane (Group IV), or heterocystous and divide in multiple planes (Group V).

1.2 Cyanobacteria as a Source of Bioactive Natural Products

In 2003, it was estimated that over 60% of cancer and infectious disease therapeutics were of natural origin.4 A more recent survey identified 1184 new chemical entities (NCE) spanning the period of 1981-June 2006, of which 52% have a natural product connection.5

Even in the years 2000-2010 the natural products field was still responsible for about 50% of all NCE small molecules,6 with 41 of the 62 small-molecule drugs approved from 2011-

12 using natural product structures as leads.7 With the increased capability of today’s biotechnology tools,8 including genomics-based investigations, advanced activity screening, the use of genetically modified organisms or cell lines, and improved synthetic biology methods, it is predicted that natural sources will provide even more interesting compounds with exciting bioactivities in the years to come.9

Therefore, it is well-known that natural sources are highly attractive for new bioactive compounds,10 and cyanobacteria are no exception.11 The ability of these organisms to inhabit almost every aquatic and terrestrial environment and their ubiquitous survival is accredited to their exceptionally diverse genetics. This in turn has led to their successful combination of metabolic pathways allowing the majority of cyanobacteria to encode a variety of unique secondary metabolites12 with powerful bioactivities.13 The exceptions to this observation are unicellular cyanobacteria such as the genera Prochlorococcus and

Synechococcus.14 Although cyanobacterial genomes vary greatly with respect to size

(~1.4-9.1 Mb),15 unicellular cyanobacteria typically possess genomes 5-6 Mb smaller than their filamentous or colonial counterparts. Consequently, unicellular cyanobacteria do not usually produce secondary metabolites.14

Cyanobacterial secondary metabolites are separated into two main categories: toxic compounds12c, 16 and those with biological activities that could have significant pharmacological value.12e Some well-known cyanobacterial toxins include the microcystins, saxitoxins, and cylindrospermopsins14 (Figure 1.1). The microcystins are one of the most extensively studied cyanobacterial natural product families, possibly due to their being the cyanobacterial toxins most commonly responsible for causing challenges

Figure 1.1 Chemical structures of well-known cyanobacterial toxic natural products. in producing safe drinking water. This family of hepatotoxic, cyclic, heptapeptides17 currently consists of over 90 congeners and has been isolated from over ten genera of cyanobacteria, but most predominantly from strains Anabaena sp. and Microcystis sp.18

One of the most frequently isolated congeners of this family, microcystin-LR (1.1), is displayed in Figure 1.1.

Saxitoxin (1.2), a tricyclic perhydropurine alkaloid,19 is the parent compound of a group of molecules known as paralytic shellfish toxins due to their neurotoxic bioactivities.20

The saxitoxin family contains more than 50 members, some of which are known to interact with ion channels including sodium, potassium, and calcium.21 Several cyanobacterial genera have been known to produce saxitoxin or its derivatives, including Anabaena,

Cylindrospermopsis, Aphanizomenon, Planktothrix, and Lyngbya.20

The cylindrospermopsins22 are another family of cyanotoxins. This family is quite small in number compared to the other examples discussed with only three naturally occurring variants. These hepatotoxic alkaloids combine a uracil fragment with a tricyclic guanidinium moiety,12d and are most known for their ability to cause human illness after being consumed through drinking water. Cylindrospermopsin (1.3) was isolated from several genera of freshwater cyanobacteria, such as Cylindrospermopsis raciborskii,

Aphanizomenon ovalisporum, and Aphanizomenon flosaquae.12a

As stated previously, not all cyanobacterial secondary metabolites are toxic. In fact, a variety of the unique natural products have engendered great pharmaceutical interest. For example, hapalosin (1.4) isolated from H. welwitschii has been shown to reverse P- glycoprotein-mediated multi-drug resistance (MDR);23 and dolastatin 10 (1.5) from

Symploca sp. inhibits the polymerization of microtubules leading to apoptosis24 (Figure

1.2). Other metabolites have displayed anticancer properties (calothrixin A and B from

Calothrix sp.,25 isomalyngamide from Lyngbya majuscula,26 and malyngamine 3 from

Lyngbya majuscula),27 antiviral properties (nostoflan from Nostoc flagelliforme,28 and

5 icthyopeptins from Microcystis ichthyoblabe),29 antibacterial properties (noscomin from

Nostoc commune),30 and anti-HIV properties (cyanovirin from Nostoc ellipsosporum,31 scytovirin from Scytonema varium,32 and sulfoglycoplipid from Scytonema sp.).33

Figure 1.2 Chemical structures of cyanobacterial natural products with therapeutic potential due to their interesting bioactivities. Hapalosin (1.4) has shown anti-MDR properties and dolastatin 10 (1.5) has shown to be cytotoxic. In addition to the examples provided above, the hapalindole-type alkaloid family of natural products displays a variety of promising bioactivities (antimycotic34 and insecticidal,35 for instance). The unique molecular architectures and interesting bioactivities of this family of compounds are discussed in Section 1.4.

1.3 Cyanobacterial Natural Product Biosyntheses Some cyanobacterial natural products are produced in very small quantities in nature.

In order to fully understand their bioactive potential, access to larger amounts of the natural products is desired. In addition, a large number of cyanobacterial secondary metabolites contain a beautiful combination of structural motifs that are challenging to recreate synthetically. Therefore, the need to explore the biosyntheses of these compounds exists.

Isolating and understanding the enzymes responsible for the chemically elegant transformations within these biosynthetic pathways allows for access to greater quantities of the natural product, while at the same time, providing additional catalysts to be exploited by synthetic biology methods (Section 1.7) to produce variants of natural products.

Traditionally, natural product biosyntheses were proposed based on experiments involving feeding plants or cell cultures with isotopically labeled precursors.36

Complications arise from such studies due to scrambling of the labeled precursor due to metabolic pathway crossing, reduced substrate uptake due to poor cell permeability, or cell death due to toxicity.

After the recent surge in microbial genome information the characterization of biosynthetic pathways, in general, has taken a different route.37 For example, conserved sequences among groups of homologous proteins can be attributed to particular enzymatic functions. With this information, degenerate PCR primers can be bioinformatically designed to probe the genome of an unsequenced organism (or metagenomic sample) for a gene (or gene fragment) responsible for a particular enzymatic activity.38 This methodology was used to locate a gene within the cylindrocyclophane gene cluster of

Cylindrospermum licheniforme (UTEX B 2014), ultimately leading to characterization of the entire cluster. An earlier feeding study39 indicated the incorporation of an acetate- derived β-methyl group into the structural scaffold of the cylindrocyclophanes. PCR was then used to probe for putative homologs of HMG-CoA synthase, an enzyme that is part of a conserved pathway responsible for the unusual incorporation of acetate-derived β- methyl groups.40 Sequencing of the PCR product and location of the predicted coding sequence within the genome sequence data of C. licheniforme (UTEX B 2014) led to

7 tentative identification of the entire cylindrocyclophane (cyl) biosynthetic gene cluster, which was attributed more definitively to the cylindrocyclophanes after several enzymatic activities were characterized in vitro.

In contrast to the investigations based on PCR results some biosynthetic inquiries may be based on bioinformatic annotation of genomic data alone, either from analysis of a newly drafted genome or from mining published genomes for particular enzymatic activities.41 PCR can then be used to isolate and amplify individual genes to be cloned into plasmids for biochemical characterization. Alternatively, plasmids can be commercially synthesized to contain the gene of interest, side-stepping PCR experiments. These approaches can expand biosynthetic knowledge of a known natural product or even shed light on metabolites that have yet to be isolated.42

Cyanobacterial genomes are repositories for it’s remarkable natural products because the genes that encode the enzymes are initially responsible for the production of these small molecules.43 By unravelling the genetic code available within these organisms, a wealth of information can be obtained towards unexplored natural products and their biosyntheses.

Figure 1.3 displays the current status of genome availability for the various subgroups of cyanobacteria. The majority of these genomes belong to unicellular species, which don’t produce secondary metabolites.2 Therefore, there is a need for additional cyanobacterial genomic data, especially from the filamentous, secondary-metabolite producing, species.

However, genomic sequencing data from other microbial sources can also be incredibly useful for bioinformatic annotations/predictions within cyanobacterial genomes, as well.

Figure 1.3 Graphical representation of available cyanobacterial genomes.

In general, cyanobacteria are better known for the production of nonribosomal peptides

(NRPs) and polyketides (PKs) and especially those of the hybrid type,44 such as hapalosin

(1.4) and dolastatin 10 (1.5) (Figure 1.2). Although the biosynthetic repertoire for these particular molecules remains to be fully elucidated, the general mechanisms of the modular polyketide synthases (PKSs) and nonribosomal peptide synthases (NRPSs) are largely understood.12a, 38, 45 In addition, ribosomally-synthesized and post-translationally-modified peptides (RiPPs) are among cyanobacterial natural products. Biosyntheses belonging to this class of compounds are also well-represented in the literature.46 In contrast, groups of monofunctional enzymes encoded within the genomes of cyanobacteria have remained underexplored.

1.4 Group V Cyanobacteria’s Hapalindole-type Alkaloid Family of Natural Products

The hapalindole family of natural products encompasses more than 80 structurally diverse congeners that have been isolated solely from filamentous Group V cyanobacteria

(taxonomic family Stigonemataceae; genera Hapalosiphon, Fischerella, Westiella, and

Westielopsis). To date, this family of indole-alkaloids has displayed bioactivities ranging from antibiotic,47 antimycotic,48 and immunosuppressive,49 to sodium channel modulators,50 anti-MDR compounds,23 and antitumor agents.24

The hapalindole-type alkaloids include the hapalindoles,34 fischerindoles,51 ambiguines,52 and welwitindolinones.53 The first of these isoprenoid indole-alkaloids, the hapalindoles, were isolated from Hapalosiphon fontinalis in the mid 1980’s while seeking to identify the antialgal component of the strain’s extract. Specifically, hapalindole A (1.6) and B (1.7) were discovered (Figure 1.4).54 Shortly thereafter, numerous hapalindole

Figure 1.4 Structures of the first isolated hapalindole-type alkaloids, hapalindole A (1.6) and hapalindole B (1.7) isolated from Hapalosiphon fontinalis.

10 analogs were isolated from other group V cyanobacteria, Table 1.1. This led to the identification of a common tetracyclic core for the hapalindole alkaloids (Figure 1.5). All hapalindoles contain common indole and cyclized isoprenoid skeletons decorated with an isonitrile or isothiocyanate moiety, as shown in Figure 1.5. Each of these structural elements offer important bioactive properties. For example, isonitrile and isothiocyanate containing compounds have been shown to possess antimalarial activity,55 while some indole alkaloids are known anti-tumor agents;56 and the combination of these structural features is thought to lead to antibacterial and antimycotic properties.34

Table 1.1 Hapalindole analogs that have been isolated from group V cyanobacteria

Hapalindole analog Strain Reference

Hapalindoles C-Q and T-V Hapalosiphon fontinalis Moore, et. al.; 1987.34 (Ag.) Bornet 12-epi-hapalindole E isonitrile, 12-epi-hapalindole C isonitrile, 12-epi- Hapalosiphon Stratmann, et. al.; 1994.53 hapalindole F isothiocyanate, welwitschii W. & G. S. 12-epi-hapalindole D West UH IC-52-3 isothiocyanate

12-epi-hapalindole H, 12- epi-hapalindole G, 12-epi- Hapalosiphon laingii Hoffman, et. al.; 1995.57 hapalindole Q isonitrile

12-epi-hapalindole F Fischerella sp. CENA 19 Etchegaray, et. al.; 2004.58

12-epi-hapalindole J Fischerella ATCC 43239 Becher, et. al.; 2007.35 isonitrile

Hapalindole X and deschloro Westiellopsis sp. (SAG Kim, et. al.; 2012.59 hapalindole I 20.93)

Figure 1.5 The common tetracyclic core of the hapalindoles includes an indole ring, an isonitrile or isothiocyanate moiety, and a cyclized isoprenoid unit.

Shortly after the initial isolation of the hapalindoles, a variety of hapalindole-type analogs were isolated from other group V cyanobacteria with structural variations derived through various chlorinations, oxidations or reductions, and cyclizations.60

One such example is the fischerindole sub-family of hapalindole-type natural products,

Figure 1.6A. Unlike the hapalindoles, which display ring fusion at C-3 and C-4 of the indole ring resulting in a six membered carbon ring fused to the indole, the fischerindoles display a five membered ring fused to the indole with ring fusion at C-2 and C-3 of the indole ring51 (Figure 1.6B). Additionally, the hapalindoles can consist of tri- or tetracyclic structural scaffolds but the fischerindoles are consistently tetracyclic. Outside of variation in cyclization patterns, the fischerindoles and the hapalindoles are stereochemically

12 similar, possess isonitrile/isothiocyanate moieties, and contain similar modifications

(chlorinations, oxidations/reductions). Fischerindole analogs are listed in Table 1.2.

tetracyclic fischerindole

Figure 1.6 A) Representative structures of the fischerindole sub-family within the hapalindole-type natural products. B) General structures for the tetracyclic hapalindoles and fischerindoles highlighting the difference in ring fusion.

Table 1.2 Fischerindole analogs isolated from group V cyanobacteria Fischerindole analog Strain Reference

Fischerindole L Fischerella muscicola Park, et. al.; 1992.51 UTEX 1829

12-epi-fischerindole G isonitrile, 12-epi- Hapalosiphon welwitschii Stratmann, et. al.; 1994.53 fischerindole U isonitrile, W. & G. S. West UH IC- 12-epi-fischerindole U 52-3 isothiocyanate, 12-epi- fischerindole I isonitrile

12-epi-fischerindole I nitrile, deschloro 12-epi- Fischerella sp. (SAG strain Kim, et. al.; 2012.61 fischerindole I nitrile, 12- number 46.79) epi-fischerindole W nitrile, deschloro 12-epi- fischerindole W nitrile

Ambiguines are more complex than their hapalindole and fischerindoles counterparts. Most notably, ambiguines contain a tert-prenyl (“reverse prenyl”) unit at C-

2 of the indole ring that can be cyclized and oxidized in some examples. Figure 1.7 shows the structures of several ambiguine congeners including the unusual and first of its kind, ambiguine G62 (1.13). Ambiguine G contains a nitrile group rather than an isonitrile/isothiocyanate unit like the other hapalindole-type alkaloids. Table 2.3 lists other ambiguine analogs.

Figure 1.7 Structures of a few members of the ambiguine sub-family of natural products; unique structural characteristics are highlighted, including the presence of a tert-prenyl unit, a cyclized tert-prenyl unit, and a nitrile moiety. The welwitindolinones are some of the most complicated hapalindole-type alkaloids. These natural products contain an oxindole core (oxidized indole) and even more unique cyclization patterns leading to distinct connectivity when compared to other members of the hapalindole-type family.63 Interestingly, all but one welwitindolinone analog contains a chlorine substituent, specifically at C-13;53 this is in contrast to the other sub-families among the hapalindole-type alkaloids, which vary in chlorination at C-13.

Table 1.3 Ambiguine analogs isolated from group V cyanobacteria

Ambiguine analog Strain Reference

Fischerella ambigua Ambiguine isonitriles A-F UTEX 1903, Hapalosiphon Smitka, et. al.; 1992.52 hibernicus BZ-3-1, Westiellopsis prolifica EN- 3-1

Ambiguine G nitrile Hapalosiphon delicatulus Huber, et. al.; 1998.62

Ambiguine H isonitrile, ambiguine I isonitrile, Fischerella sp. Raveh, et. al.; 2006.48 ambiguine J isonitrile

Figure 1.8 shows the structures of some of the exceptionally distinct welwitindolinones.

Welwitindolinone A (1.14) contains a rare cyclobutane ring within its tetracyclic backbone.

N-Methylwelwitindolinone C isothiocyanate (1.15) is an analog well-known for its MDR reversing activity. Lastly, N-Methyl-welwitindolinone D isonitrile (1.16) is the only known non-chlorinated congener within the welwitindolinones. Table 1.4 lists additional welwitindolinones.

Cyclobutane ring C-13 chlorine functionality is absent

Figure 1.8 Structures of some of the structurally intriguing and biologically active welwitindolinones.

Table 1.4 Welwitindolinone analogs isolated from group V cyanobacteria Welwitindolinone analog Strain Reference

Welwitindolinone A, welwitindolinone B isothiocyanate, N- methylwelwitindolinone B isothiocyanate, 3-epi- welwitindolinone B isothiocyanate, Hapalosiphon welwitchii Stratmann, et. al.; 1994.53 welwitindolinone C isothiocyanate, N- methylwelwitindolinone C isonitrile, N- methylwelwitindolinone C isothiocyanate

3-hydroxy-N- Fischerella muscicola (HG- Jimenez, et. al.; 1998.64 methylwelwitindolinone C 39-5) isonitrile

3-hydroxy- N-methylwelwitindolinone C Fischerella major (HX-7-4) Jimenez, et. al.; 1998.64 isothiocyanate, N- methylwelwitindolinone D isonitrile

The intricate combination of structural features within the hapalindole-type alkaloids have stimulated the efforts of many synthetic chemists towards numerous synthetic targets65 directed toward these unprecedented molecular architectures. Total synthesis has resulted in scalable quantities of the hapalindole-type alkaloids via novel, state of the art organic syntheses.63 However, the synthetic approaches can be time consuming and may involve traces of toxic transition-metal based reagents. Today’s synthetic biology tools offer complementary alternatives to traditional organic synthesis methods66 by enhancing the understanding and manipulation of nature’s biocatalytic

17 toolbox. Investigation into the biosynthesis of the hapalindole family of natural products would pave the way for biocatalytic production of the natural products in an environmentally conscious, time and cost effective manner. In addition, access to undiscovered analogs or the production of unnatural variants of these alkaloids could be possible.

1.5 Hapalindole-type Alkaloid Biosynthesis

Three years after the isolation of the first hapalindole,54 investigation into the biosynthesis of this family of molecules was pursued using isotopically labelled precursors.67 The study focused on elucidating the source of the isonitrile moiety. The origins of isonitrile functionalities within natural products, in general, had challenged chemists for decades. Figure 1.9 shows the chemical structures of some of these natural products and highlights putative origins of the isonitrile nitrogen and carbon. Xanthocillin

(1.17) biosynthetic inquiries68 suggested the involvement of L-tyrosine as the donor of the

L-tyrosine derived Cyanide derived

L-methionine derived

Figure 1.9 Structures of xanthocillin, diisocyanoadociane, and hazimicin. Proposed sources of the isonitrile nitrogen and carbon are highlighted.

19 isonitrile nitrogen, but several investigations into the source of the isonitrile carbon had provided a variety of outcomes for several natural products. For example, the marine sponge-derived diisocyanoadociane (1.18) utilized [14C]cyanide as a source of the isonitrile carbon;69 and the hazimicins70 (1.19) isolated from the bacterium Micromonospora incorporated L-[methyl-13C]methionine. However, xanthocillin (1.17) investigations71 suggested that cyanide and methionine, among other tetrahydrofolate (THF) metabolism- related C1 donors, were not sources of the isonitrile carbon.

The Moore67 group sought to determine the source of the isonitrile carbon within the major hapalindole of H. fontinalis (hapalindole A) by feeding [14C]-labelled C1 donors related to THF metabolism to the native cyanobacterial cells. Positive outcomes for those feeding studies led to the finding that [2-13C,15N]glycine feeding resulted in intact incorporation of C(2)-N of glycine into the isonitrile moiety of hapalindole A. In addition, the source of the indole nitrogen was confirmed to be tryptophan through feeding of DL-

[methylene-14C]tryptophan. In addition, the role of geranyl diphosphate as a substrate was proposed, but not confirmed. The specific involvement of geranyl diphosphate in a chloronium-ion induced condensation72 event was later suggested.53 These biosynthetic investigations and hypotheses are summarized in Figure 1.10.

Figure 1.10 Summary of biosynthetic investigations and hypotheses towards hapalindole biosynthesis. L- tryptophan and L-glycine are putative substrates for formation of indole-isonitrile, which is proposed to combine with geranyl diphosphate (pathway A) or a derivative thereof, (Z)-3,7-dimethyl-1,3,6-octatriene through a chloronium-ion induced condensation (pathway B). Interestingly, it was thought that the hapalindoles were structural scaffolds upon which higher order pathways operate to produce the fischerindoles, ambiguines, and welwitindolinones51, 53-54 (Figure 1.11). In other words, the organisms that produce these fascinating alkaloids were predicted to share a common set of biosynthetic genes responsible for this unique family of alkaloids. However, limited genetic research hindered the evaluation of these ideas until more recently.

Fischerindoles

Welwitindolinones

Hapalindoles

Ambiguines

Figure 1.11 Relationship of the hapalindole-type alkaloids; the family is thought to share a common set of genes that biosynthesize the hapalindole structural scaffold which is then modified in higher order pathways, leading to the more complex hapalindole-type alkaloids. Based on the above information, Group V cyanobacteria’s family of hapalindole- type alkaloids were considered to be an excellent candidate for biosynthetic elucidation.

The Viswanathan group hypothesized that unambiguous characterization of a hapalindole- type alkaloid biosynthetic gene cluster would lead to the elucidation of a set of novel genetically-encoded enzymes. Additionally, we hypothesized that enzymes responsible for the biosynthesis of this family of compounds could be heterologously expressed and exploited as biocatalysts in substrate-directed biosynthesis investigations, resulting in novel natural product analogs. A synthetic biology-based approach to these inquiries was deemed most appropriate based on genomic data availability and analogous investigations in the literature.

1.6 Synthetic Biology Approach to Bioactive Natural Products

1.6.1 Introduction to Synthetic Biology as a Tool for Biosynthetic Investigations

Simply stated, synthetic biology is a field that utilizes engineering to manipulate biological systems.73 Many in vitro synthetic biology endeavors involve the use of E. coli to produce biocatalysts for use as purified enzymes or in E. coli cell lysate form.74 The use of E. coli, or an alternative host, to express a gene foreign to its native genome is termed heterologous expression.75 This technique is often used in protein overexpression and E. coli76 is a common host due to its easy manipulation and low cost. BL21(DE3), a specific strain of E. coli, is particularly useful for high-level protein production due to its deficiency of certain proteases and compatibility with the T7 lac operon promoter system.77-78 The lac operon can be inserted into plasmid DNA preceding the gene of interest. The T7 promoter system can then allow for overexpression of a particular gene through induction with IPTG (isopropyl β-D-1-thiogalactopyranoside), a molecular mimic of allolactose, which triggers transcription of the lac operon.

When using synthetic biology to investigate the biosynthesis of a natural product for the first time, some bioinformatic analysis is necessary in order to successfully express and purify a new biocatalyst. Biocatalysts with homologous sequences and/or similar active sites may function under similar chemical conditions. As a result, bioinformatic inquiries can provide insight towards the optimal E. coli growth temperature and incubation time for overexpression of a particular gene. Additionally, bioinformatics can help identify putative substrates, required cofactors/coenzymes, and enzymatic assay environments (such as pH and salt concentration), depending on the information available in the literature.

Useful bioinformatic resources available online include the Swiss Institute of

Bioinformatics (SIB) ExPASy (Expert Protein Analysis System) Bioinformatics Resource

Portal79 and the National Center for Biotechnology Information’s (NCBI) Basic Local

Alignment Search Tool (BLAST).80 ExPASy includes useful tools such as “Translate,” which translates nucleotide sequences to protein sequences. Another useful tool,

“ProtParam,” predicts physical and chemical parameters of proteins when provided the amino acid sequence. ProtParam can be used to obtain estimated molecular weights, extinction coefficients, and pIs of unknown proteins.

A protein BLAST (BLASTP) performed with the amino acid sequence of an unknown protein can result in the identification of homologous protein sequences within the database. Some proteins in the database may be hypothetical or uncharacterized; however, some results may be of characterized enzymes, which can provide useful information applicable to the new biocatalytic system in question.

Once enough information has been gathered through bioinformatic analyses, protein overexpression of the uncharacterized biocatalyst can be performed with E. coli.

Enzymatic function can then be assessed using enzymatic assays.

1.6.2 Introduction to Synthetic Biology as a “Green” Synthesis Methodology Toward Novel Natural Product Analogs

Synthetic biology tools have enhanced the structural and functional innovation of natural products by recruiting a diverse set of enzymes from secondary metabolic pathways.81 In turn, this has spurred new interest in engineering the novelty offered by these green bond-building machines toward the prospect of developing therapeutics possessing enhanced function.82

While total chemical synthesis continues to be a tour-de-force for access to scalable quantities of fine chemicals and pharmaceuticals,83 recent advances in synthetic biology offer complementary tools to execute chemoenzymatic approaches to clinically-relevant targets.84 Biocatalysts, in general, are green85 and cost-effective options to employ for synthesis. They could serve as alternatives in step-wise synthesis and can even replace toxic transition metal-based stoichiometric reagents, provided newer enzymes with unique bond-forming repertoires continue to be discovered.

Synthetic biology used as a methodology towards novel natural product analogs employs engineered biocatalysts84b to expand the structural diversity of the natural product treasure trove.86 This approach is sometimes referred to as “combinatorial biosynthesis”.87

The idea of this technique is to exploit the substrate promiscuity of the system in question by providing non-native substrates to the biocatalyst for possible incorporation into natural product structures.88

1.7 Overview of Investigations

1.7.1 Heterologous Expression and in vitro Reconstitution of Isonitrile Synthase (WelI1) and Fe(II)-α-Ketoglutarate Dependent Oxygenase (WelI3)

Chapter 2 describes a synthetic biology approach utilized to characterize two enzymes involved in hapalindole-type alkaloid biosynthesis. A putative biosynthetic gene cluster was identified to be involved in welwitindolinone biosynthesis in Westiella intricata (HT-29-1) and two genes, welI1 and welI3, were hypothesized to encode for biocatalysts responsible for producing an intermediate en route to the hapalindole-type alkaloids. Bioinformatic analysis revealed homology of WelI1 and WelI3 to an isonitrile synthase and an oxidative decarboxylase, respectively. Within this investigation, the two enzymes putatively involved in isonitrile biosynthesis were heterologously expressed and the E. coli cell lysates were utilized in assays of the enzymatic activities. Reconstitution of the first two enzymatic steps toward the hapalindole-type alkaloid biosynthetic pathway led to the production of the first stable intermediate, an indole isonitrile. Products from the enzyme assay were verified through GC-MS, LC-MS, and HPLC analyses by comparison to synthetically prepared authentic standards. The enzymatic steps catalyzed by WelI1 and

WelI3 were found to proceed with a basal level of promiscuity, as the products constituted both cis and trans isomers of indole isonitrile.

1.7.2 Substrate Tolerance of Isonitrile Synthase and Fe(II)-α-Ketoglutarate Dependent Dioxygenase affords Unnatural Variants of Cyanobacterial Hapalindole Pathway Intermediate

Chapter 3 describes an investigation probing the substrate promiscuity of WelI1 and WelI3, indole-isonitrile producing biocatalysts whose characterization is described in

Chapter 2. These enzymes (or homologs thereof) work in tandem to synthesize cis and trans indole-isonitrile encoded within the hapalindole-type alkaloid (hpi/wel/amb) pathways. WelI1 and WelI3 were reconstituted in vitro, and their substrate versatility was evaluated utilizing seven tryptophan derivatives, including one derivative (C2-methyl L- tryptophan) biosynthesized by a third biocatalyst, the tryptophan synthase TmTrpB1. All seven tryptophan derivatives yielded corresponding cis and trans indole-isonitrile analogs.

The assay extracts were characterized by HPLC for turnover, and product identities were established by LC-HRMS and MS/MS. Quantitative distribution between the cis and trans indole-isonitrile of the analogs was determined by comparison of HPLC results with those of a synthetic standard for the L-trp-derived indole-isonitrile. The relaxed substrate specificity of WelI3 was rationalized based on a 2.1 Å crystal structure of one of its homologs, PaPvcB.

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Chapter 2: Heterologous Expression and in vitro Reconstitution of Isonitrile Synthase (WelI1) and Fe(II)-α-Ketoglutarate Dependent Oxygenase (WelI3)

Portions of the investigation described in this chapter were published under the following citation:

Melinda L. Micallef,‡ Deepti Sharma,‡ Brittney M. Bunn, Lena Gerwick, Rajesh Viswanathan,*

Michelle C. Moffitt.* "Comparative analysis of hapalindole, ambiguine and welwitindolinone gene clusters and reconstitution of indole-isonitrile biosynthesis from cyanobacteria." BMC

Microbiology, 14, 213, 2014.

‡ Indicates first author contribution.

*Indicates corresponding author.

2.1 Introduction

The chemical diversity of the family of hapalindole-type alkaloids was discussed at length in Chapter 1 (Section 1.4) and biosynthetic endeavors on this family were discussed in Section 1.5.

Using synthetic biology tools such as bioinformatic analysis, heterologous expression, and biocatalytic characterization through enzymatic assays, the Viswanathan group sought to unravel the knowledge underlying the biosynthesis of the hapalindole-type alkaloids. Specifically, this thesis focuses on exploring the biocatalysis of indole isonitrile intermediates (that are the first stable intermediates leading to the hapalindoles). We wanted to gain a better understanding of the unique hapalindole-type natural products by identifying the gene clusters encoded by the producing organisms, followed by characterizing specific enzymatic activities resident in the cluster(s). By doing so, we would have access to the biocatalytic prowess of the enzymes which encode this family of unique natural products. Figure 2.1 outlines the synthetic biology approach utilized for the investigation described in this chapter. Once putative biosynthetic genes were identified within a cyanobacterial genome of the natural product producing strain,1 plasmids were constructed hosting the respective genes for heterologous expression in E. coli. Bioinformatic analysis and homology-based 3D modeling led to a biosynthetic hypothesis to be evaluated with E. coli cell lysates expressing cyanobacterial proteins.

Catalysis was evaluated through an enzymatic assay with characterization of biosynthetic products via GC-MS, LC-MS, and HPLC, leading to the in vitro reconstitution of two steps of the hapalindole-type alkaloid biosynthetic pathway.

Cell Lysate

Figure 2.1 Synthetic biology approach utilized to unambiguously characterize the biocatalytic activity of enzymes responsible for hapalindole-type alkaloid biosynthesis.

2.2 Results and Discussion 2.2.1 Identification of the Putative Hapalindole-type Alkaloid Biosynthetic Gene ClusterA

Through whole genome sequencing, five putative gene clusters for the hapalindole- type alkaloids were identified within the genomes of several terrestrial and freshwater group V cyanobacteria,1 specifically Westiella intricata UH strain HT-29-1, Hapalosiphon welwitschii UH strain IC-52-3, Fischerella ambigua UTEX 1903 and Fischerella sp.

ATCC 43239 (Appendix 1.1, Figure A1.1). Prior to the publication of this work,2 but subsequent to the findings, two investigations3 were published that dictated the naming of the genes and enzymes described herein; however, the findings of those investigations will not be described until the end of this chapter.

The wel cluster putatively encoding the welwitindolinone biosynthetic pathway within the genome of Westiella intricata UH strain HT-29-1 was chosen as the candidate cluster for further verification. The 59.3 kb cluster, Figure 2.2, was predicted to encode three genes involved in isonitrile biosynthesis (specifically welI1, welI2, and welI3), the first step proposed to be involved in the production of the hapalindole-type alkaloids. welI2 was determined to be identical to welI3. Therefore, biosynthetic endeavors within the investigation described in this chapter focused solely on welI1 and welI3. However, the functional redundancy of welI2 and welI3 remains a subject of curiosity at this time.

A Identification of the putative gene clusters was accomplished by Deepti Sharma and Melinda L. Micallef.

Figure 2.2 Illustration of the wel gene cluster from Westiella intricata UH strain HT-29-1. This investigation focuses on the isonitrile synthase genes welI1 and welI3.

2.2.2 Bioinformatic Analysis of WelI1/3 & Homology to IsnA/B and PvcA/B

Translation of the two genes into protein sequences using ExPaSy’s translate tool

(Appendix 1.2) allowed for a BLASTP inquiry (results are presented in Appendix 1.3), which led to two sets of characterized proteins showing homology to WelI1/3, among other uncharacterized or hypothetical proteins. WelI1 and WelI3 displayed 43% and 45% maximum identity to characterized isonitrile synthase IsnA and IsnB (from an eDNA metagenomic-derived sample),4 respectively (Appendix 1.3, Tables A1.1 and A1.2), and

46% maximum identity to PvcA and PvcB proteins related to those characterized from

Pseudomonas aeruginosa,5 respectively (Appendix 1.3, Tables A1.1 and A1.2).

Genes isnA and isnB were found within an eDNA clone determined to produce indole-isonitrile.4 The genes were predicted to be an isonitrile synthase (isnA) and a non- heme iron α-ketoglutarate dependent oxygenase (isnB). Heterologous expression of IsnA and IsnB sequenced with appropriate enzymatic assays confirmed these hypotheses, and feeding studies with a tryptophan deficient strain of E. coli and 15N-tryptophan established the role of tryptophan (2.1) in the biosynthesis of indole-isonitrile. A subsequent study verified the source of the isonitrile carbon as ribulose-5-phosphate or its tautomers, ribose-

5-phosphate or arabinose-5-phosphate.6 Scheme 2.1A shows the reaction catalyzed by

IsnA and IsnB.

The pvc gene cluster was originally linked to pyoverdine biosynthesis in

Pseudomonas aeruginosa;5a however, reevaluation of the genes identified them to be involved in the formation of the isonitrile moiety within isocyano derivatives of amino acids, like paerucumarin.7 PvcA was determined to be an isonitrile synthase that utilizes tyrosine (2.4) and a sugar phosphate as its substrates and PvcB was determined to be a non- heme iron α-ketoglutarate dependent oxygenase5b (Scheme 2.1B). The crystal structures of PvcA/B were obtained to 3.0 and 2.6 Å, respectively.

or Ribulose-5-phosphate

Scheme 2.1 A) IsnA catalyzes the reaction between L-tryptophan (2.1) and ribose-5-phosphate, which yields an unstable intermediate (2.2). The intermediate is oxidatively decarboxylated by Isn B, which requires Fe(II) and α-ketoglutarate to yield trans indole isonitrile (2.3a). B) PvcA catalyzes the reaction between L-tyrosine (2.4) and ribulose-5-phosphate, which yields an unstable intermediate (2.5). The intermediate is oxidatively decarboxylated by Isn B, which requires Fe(II) and α-ketoglutarate to yield an oxidized tyrosine-isonitrile (2.6).

An alignment of PvcA with a group of homologous proteins, including IsnA, identified six well-conserved regions within the proteins of that family that are proposed to contribute to phosphate binding and catalytic activity. Through a Clustal Omega8 alignment, these six motifs were identified in WelI1 (Figure 2.3A). A homology based 3- dimensional model of WelI1,9-10 based on PvcA, highlights the amino acid residues located in a cavity proposed to be involved in hydrogen bonding or catalytic activity (TPWH),5b

(Figure 2.3B).B Therefore, it was hypothesized that the cyanobacterial WelI1 would behave similarly to IsnA and PvcA by utilizing an amino acid (tryptophan, in particular, due to the presence of the indole ring within the hapalindole-type alkaloids) and a sugar phosphate as substrates.

B Homology-based three dimensional modeling was performed by William Lang.

Figure 2.3 A) Clustal Omega alignment of WelI1, IsnA, and PvcA. Clustal Omega key: *(asterisk): fully conserved residue, : (colon): groups with highly similar properties, . (period) groups with weak similarity. Conserved domains within the three sequences are highlighted. B) Homology based 3-D model of WelI1 with the potential active site highlighted.

Both IsnB and PvcB were found to be Fe(II)-dependent α-ketoglutarate dependent oxygenases. Most α-ketoglutarate dependent oxygenases are known to couple oxidative decomposition of α-ketoglutarate to succinate and CO2 with hydroxylation of a cosubstrate.11 Although the exact mechanism of these enzymes is unknown and may not be uniform among the family, this group is distinguished based on the requirement for

Fe(II).5b, 11 A metal binding motif consisting of His-X-Asp/Glu-X-His is common among the enzymes. WelI3 possess this motif along with IsnB and PvcB, as shown in the Clustal

Omega8 alignment (Figure 2.4A). Using the crystal structure of PvcB and homology- based three dimensional modeling,9-10 this motif was identified and is highlighted in Figure

2.4B.C Due to sequence similarity, it was hypothesized that WelI3 is also a member of the

Fe2+/α-ketoglutarate-dependent family of oxygenases.

C Homology-based three dimensional modeling was performed by William Lang.

Figure 2.4 A) Clustal Omega alignment of WelI3, IsnB, and PvcB. Clustal Omega key: *(asterisk): fully conserved residue, : (colon): groups with highly similar properties, . (period) groups with weak similarity. The amino acids comprising the metal binding motif common to α-ketoglutarate dependent oxygenases are indicated. B) Homology based 3-D model of WelI3. The Fe (II)-binding motif is highlighted.

Based on the above bioinformatic analyses, a biosynthetic pathway to indole isonitrile utilizing WelI1 and WelI3 was proposed, Figure 2.5. L-Tryptophan (2.1) and ribose-5-phosphate (2.7) are utilized by WelI1, which yields an unstable, indole-isonitrile intermediate (2.2). The intermediate (2.2) is oxidatively decarboxylated by WelI3, which is predicted to require an iron source and α-ketoglutarate (2.8) to yield cis indole-isonitrile

(2.3b).

Figure 2.5 Biosynthetic hypothesis towards cis indole-isonitrile (2.3b) utilizing biocatalysts WelI1 and WelI3, substrates L-tryptophan (2.1) and ribose-5-phosphate (2.7), and cofactor/cosubstrate ammonium iron (II) sulfate and α-ketoglutarate (2.8).

2.2.3 In vitro Reconstitution and Unambiguous Characterization of WelI1 and WelI3

In order to test the biosynthetic hypothesis outlined in Figure 2.5, welI1 and welI3 were submitted to DNA2.0 for codon optimization and plasmid construction. A 6x-His tag was included at the C-terminus of the biosynthetic gene, to facilitate purification, preceded by a factor Xa protease recognition site. The ribosomal binding site (RBS) was customized to contain an NdeI restriction site and an XhoI restriction site was included after the stop codon for cloning purposes. The vector map for the plasmids containing welI1 and welI3 is shown in Figure 2.6.

Figure 2.6 Vector map for plasmids hosting welI1 and welI3; the plasmids contain genes for kanamycin resistance and are under control of the T7 promoter.

The two cyanobacterial genes were then transformed into E. coli BL21(DE3) for heterologous expression. Protein overexpression methods were based on previous work on IsnA/B4 and PvcA/B.5b E. coli BL21(DE3) hosting the gene of interest was incubated at 37 °C until an OD600 of approximately 0.6 was obtained. Protein overexpression was induced by the addition of 1.0 mM IPTG and the cells were incubated at 16 °C overnight.

At this time, a number of protein purification attempts yielded low quantities of both proteins, therefore the biosynthetic investigation was further pursued with E. coli cell lysates containing the proteins of interest, Figure 2.7. Note that molecular weights of the proteins were estimated using ExPASy’s “Protparam” tool (WelI1: ~38 kDa, WelI3: ~32 kDa).

kDa

WelI1

WelI3

Figure 2.7 SDS-PAGE analysis of E. coli BL21(DE3) cell lysates hosting WelI1 and WelI3.

An enzymatic assay was developed in order to establish the biosynthetic roles of

WelI1 and WelI3. E. coli cell lysates containing WelI1 and WelI3 were mixed with the proposed assay components (L-tryptophan (2.1), a sugar phosphate (2.7), α-ketoglutarate

(2.8), and an iron source) in a Tris buffer containing 150 mM NaCl. An assay containing both enzymes was preferred to two separate assays based on the instability of the intermediate produced by WelI1, in view of previous unsuccessful isolation attempts.4

Assay mixtures were incubated for 3 hours at 37 °C, extracted with 1:1 ethyl acetate/hexanes and subjected to GC-MS analysis. Assay results were compared to the spectra of synthesized standardsD, 1a of the cis (2.3b) and trans (2.3a) isomers of indole- isonitrile (characterization of standards: Appendix 2, GC-MS spectrum of standards:

Appendix 4).

Assay mixtures containing both enzymes appeared to produce both the cis (2.3b) and trans (2.3a) isomers of indole-isonitrile, as shown in the GC spectrum, Figure 2.8A.

The peak at Rt 6.42 min correlates to the trace of the cis isomer (2.3b) standard and the peak at Rt 6.70 min correlates to the trace of the trans isomer (2.3a) standard. The mass spectra of the two major peaks are shown in 2.8B and 2.8C, providing additional support for the identity of products to be the two isomers. Furthermore, indole-isonitrile formation was established via LC-MS (Appendix 3, Figure A3.1).E

After these findings were made, an investigation reporting the ambiguine biosynthetic pathway3a (amb) was published. Within this investigation, isonitrile synthase

AmbI1 and Fe2+/α-ketoglutarate-dependent oxygenase AmbI3 were biochemically characterized via heterologous expression and enzymatic activity assays. Interestingly,

D Standards were synthesized and characterized by Deepti Sharma. E LC-MS analysis was performed by Deepti Sharma.

54 these enzymes were found to produce solely the cis isomer (2.3b) of indole-isonitrile.

Other details of this investigation will be discussed in the conclusion, Section 2.3.

Although it was suggested through the GC-MS (2.8A) and LC-MS results

(Appendix 3, Figure A3.1) that indole isonitrile was being biosynthesized by WelI1 and

WelI3, additional confirmation was necessary in order to accurately determine whether both isomers of the small-molecule were being produced enzymatically. To verify that both isomers were being produced enzymatically by WelI1 and WelI3, and not the result of thermal degradation/isomerization from the high temperatures of the GC, HPLC analysis was conducted.

Figure 2.8 A) GC of assay extract indicating the production of both the cis (2.3b) and trans (2.3a) isomers of the indole-isonitrile. B) MS of the peak at Rt 6.42 min from the GC; notice the parent peak with a mass of 168.06. C) MS of the peak at Rt 6.70 min from the GC; notice the parent peak with a mass of 168.06.

For HPLC analysis, the enzymatic assay was conducted once more. The assay components were mixed as previously described and incubated at 25 °C for 16 hours, rather than 3 hours at 37 °C. Note that the temperature and incubation adjustments were made based on the AmbI1/AmbI33a investigation. To ensure an E. coli-derived isomerase was not responsible for either the cis (2.3b) or trans indole-isonitriles, assays were conducted with E. coli cell lysates lacking WelI1 and WelI3 as negative controls. Figure 2.9 shows the results of the HPLC analysis of the assay extract (with 1:1 isopropanol/hexanes).

Traces 1 and 2 show the retention times for the synthesized cis (2.3b) and trans indole- isonitrile standards, 8.8 min and 13.1 min, respectively. The standards display the same retention times when co-injected as shown in Trace 3. Trace 4 shows the results of the assay conducted with an E. coli cell lysate without WelI1 and WelI3; the absence of product peaks confirms that the assembly of indole-isonitrile cannot be attributed to proteins in the lysate derived from the genome of E. coli. The results of the assay containing WelI1 and

WelI3 are shown in Trace 5, which clearly indicates the presence of both isomers of indole isonitrile, favoring the trans isomer. To ensure that an E. coli-derived isomerase was not responsible for the presence of both isomers, aliquots of the assay conducted with an E. coli cell lysate without WelI1 and WelI3 were spiked with the cis isomer (2.3b) of indole- isonitrile and incubated for 3 and 16 hours at 37 °C. Results of these analyses are shown in Traces 6 and 7, which unambiguously confirm that the trans isomer is not produced by isomerization of the cis isomer, but rather is produced directly by the WelI1/3 system.

Figure 2.9 HPLC was analyzed at 310 nm with a UV detector. X-axis – retention time in minutes (min). Y- axis - intensity in arbitrary units. Presented as a stacked Y-plot and is drawn to relative intensity units. Peaks show only relative intensities and are not normalized for concentration of metabolites. 1) Synthesized cis indole-isonitrile only (2.3b) (Rt = 8.8 min). 2) Synthesized trans (2.3a) indole-isonitrile only (Rt = 13.1 min). 3) Co-injection of synthetic standards of cis (2.3b) and trans indole-isonitrile (2.3a). 4) Control for enzyme assay where cell lysates of E. coli BL21(DE3) were subjected to assay conditions without WelI1 and WelI3. 5) WelI1 and WelI3 enzyme assay after 16 h incubation at 25°C. 6) Control sample (4) spiked with cis indole- isonitrile (2.3b) after 3 h incubation. 7) Control sample (4) spiked with cis indole-isonitrile (2.3b) after 16 h incubation. 8) Co-injection of cis (2.3b) and trans (2.3a) indole-isonitrile with enzyme assay mixture.

2.3 Conclusions

Through whole genome sequencing, a putative wel biosynthetic gene cluster was located within the genome of Westiella intricata UH strain HT-29-1. This biosynthetic gene cluster is conserved to varying degrees among other hapalindole-type alkaloid producing Group V cyanobacteria.2 This chapter discussed how utilizing a synthetic biology approach allowed for unambiguous characterization of two enzymes from the wel biosynthetic gene cluster. Through heterologous expression and an enzymatic assay using

E. coli cell lysates, evidence was obtained that WelI1 and WelI3 are an isonitrile synthase and an Fe2+/α-ketoglutarate-dependent oxygenase, respectively. Assay results were verified via GC-MS, LC-MS, and HPLC.

As stated previously, by the time of publication this investigation complemented several other investigations, reported by the Liu group,3 which augmented the biosynthetic repertoire towards the hapalindole-type alkaloids. The ambiguine (amb) cluster from

Fischerella ambigua was the first hapalindole-type alkaloid biosynthetic gene cluster published and AmbI1/AmbI3 were the first isonitrile biosynthetic enzymes characterized within the hapalindole-type alkaloid-producing strains.3a Both AmbI1 and AmbI3 were characterized as purified proteins following heterologous expression in E. coli. In addition to these biocatalysts, the same investigation characterized AmbP2 and AmbP3, a GPP synthase and dimethylallyl transferase, respectively, involved in the biosynthesis of the ambiguines.

In a subsequent publication, the same group identified the wel biosynthetic gene cluster in the genome of Hapalosiphon welwitschii UTEX B1830.3b In order to correlate this cluster to the welwitindolinones with certainty, WelI1, WelI3, WelP2 (geranyl

58 diphosphate synthase), and WelM (SAM-dependent methyltransferase) were characterized through heterologous expression and enzymatic assay verification. Shortly thereafter,

WelO5 was characterized as an Fe2+/α-ketoglutarate-dependent halogenase responsible for the chlorination observed at C-13 of hapalindole-type molecules.12

Enzymatic assays conducted with the AmbI1/I3 system and the H. welwitschii

WelI1/I3 system were similar to those conducted within the investigation described in this chapter, with minor differences. Detection of both isomers of indole-isonitrile with the W. intricata WelI1/3 system is not attributed to the slight variation in enzymatic assay methodologies. A possible explanation for this observation can be developed using the structural variations of the hapalindole-type alkaloids, Figure 2.10; hapalindole scaffolds contain either cis or trans stereochemistry across C10-C11, whereas ambiguines contain solely cis connections across C10-C11, and some hapalindole-type alkaloids lose the stereochemistry across this bond, as in N-methyl welwitindolinone C isothiocyanate (2.15).

Perhaps clusters for some of the hapalindole-type alkaloids produce both isomers while others do not. In order to draw any conclusions on this topic, however, more investigations are necessary.

Loss of Stereochemistry at C10

Figure 2.10 Depiction of various C10-C11 stereochemistries among the hapalindole-type alkaloids.

2.4 Experimental Section 2.4.1 Cyanobacterial culturing2

The cyanobacterial strains WI HT-29-1 and HW IC-52-3 were obtained from the

University of Hawaii cyanobacterial culture collection, FS ATCC43239 from American

Type Culture Collection and FA UTEX1903 from Culture Collection of Algae at the

University of Texas at Austin. All cyanobacterial cultures were maintained in Blue-Green

11 (BG-11) medium13 (Fluka, Buch, Switzerland). WI HT-29-1 and HW IC-52-3 cultures were maintained at 24 °C with 12 h light/dark cycles illuminated with 11 µmol m-2 s-1 of photons. FS ATCC43239 and FA UTEX1903 were illuminated with 80-100 µmol m-2 s-1 of photons on a 18:6 h light/dark cycle at 22 °C.

2.4.2 Genomic DNA extractionF, 1, 2

Prior to genomic DNA (gDNA) extraction, WI HT-29-1 and HW IC-52-3 cyanobacterial cells were first filtered using a 3 µm nitrocellulose membrane (Millipore,

North Rhyde, Australia) to remove heterotrophic bacteria and washed with 200 mL of sterile BG-11 media. gDNA was extracted from WI HT-29-1 and HW IC-52-3 cyanobacterial cells following the protocol outlined in Morin et al.14 RNA was removed using 2 µL of ribonuclease A (≥70 Kunitz U/mg) and incubation at room temperature for

15 min. Phenol:chloroform:isoamyl alcohol extraction was performed and gDNA was precipitated using isopropanol and resuspended in TE buffer. Additional polysaccharides were removed following the protocol outlined in Wilson.15 FS ATCC43239 gDNA was isolated following the protocol described in Ausubel et al.16 and FA UTEX1903 gDNA was extracted following a protocol described in Mustafa.17

F Genomic DNA extraction was performed by Deepti Sharma and Melinda L. Micallef.

2.4.3 Whole genome sequencing and bioinformaticsG, 1, 2

High molecular weight gDNA from WI HT-29-1 and HW IC-52-3 was sent to BGI

(Beijing Genome Institute, China) for genome sequencing via high throughput Illumina sequencing technology. BGI performed genome assembly and gene annotation using

Glimmer v3.0.

Extracted gDNA from FA UTEX1903 and FS ATCC43239 was submitted to Case

Western Reserve Genomics Core Facility for whole genome sequencing. Paired end DNA libraries were obtained by using Nextera DNA sample preparation kit and sequenced using the Illumina GAIIx platform. Raw reads quality was assessed using FastQC 0.10.1

(Babraham Bioinformatics) with default settings and trimmed with Seqyclean 1.3.12

(http://cores.ibest.uidaho.edu/software/seqyclean). Filtered reads were assembled de novo using the velvet package (Version 1.2.08) and a kmer range between 55-63. The optimal assembly based on expected genome size, N50 and contig number was used for downstream annotation and analysis. Gene annotation was performed by BGI using Glimmer v3.2. A

Basic Local Alignment Search Tool(BLAST) search was performed to identify the putative function of proteins based on sequence similarity.16

Nucleotide and protein sequences were organised and visualised using Geneious v6.1.7 created by Biomatters, available from http://www.geneious.com/. Nucleotide alignments were performed using Geneious Alignment with default settings. For protein alignments, Clustal Omega (Version 1.2.1) was used with default settings, except order changed from aligned to input.18

G Whole genome sequencing and bioinformatics were performed by Deepti Sharma and Melinda L. Micallef.

2.4.3.1 Homology Based Model GenerationH, 9

PvcA and PvcB 3D structures were obtained from published Protein Data Bank codes (35E9 and 3EAT respectively).5b Amino acid sequences for Isn A/IsnB4 and

WelI1/WelI32 were obtained from recently sequenced genomes. 3D protein structures were created through the PHYRE210 (protein homology/analogy recognition engine) server. Protein modeling software Pymol (v1.5.0.5)19 was used to visualize structures.

2.4.3.2 Nucleotide Accession Numbers

The nucleotide sequences of the gene clusters were deposited to NCBI GenBank under the following accession numbers: KJ742064 for FS ATCC43239, JK742065 for FA

UTEX1903, KJ767018 for WI HT-29-1 and KJ767017 for HW IC-52-3. The nucleotide sequence of the 16S ribosomal RNA gene was also deposited to NCBI GenBank under the following accession numbers: KJ768872 for FS ATCC43239, KJ768871 for FA

UTEX1903, KJ767016 for WI HT-29-1 and KJ767019 for HW IC-52-3.

2.4.4 Gene Cloning for heterologous expression2

The pJexpress411-T7-kan plasmids (with C- terminal His6-tag) harboring the codon-optimized genes of welI1 and welI3 from WI HT-29-1 were designed and subsequently purchased from (DNA2.0, Inc, USA).

2.4.5 Heterologous expression of WelI1 and WelI32

A 50% (v/v) glycerol stock of BL21(DE3) transformed with the gene of interest was used to inoculate a flask containing 25 mL LB broth supplemented with 50 µg/mL

H Homology-based three dimensional modeling was performed by William Lang.

63 kanamycin. The flask was incubated at 37 °C with shaking at 180 rpm for 6-8 h. This culture was added to a flask containing 1 L of LB broth supplemented with 50 µg/mL kanamycin and incubated at 37 °C until an OD600 of approximately 0.6 was obtained. The cells were then induced with 1 mM IPTG and grown at 16 °C overnight. The cells were centrifuged at 6,084 x g for 10 min and frozen at -20 °C. The cell pellet was thawed on ice and resuspended in 50 mM Tris buffer (pH 7.5) containing a cocktail of protease inhibitors

(Sigma Aldrich, SIGMAFAST™ protease inhibitor cocktail tablets, EDTA-free for use in purification of histidine-tagged proteins), 0.2 mM TCEP, 250 mM NaCl, and 10 % (v/v) glycerol. Lysozyme was added to a final concentration of 1 mg/mL and stirred until a viscous suspension was obtained. The sample was sonicated (Fisher Scientific™ Model

120 Sonic Dismembrator, power: 120 W, frequency: 20 kHz, amplitude: 40%, probe: model CL-18) under the following cycle: [(10 s pulse + 1 s pause) x 5, 1 min cooling period] repeated five times and the cellular debris was removed by centrifugation at 57,000 x g for 1 h at 4 °C.

2.4.6 Enzymatic assay with cell lysates containing WelI1 and WelI3 for GC-MS and LC-MS2

Assay components were mixed to a final reaction volume of 10 mL (2 mL WelI1 cell lysate, 2 mL WelI3 cell lysate, 25 mM Tris (pH 7.0), 150 mM NaCl, 0.8 mg/mL L- tryptophan, 0.8 mg/mL ribose-5-phosphate, 0.8 mg/mL α-ketoglutaric acid, 25 µM

(NH4)2Fe(SO4)2, incubated at 37 °C for 2-3 hours and extracted with ethyl acetate/hexanes

(1:1). Extracts were analyzed by GC-MS.

2.4.7 GC-MS analysis2

A GC instrument coupled to a DSQII MS probe was employed for analyses reported in this study. Assay extracts were analyzed on a 30 m x 0.25 mm x 0.25 µm HP-

5 capillary column (Agilent Technologies) with a temperature gradient (ramp-up) from

50 ºC to 275 ºC at 30 ºC per min, a hold at 275 ºC for 2 mins, followed by a temperature gradient (ramp-down) of 275 ºC to 60 ºC at 60 ºC per min, with a He flow rate of 1 ml/min. The overall time for each assay was 16 mins. For MS analyses of the Gas chromatogram, the method scanned a mass range of 30-600 amu. with the ion source kept stable at 250 ºC. Both positive and negative ionization mode were run sequentially for each sample. All GC / GC-MS analyses were carried out under the same conditions.

2.4.8 LC-MS analysisI, 1a, 2

Accurate LC-MS data was recorded with a Waters Acquity I-Class UPLC system and a Waters Synapt G2 HDMS mass spectrometer. A 2.1x50 mm column packed with

BEH C18 1.7 μm particles (Waters) was held at 45 °C throughout the separation; mobile phase A was 5% v/v Omnisolve grade CH3CN (EMD Millipore, Billerica, MA), 0.1% v/v formic acid (Sigma Aldrich) in Omnisolve grade water (EMD). Mobile phase B was 5% v/v Omnisolve water, 0.1% v/v formic acid in acetonitrile and the flow rate was maintained at 0.3 mL/min. The gradient profile was: Start at 10% B, linear gradient to 100% B over

30 minutes, hold 3 minutes at 100% B, and a linear gradient to 0% B over two minutes followed by a 3 minute re-equilibration period between injections. All effluent was directed into the ESI source of the G2 (3.0 kV on capillary, 120 °C source temperature, 850

I LC-MS analysis was performed by Deepti Sharma

L/h of nitrogen desolvation gas @ 600 °C, 20 L/h of cone gas, 40 V on sample cone, 4 V on extraction cone) which was used in resolution mode (20,000 resolving power).

2.4.9 Enzymatic assay with cell lysates containing WelI1 and WelI3 for HPLC2

Each cell lysate containing a protein of interest (WelI1 or WelI3) totaled approximately

10 mL (resulting from 1 L of culture). Assay components were mixed to a final reaction volume of 5 mL (1 mL WelI1 cell lysate, 1 mL WelI3 cell lysate, 25 mM Tris (pH 7.0),

150 mM NaCl, 0.8 mg/mL L-tryptophan, 0.8 mg/mL ribose-5-phosphate, 0.8 mg/mL α- ketoglutaric acid, 25 µM (NH4)2Fe(SO4)2, Samples were then incubated for 16 h at 25 °C and extracted with 1:1 isopropanol/hexanes. Following extraction, samples were analyzed by HPLC. A negative control was performed with E. coli BL21 (DE3) cell lysate hosting no plasmid.

2.4.9 HPLC analyses2

HPLC analyses for synthetic intermediates were performed using a Shimadzu LC-

20-AT Series separations module equipped with Shimadzu SPD-M20A PDA (photo diode array) multiple wavelength detectors (180nm-800nm). For indole-isonitrile compounds,

UV detector was set at 310 nm with a 5 nm slit-width. The overall system, CBM-20 was controlled using LC Solutions software. Raw data was plotted using the Origin® software program after exporting absorbance data as an ASCII-formatted file. Analytical separations of stereoisomers (of cis and trans) mixtures were carried out on Daicel® (normal phase)

AS chiral column. A 10 % isopropanol/ 90 % hexanes mixture was used as elution medium with a flow rate of 1 mL/min in an isocratic mode. Individual retention times for indole- isonitriles are reported along with analytical data for each isomer.

2.4.10 Synthesis and spectroscopic analysis of indole-isonitrileJ,1a, 2

Anhydrous tetrahydrofuran was obtained from an mBraun solvent purification system (A2 alumina). Reactions were monitored by thin-layer chromatography (TLC) on silica gel plates (60 F254) with a fluorescent indicator, and independently visualized with

UV light. Preparatory thin-layer chromatography (TLC) was performed on glass plates (7.5 x 2.5 and 7.5 x 5.0 cm) pre-coated glass plates coated with 60 Å silica gel (Whatman).

Separations of isonitrile intermediates were carried out using flash chromatography (Silica gel grade: 200-400 mesh, 40-63 μm) at medium pressure (20 psi). NMR spectra were

1 recorded at 400 MHz in CDCl3 and chemical shift values (δ) are reported in ppm. H NMR spectra are reported in parts per million (δ) relative to the residual (indicated) solvent 1H peak. Data for 1H NMR are reported as follows: chemical shift (δ ppm), multiplicity (s = singlet, brs = broad singlet, d = doublet, t = triplet, q = quartet, ddd = double double doublet, m = multiplet, cm = complex multiplet), integration, and coupling constants in Hz. 13C

NMR spectra were obtained on 400 MHz spectrometers (100 MHz actual frequency) and are reported in parts per million (δ) relative to the residual (indicated) solvent peak.

HRESI-MS data for synthetic compounds were obtained by direct infusion of methanolic solutions on a Waters Synapt HDMS QTOF mass spectrometer (Waters Corporation,

Milford, MA). The HRMS spectra for a sample containing a mixture of cis and trans isomers of indole isonitrile is presented in Appendix 2.2, Figure A2.5.

J Indole-isonitrile standards were synthesized and characterized by Deepti Sharma.

2.4.11.1 Synthesis of Cis and Trans Isomers of Indole-isonitrile

2.4.11.1.1 Synthesis of 3-indolecarbaldehyde (Precursor for Indole-isonitrile Synthesis)

To 7 mL of N,N-dimethylformamide (DMF), 1 mL of phosphorus oxychloride

(POCl3) was added dropwise at 0 ºC. The mixture was stirred for 20 min, after which 3 mL of 10 mmol indole in DMF was added dropwise to the mixture. After the mixture was stirred at 35 oC for 1 h, crushed ice was added, followed by 20% aq. sodium hydroxide

(NaOH) and the mixture was refluxed for 6 h. On cooling, the mixture was poured into ice water, and the precipitated product was collected, washed by water, and dried. 3- indolecarbaldehyde was the sole product and was isolated in 84 % yield. This product was sufficiently pure for subjection to the subsequent isonitrile formation step as shown from

1 1 its H NMR spectrum. H NMR (400MHz, DMSO-d6): δ 9.97 (s, 1H), 8.33 (s, 1H), 8.13

(d, J = 7.6 Hz, 1H), 7.55 (d, J = 7.2 Hz, 1H), 7.31–7.24 (m, 2H).

2.4.11.1.2 Synthesis of indole-isonitrile (3-(2-isocyanovinyl)indole) A 5 mL tetrahydrofuran (THF) solution containing 584 mg (3.3 mmol, 1.1 equiv.) of diethyl (isocyanomethyl) phosphonate was added drop wise to a stirred solution containing 839 mg (4.57 mmol, 1.5 equiv.) of sodium bis (trimethylsilyl)amide in 5 mL of

THF at – 78 °C. The resulting mixture was stirred for 15 min and then treated with a solution of 436 mg (3.0 mmol, 1.0 equiv.) of 3-indolecarbaldehyde in 30 mL of THF. The solution was allowed to warm to 4 °C and allowed to stir for an additional 48 h. Acetic acid (198 mg, 3.3 mmol) of acetic acid in 1.5 mL of THF was added to quench the reaction. The solvent was removed in vacuo, the residue was dissolved in 30 mL of ethyl acetate, washed with 15 mL of 0.1 M phosphate buffer (pH = 7.2), then with 15 mL of H2O and the resulting organic layer was dried on a bed of MgSO4. The collected organic layer was evaporated to

68 obtain the crude product which upon purification through chromatography (silica gel) eluting with a gradient of 10-12% ethyl acetate in hexane yielded a mixture of trans (196 mg, 45%) and cis (106 mg, 25%) indole-isonitrile in an almost 2:1 ratio as indicated by 1H

NMR analysis and in 70% overall yield.

2.4.11.2 Spectroscopic Analysis of Indole-isonitrile

2.4.11.2.1 Cis Indole-isonitrile 1H and 13C NMR Data

1 H NMR (400 MHz, CDCl3) δ 8.56 (brs, 1H), 8.15 (d, 2.8 Hz, 1H), 7.68 (d, 7.9 Hz,

1H), 7.44 (d, J = 7.9 Hz, 1H), 7.32-7.20 (m, 2H), 6.84-6.75 (m, 1H), 5.75 (d, J = 8.8 Hz,

13 1H). C NMR (100 MHz, CDCl3) δ 169.1, 135.2, 126.9, 126.5, 124.2, 123.4, 121.1, 118.2,

111.6, 110.3, 104.6 (Appendix 2.1, Figure A2.1 and Figure A2.2)

2.4.11.2.2 Trans Indole-isonitrile 1H and 13C NMR Data

1 H NMR (400 MHz, CDCl3) δ 8.35 (brs, 1H), 7.69 (d, 7.9 Hz, 1H), 7.44-7.40 (m,

1H), 7.35 (d, J = 2.6 Hz, 1H), 7.32-7.21 (m, 2H), 7.14 (d, J = 14.2 Hz, 1H), 6.36 (d, J =

13 14.2 Hz, 1H). C NMR (100 MHz, CDCl3) δ 163.3, 137.0, 130.3, 126.4, 124.8, 123.6,

121.6, 120.1, 112.0, 111.3, 107.3 (Appendix 2.1, Figure A2.3 and Figure A2.4)

2.5 References

1. (a) Sharma, D. Harnessing Genomes and Building Molecules for Investigating

Biosynthetic Mechanisms in Model Group V Cyanobacteria. Ph. D. Thesis, Case Western

Reserve University, 2016; (b) Micallef, M. L. Identification and Characterisation of

Biosynthetic Gene Clusters from Subsection V Cyanobacteria. Ph. D. Thesis, University of Western Sydney, 2015.

2. Micallef, M. L.; Sharma, D.; Bunn, B. M.; Gerwick, L.; Viswanathan, R.; Moffitt,

M. C., Comparative analysis of hapalindole, ambiguine and welwitindolinone gene clusters and reconstitution of indole-isonitrile biosynthesis from cyanobacteria. BMC Microbiol.

2014, 14 (213), 1-18.

3. (a) Hillwig, M. L.; Zhu, Q.; Liu, X., Biosynthesis of Ambiguine Indole Alkaloids in Cyanobacterium Fischerella ambigua. ACS Chem. Biol. 2014, 9 (2), 372-377; (b)

Hillwig, M. L.; Fuhrman, H. A.; Ittiamornkul, K.; Sevco, T. J.; Kwak, D. H.; Liu, X.,

Identification and Characterization of a Welwitindolinone Alkaloid Biosynthetic Gene

Cluster in the Stigonematalean Cyanobacterium Hapalosiphon welwitschii. ChemBioChem

2014, 15 (5), 665-669.

4. Brady, S. F.; Clardy, J., Cloning and heterologous expression of isocyanide biosynthetic genes from environmental DNA. Angew. Chem. Int. Ed. Engl. 2005, 44 (43),

7063-5.

5. (a) Clarke-Pearson, M. F.; Brady, S. F., Paerucumarin, a new metabolite produced by the pvc gene cluster from Pseudomonas aeruginosa. J. Bacteriol. 2008, 190 (20), 6927-

30; (b) Drake, E. J.; Gulick, A. M., Three-dimensional Structures of Pseudomonas

70 aeruginosa PvcA and PvcB, Two Proteins Involved in the Synthesis of 2-Isocyano-6,7- dihydroxycoumarin. J. Mol. Biol. 2008, 384 (1), 193-205.

6. Brady, S. F.; Clardy, J., Systematic investigation of the Escherichia coli metabolome for the biosynthetic origin of an isocyanide carbon atom. Angew. Chem. Int.

Ed. 2005, 44 (43), 7045-8.

7. Brady, S. F.; Bauer, J. D.; Clarke-Pearson, M. F.; Daniels, R., Natural Products from isnA-Containing Biosynthetic Gene Clusters Recovered from the Genomes of

Cultured and Uncultured Bacteria. J. Am. Chem. Soc. 2007, 129 (40), 12102-12103.

8. (a) Sievers, F.; Wilm, A.; Dineen, D.; Gibson, T. J.; Karplus, K.; Li, W.; Lopez, R.;

McWilliam, H.; Remmert, M.; Söding, J.; Thompson, J. D.; Higgins, D. G., Fast, scalable generation of high‐quality protein multiple sequence alignments using Clustal Omega.

Mol. Syst. Biol. 2011, 7 (1); (b) Goujon, M.; McWilliam, H.; Li, W.; Valentin, F.;

Squizzato, S.; Paern, J.; Lopez, R., A new bioinformatics analysis tools framework at

EMBL-EBI. Nucleic Acids Res. 2010, 38 (Web Server issue), W695-9.

9. Lang, W.; Bunn, B. M.; Viswanathan, R., Isonitrile Synthases (Wwi-IsnA and

Wwi-IsnB) of Terrestrial Cyanobacteria: Mechanistic Insights from Homology-Based

Three Dimensional Structures. Intersections: SOURCE Symposium and Poster Session,

Case Western Reserve University. 2012.

10. Kelley, L. A.; Sternberg, M. J. E., Protein structure prediction on the Web: a case study using the Phyre server. Nat. Protocols 2009, 4 (3), 363-371.

11. Hausinger, R. P., FeII/alpha-ketoglutarate-dependent hydroxylases and related enzymes. Crit. Rev. Biochem. Mol. Biol. 2004, 39 (1), 21-68.

12. Hillwig, M. L.; Liu, X., A new family of iron-dependent halogenases acts on freestanding substrates. Nat. Chem. Biol. 2014, 10 (11), 921-923.

13. Rippka, R.; Deruelles, J.; Waterbury, J. B.; Herdman, M.; Stanier, R. Y., Generic

Assignments, Strain Histories and Properties of Pure Cultures of Cyanobacteria. J. Gen.

Microbiol. 1979, 111, 1-61.

14. Morin, N.; Vallaeys, T.; Hendrickx, L.; Natalie, L.; Wilmotte, A., An efficient DNA isolation protocol for filamentous cyanobacteria of the genus Arthrospira. J. Microbiol.

Meth. 2010, 80 (2), 148-154.

15. Wilson, K., Preparation of Genomic DNA from Bacteria. In Current Protocols in

Molecular Biology, John Wiley & Sons, Inc.: 2001.

16. Ausubel, F. B., R.; Kingston, R.; Moore, D.; Seidman, J.; Smith, J.; Struhl, K., Short protocols in molecular biology. third ed.; John Wiley & Sons, New York. : 1996; Vol. 24, p 68-68.

17. Mustafa, E. Ambigols A-C and tjipanazole D: bioinformatic analysis of their putative biosynthetic gene clusters. University of Bonn, Institute for Pharmaceutical

Biology, 2011.

18. McWilliam, H.; Li, W.; Uludag, M.; Squizzato, S.; Park, Y. M.; Buso, N.; Cowley,

A. P.; Lopez, R., Analysis Tool Web Services from the EMBL-EBI. Nucleic Acids Res.

2013, 41 (Web Server issue), W597-W600.

19. The PyMOL Molecular Graphics System, Version 1.5.0.4 Schrödinger, LLC.

Chapter 3: Substrate Tolerance of Isonitrile Synthase and Fe(II)--Ketoglutarate Dependent Dioxygenase affords Unnatural Variants of Cyanobacterial Hapalindole Pathway Intermediate

The results and investigation described in this chapter are currently under review as the following citation:

Brittney M. Bunn, Kathryn J. Howard, Jonathan A. Karty and Rajesh Viswanathan*

“Substrate Tolerance of Isonitrile Synthase and Dioxygenase affords Sustainable Synthesis of Rare Isonitrile Antibiotics from Cyanobacterial Hapalindole Pathway” RSC Chemical

Science, Under Review, Submitted: October 28, 2015.

*Indicates corresponding author.

3.1 Introduction

3.1.1 Assessing the Substrate Promiscuity of WelI1 and WelI3

Isocyanide (or isonitrile) containing natural products1 and their intriguing biosynthetic pathways2 continue to inspire interest. Isonitriles have shown promise towards imaging3 and the rich chemical repertoire of these unique natural products continues to expand.4 The exceptional molecular architectures of the isonitrile/isothiocyanate- containing family of hapalindole-type alkaloids were discussed in Section 1.4 and biosynthetic investigations on this family were discussed in Section 1.5. Chapter 2 described the in vitro, cell-lysate based reconstitution of WelI1 and WelI3, leading to the characterization of an isonitrile synthase and an Fe(II)-α-ketoglutarate dependent oxygenase, respectively, from Westiella intricata UH strain HT-29-1. The investigation described in Chapter 2 complemented additional studies on hapalindole-type alkaloid biosynthesis, explained in Section 2.3.

The feasibility of generating hapalindole-type analogs by probing substrate promiscuity of heterologously expressed biocatalysts was established in previous investigations5 by the Liu group. The substrate tolerance of WelM,5a a SAM-dependent methyltransferase, and WelO5,5b an Fe2+/α-ketoglutarate-dependent halogenase, from the wel pathway from H. welwitchii had been assessed. WelM only accepted one of the six substrates tested; however, WelO5 displayed slight promiscuity by successfully chlorinating two different substrates.

In contrast to the enzymes discussed above that are involved in later stages of the biosynthetic pathway, the initial biocatalytic events within the hapalindole-type alkaloid

74 biosynthetic pathway had not been investigated (in regard to substrate versatility). WelI1-

3 (alongside their Hpi and Amb counterparts) are the only known isonitrile synthases reported in hapalindole-producing cyanobacteria, and are responsible for the production of indole-isonitrile. Indole-isonitrile possesses bioactivity itself (antibacterial)6; but more importantly, is the first stable intermediate in the biosynthetic pathway leading to the family of hapalindole-type alkaloids.

To date, there are no reports on explorations of substrate tolerance on any of the isonitrile synthase family of enzymes. Therefore, the premise of the investigation described in this chapter was to explore the possible production of novel hapalindole natural product analogs with potentially exciting therapeutic applications by exploiting the initial biocatalysts of the pathway. This chapter describes the assessment of the substrate promiscuity of WelI1 and WelI3 through heterologous expression and purification, followed by enzymatic assays with seven distinct tryptophan derivatives. Through bioinformatic alignment and substrate docking, a rationale for the substrate specificity of

WelI3 will be provided.

Chapter 1 (Section 1.6) previously highlighted the advantages that synthetic biology offers to the production of novel natural products when compared to traditional synthetic methodologies. Figure 3.1A-B illustrates the advantages of biocatalytic conversions by comparison over existing synthesis methods used for constructing indole isonitriles such as 3.3.

Figure 3.1 A) Existing synthetic methodology for production of hapalindole biosynthesis intermediate, indole isonitrile (3.3); B) This work employs sequential biocatalysis of TmTrpB1 and WelI1/3 in order to produce indole-isonitrile (cis and trans-3.3), which could be carried through the reminder of the biosynthetic pathway to produce novel hapalindole-type alkaloids.

3.1.2 Tri-Catalytic Biosynthetic Methodology to Access Novel Natural Product Analogs

To establish feasibility toward a successive assembly of these alkaloids, the two- enzyme-single-assay protocol (with WelI1-WelI3) was employed with a third biocatalyst,

TmTrpB1, a tryptophan synthase from the hyperthermophilic Thermotoga maritima.7

Milligram quantities of commercially available tryptophan derivatives can be quite costly. In addition, the prospect of a green methodology utilizing three biocatalysts to produce hapalindole-type analogs from indole derivatives (much less expensive compared to tryptophan derivatives), sparked our interest. In the presence of indole (3.6),

L-serine (3.7), and pyridoxal 5'-phosphate (PLP), TmTrpB1 catalyzes the formation of a tryptophan derivative8 (3.8), illustrated in Figure 3.2. In this chapter, an investigation is described which utilized all three biocatalysts to produce a novel indole isonitrile natural product analog.

Figure 3.2 Reaction catalyzed by TmTrpB1

3.2 Results and Discussion

3.2.1 Heterologous Expression and Purification of WelI1 and WelI3

Considering this was the first evaluation of the substrate promiscuity of these enzymes, a purified enzymatic system was necessary. Therefore, WelI1 and WelI3 were heterologously expressed as previously described in Chapter 29 and were purified to homogeneity, Figure 3.3.

Figure 3.3 A) SDS-PAGE analysis of fractions from the Ni-NTA purification of 6x-His tagged WelI1. B) SDS-PAGE analysis of fractions from the Ni-NTA purification of 6x-His tagged WelI3.

Once pure WelI1 and WelI3 were in hand, Figure 3.4, a tandem enzymatic assay was set up based on previous experiments with L- tryptophan as the natural substrate.9-10

Figure 3.4 SDS-PAGE analysis of purified WelI1 and WelI3.

3.2.2 Heterologous Expression, Purification, and Enzymatic Assay of TmTrpB1

In this investigation, seven distinct L-tryptophan derivatives were tested as possible alternatives to the natural substrate for the WelI1/3 system. Of the seven tryptophan derivatives analyzed, six were obtained from commercial sources. The seventh derivative,

2-methyl-L-tryptophan, was biosynthesized using the tryptophan synthase TmTrpB1.

Therefore, E. coli BL21-(DE3)-RIPL cells containing pET28a-TmTrpB1 were cultured and induced to overexpress TmTrpB1, which was subsequently purified by Ni-NTA chromatography, Figure 3.5.

Figure 3.5 SDS-PAGE analysis of purified TmTrpB1

An assay was set up containing L-serine (3.7), PLP, TmTrpB1, and 2-methyl indole.

The reaction mixture was incubated at 80 °C for approximately 48 hours. The reaction was monitored by TLC and the product was purified by silica gel flash chromatography. The production of 2-methyl-L-tryptophan was confirmed by 1H and 13C NMR spectra

(Appendix 5, Figure A5.1 and Figure A5.2).

3.2.3 Assessment of Substrate Promiscuity of WelI1 and WelI3

2 1 R R 3.6 3.8 3.9 3.10 3.11 cis 3.3 trans 3.3

Figure 3.6 Sequential biocatalysis involving three enzymatic steps utilized in this investigation Assay components included the enzymes (21-28 µM WelI1 and 11-19 µM WelI3, based on A280 data and extinction coefficients predicted using ExPaSy’s Protparam feature) in addition to 2 mg of L-tryptophan or tryptophan derivative (3.8) (2-methyl-L tryptophan was biosynthesized as described above, all other tryptophan derivatives were purchased from commercial sources). The assay buffer consisted of 2.5 mM ribose-5-phosphate (3.9),

250 µM α-ketoglutaric acid (3.11), 100 µM (NH4)2Fe(SO4)2), and 5% (v/v) glycerol.

Figure 3.6 displays the biocatalytic sequence employed in this investigation, including the use of TmTrpB1 for generation of an L-tryptophan derivative, 3.8. After incubation at 37

°C for 18 hours, the assays were extracted with ethyl acetate (v/v: 1:1) and analog production was verified by HPLC, LC-HRMS, and confirmed through MS/MS fingerprinting of the LC-derived data. Note that the temperature of this assay was based on the initial conditions of the investigations described in Chapter 2.

For each of the assays, HPLC analysis of the organic extract showed successful production of the expected indole-isonitrile product with relatively high percent yields (See

Figure 3.7 for the LC trace of the major product within each spectrum, Appendix 6.1 for full spectra of each assay analysis, and Table 3.1 for percent yields). The extracts were

81 also analyzed by HRMS and the results are also displayed in Figure 3.7 (see Appendix

6.2 for full LC and HRMS spectra).

Figure 3.7 List of substrate analogs (3.8a-h) incorporated into the Wel-I1-3 catalysis, resulting in isonitriles 3.3 a-h. HRMS results and cis:trans analysis are also displayed.

HPLC analysis indicated the presence of both cis and trans isomers of the natural product analogs, and their ratios were quantified (Figure 3.7). In the case of 3.8a, the results with purified WelI1-I3 reflected production of cis-3.3a and was therefore in agreement with results published by the Liu group.5a However, this result contrasts with results discussed in Chapter 2,9 which showed that the use of a lysate system generated a mixture of cis and trans isomers of 3.3. The exact reason causing this inconsistency remains to be established. A plausible explanation is that impurities (of unknown identity) present in the cell lysate, compared to the purified protein solution, could cause this discrepancy.

The use of synthesized standards (Appendix 6.1, Figure A6.1) to establish structural confirmation for assay products from 3.8a was conducted in a similar manner to the investigation described in Chapter 2.

Interestingly, tryptophans with the substituents 5-methoxy (3.8g) and 2-methyl (3.8b) produced a >10:1 ratio favoring the cis isonitrile (3.3) upon turnover by WelI1-3. All other

L-trp derivatives (3.8c, d, e, f and h) produced isonitrile analogs (3.3c, d, e, f and h) with less favorability for the cis isomer (Figure 3.7), typically in approximately a 5:1 ratio, or even an equimolar mixture as in 3.3e and 3.3h. Intriguingly, the L-trp-derived cis indole isonitrile (3.3a) was identified in the HPLC spectra of assays that were run on derivatives

3.8b-h (as evidenced in Appendix 6.1, Figure A6.1). This is attributed to plausible impurities present within the substrate sources.

Elemental composition of major products and MS2 fragmentation patterns (Table 3.1) further supported the production of seven analogs of indole-isonitrile (3.3b-h). Full LC traces and MS fragmentation patterns are provided in Appendix 6.2.

Table 3.1 List of LC-MS retention times, molecular formulas, percent yields, and MS2 fragmentation patterns for products (3.3a-h).

Tryptophan/ LC-MS Molecular Elemental % MS2 fragmentation derivative Retention formula of Composition yield pattern (Substrate) time (min) expected [M + H]+ (Product) product

L-Trp (3.8a) 3.83 C11H8N2 C11H9N2 85% 169.0766, 142.0653, 116.0538

2-methyl L- 5.17 C12H11N2 C12H11N2 90% 183.0830, 168.0682, Trp (3.8b) 154.0651

1-methyl L- 4.00 C12H10N2 C12H11N2 90% 183.0907, 186.0670, Trp (3.8c) 154.0677, 141.0577, 140.0499

4-fluoro L-Trp 5.23 C11H7N2F C11H8N2F 85% 187.0668, 158.0034 (3.8d)

5-methyl L- 4.45 C12H10N2 C12H11N2 75% 183.0906, 168.0661, Trp (3.8e) 154.0614, 141.0589, 140.0453

6-methyl L- 4.40 C12H10N2 C12H11N2 85% 183.0922, 168.0697, Trp (3.8f) 154.0667, 141.0595

5-methoxy L- 3.95 C12H10N2O C12H11N2O 65% 199.0845, 184.0613, Trp (3.8g) 156.0665, 130.0581

5-hydroxy L- 4.31 C11H8N2O C11H9N2O 60% 184.0630, 167.0607 Trp (3.8h)

3.2.4 Homology-based Model Building and in silico Docking

Based on these results, WelI1 and WelI3 display a broad substrate selectivity, acting on a range of L-trp derivatives substituted on the indole ring. Homology-based model building and in silico docking were employed in order to rationalize this broad versatility, given that neither WelI1 nor WelI3 have been crystallized.

As discussed in Chapter 2 the structure of paercumarin, an isn-derived metabolite, was identified in Pseudomonas aeruginosa and the mechanistically related IsnA/B homologs,

PaPvcA and PaPvcB, were biochemically characterized.11 Originally, the three dimensional structures of PaPvcA and PaPvcB were reported by Drake and Gulick.12

Recent mechanistic investigations and an improved crystallographic structure of PaPvcB

(to 2.1 Å), led to the discovery of additional homologs (XnPvcB and EaPvcB) with divergent biochemical pathways to distinct products.13

WelI3 shares significant active site similarity to PaPvcB (Appendix 7.1); therefore,

PaPvcB’s 2.1 Å structure was used to reconstruct a homology model of WelI3, along with clavaminate synthase14 as a model for extracting coordinates of Fe(II), as done previously by Gulick.13 The model built for WelI3 significantly superimposes with the structure of

PaPvcB with a Phyre TM-score of 0.0 (Appendix 7.1). This information, combined with the pairwise multiple sequence alignment, Figure 3.8, offered a higher degree of confidence in predicting molecular level interactions.

Using the homology-based model of WelI3, the L-tryptophan isonitrile intermediate

(3.10) was docked within the active site (Figure 3.9) in order to explain the flexible substrate specificity observed in this investigation. Residues that help define substrate binding and channeling, as defined for PaPvcB, were highly conserved in WelI3; these

Figure 3.8 Pairwise Clustal alignment between biochemically characterized PaPvcB and WelI3.

WelI3 PaPvcB

B C

Figure 3.9 A) Image of WelI3 docked with L-tryptophan isonitrile (3.10). WelI3’s active site components are shown as sticks, the docked substrate is shown in magenta, and the Fe(II) is shown in orange. PaPvcB’s hydrophobic pocket residues (Met114-Tyr115-Leu-116) are shown for comparison to WelI3’s smaller triad Ala114-Phe115-Ala116. B) Side view of WelI3 docked with L-tryptophan isonitrile (3.10). C) Top view of WelI3 docked with L-tryptophan isonitrile (3.10).

included: Trp83, Arg274, Leu89, Leu91, and Arg270. Additionally, His110, His259 and

Asp112, which bind to Fe(II) in PaPvcB, are conserved in WelI3. A noticeable difference between PaPvcB and WelI3, based on the Clustal alignments, is that PaPvcB possesses a hydrophobic pocket (Met114-Tyr115-Leu-116), which is absent in WelI3. Instead,

WelI3 has a smaller pocket filled with a Ala114-Phe115-Ala116 triad. The hydrophobic pocket resides of PaPvcB and the smaller triad of WelI3 are displayed in Figure 3.9.

The results derived from bioinformatic alignment and docking suggest the expanded substrate profile displayed by WelI3 could be attributed to three relatively small residues

(Ala-Phe-Ala compared to Met-Tyr-Leu) opening up the active site for indole-based bulkier groups. Furthermore, results discussed within this section suggest the mechanism of WelI3 could closely resemble those observed for Pvc homologs (PaPvcB, for example). Based on this reasoning, the pathway to formation of cis and trans isomers of indole isonitrile (3.3) by WelI1/I3 is proposed to be caused by constraints of stereospecifity imposed by WelI3 during the decarboxylative/elimination step (indicated by elimination of the-OH group) as represented in Scheme 3.1. In the presence of ribose-5-phosphate (3.9) (or its tautomer, ribulose-5-phosphate) WelI1 catalyzes the incorporation of the isonitrile carbon onto the

L-trp (3.8) N-terminus, resulting in a (relatively unstable) tryptophan-isonitrile intermediate (3.10). This intermediate is then presumably hydroxylated in situ at the

Cposition by WelI3 and simultaneously decarboxylated and desaturated, resulting in indole-isonitrile products (3.3).

cis 3.3

3.10 trans 3.3 B

3.12 3.13

Scheme 3.1 A) Possible mechanism of WelI3 based on its analogy to PaPvcB. B) PaPvcB mechanism.

3.3 Conclusions Natural products biogenetically arising from L-trp derived metabolic pathways continue to spur novel concepts related to enzymatic mechanism.15 In that regard, the hapalindole pathway has offered a suite of mechanistically novel enzymes, many of which are Rieske-type non-heme iron-dependent dioxygenases.16 The field of catalysis continues to generate interest in the novel mechanisms of these NHI oxygenases.17 In this chapter, biosynthetic potential of enzymes that encode for this family of natural products was harnessed by probing their relative substrate tolerance. Specifically, the substrate promiscuity of WelI1 (an isonitrile synthase) and WelI3 (an Fe(II)--ketoglutarate- dependent dioxygenase) encoded by the wel pathway in W. intricata was explored through a direct, one-step tandem enzymatic assay. Seven analogs of the product of these biocatalysts were produced, including one analog constructed through an additional biocatalytic event with the use of TmTrpB1 to yield the WelI1/I3 substrate, 2-methyl-L- tryptophan. A rationale for the flexible substrate selectivity of WelI3 was provided and a plausible mechanism for WelI3 was hypothesized, both based on homologous enzymes within the literature. However, the fact that both WelI1 and WelI3 allow a broader substrate diversity opens doors for mechanistic investigations in the future.

This investigation complements evidence towards the successful biocatalytic production of hapalindole-type analogs. Additionally, the results discussed here provide the first support towards utilizing a tri-catalytic biosynthetic methodology to produce such analogs in a more environmentally friendly, cost-effective, time efficient manner. Future directions of this work are discussed in Chapter 4.

3.4 Experimental Section

3.4.1 WelI1 and WelI3

3.4.1.1 Vector Selection9

The gene of interest was submitted to DNA2.0 and optimized for expression in E. coli. A 6x-His tag was included at the C-terminus for purification purposes preceded by a factor Xa protease recognition site. The RBS was customized to contain an NdeI restriction site and an XhoI restriction site was included after the stop codon for cloning purposes.

3.4.1.2 Transformation

Approximately 100 ng of plasmid was added to an aliquot of BL21(DE3) cells

(New England BioLabs) and incubated on ice for 30 minutes. The cells were submitted to heat shock at 42 °C for 30 seconds, then placed immediately on ice for at least 2 minutes.

Addition of S.O.C. media was followed by incubation at 37 °C for 45 minutes with shaking at 180 rpm. The cells were plated on an agar plate supplemented with 50 µg/mL kanamycin and incubated at 37 °C overnight.

3.4.1.3 Culturing and Induction9

A 50% (v/v) glycerol stock of BL21(DE3) transformed with the gene of interest was used to inoculate a 25 mL flask containing LB broth supplemented with 50 µg/mL kanamycin. The flask was incubated at 37 °C with shaking at 180 rpm for 6-8 hours. This culture was added to a 2 L flask of LB broth supplemented with 50 µg/mL kanamycin and incubated at 37 °C until an OD600 of approximately 0.6 was reached. The cells were then induced with 1.0 mM IPTG and grown at 16 °C overnight.

3.4.1.4 Lysis9

After the induction period, the cells were centrifuged at 6,084 x g for 10 minutes and frozen at -20 °C. The cell pellet was thawed on ice and resuspended in approximately

30 mL of 50 mM Tris buffer (pH 7.5) containing a cocktail of protease inhibitors (Sigma

Aldrich, SIGMAFAST™ protease inhibitor cocktail tablets, EDTA-free for use in purification of histidine-tagged proteins), 0.2 mM TCEP, 250 mM NaCl, and 10% v/v glycerol. Lysozyme was added to a final concentration of 1 mg/mL and stirred until a viscous suspension was obtained. The sample was sonicated (Fisher Scientific™ Model

120 Sonic Dismembrator, power: 120 W, frequency: 20 kHz, amplitude: 40%, probe: model CL-18) (30 second pulse/30 second pause) for 6 minutes and the cellular debris was removed by centrifugation at 57,000 x g for 1 hour at 4 °C.

3.4.1.5 Purification

The lysis procedure was followed as described above. Once the cellular debris was removed from the sonicate, imidazole was added to a final concentration of 10 mM. The sample was incubated with approximately 10 mL of Ni-NTA resin (Qiagen) at 4 °C for 1 hour. The Ni-NTA resin/sample mixture was then poured into a column, which was washed with 50 mM Tris buffer (pH 7.5) containing 25 mM imidazole, 0.2 mM TCEP, 250 mM NaCl, and 10% v/v glycerol. The column was submitted to a step gradient of imidazole (50 mM-500mM) and eluent was collected in fractions. The fractions were analyzed by SDS-PAGE for the presence of the desired protein. Buffer composition of peak protein fractions was changed during concentration (Millipore centrifugal concentrators). The final buffer consisted of 50 mM Tris (pH 7.5), 0.2 mM TCEP, 250 mM NaCl, and 10% v/v glycerol.

3.4.2 Enzymatic Assay with Purified WelI1 and WelI3 3.4.2.1 General Procedure:

Assay components were mixed to various final reaction volumes, depending on the volume of protein solution added to the reaction, Table 3.2. The assay buffer contained

25 mM Tris (pH 7.5), 125 mM NaCl, 0.1 mM TCEP, 2.5 mM ribose-5-phosphate, 250 µM

α-ketoglutaric acid disodium salt dihydrate, 100 µM (NH4)2Fe(SO4)2), and 5% (v/v) glycerol. Individual assays contained between 21 to 28 µM WelI1 and between 11 to 19

µM WelI3 (Table 3.2) in addition to 2 mg of L-tryptophan or tryptophan derivative (2- methyl-L-trp was biosynthesized using TmTrpB1 and all others were commercially available). Once all components were mixed to homogeneity, assays were incubated at 37

°C for 18 hours. After incubation, assay mixtures were extracted with ethyl acetate for

HPLC, LC-HRMS, and MS/MS analysis.

Table 3.2 Total volume, protein volume, and protein concentration data for assays with purified WelI1 and WelI3.

Total volume Total Total volume of WelI3 in Tryptophan volume of of WelI1 in Final [WelI3] Final [WelI1] assay (mL) derivative assay (mL) assay (mL) in assay in assay L-trp 2.25 0.750 27.58 0.375 11.46 6-methyl-L-trp 2.25 0.625 23.36 0.500 14.11 5-methyl-L-trp 3.00 0.750 21.19 0.750 18.19 2-methyl-L-trp 3.00 0.750 25.12 0.750 18.53 5-hydroxy-L-trp 2.25 0.750 27.58 0.375 11.46 1-methyl-L-trp 2.25 0.625 23.36 0.500 14.11 5-methoxy-L- trp 3.00 0.750 21.19 0.750 18.19 4-fluoro-L-trp 3.00 0.750 25.12 0.750 18.53

3.4.2.2 HPLC Analysis of Enzymatic Assay Extracts

HPLC analyses were performed using a Shimadzu LC-20-AT Series separations module equipped with Shimadzu SPD-M20A with UV dual channel wavelength detector

(set at 254 and 310 nm). The overall system, CBM-20 was controlled using LC Solutions software. Raw data was plotted using Origin® software program after exporting absorbance data as an ASCII-formatted file from LC Solutions. Controls (without enzymes, and without substrates) were performed for each assay condition and UV absorbance contributed from enzyme cofactors and essential media components were subtracted from initially obtained absorbance/time profiles. Once processed, the resulting plots were interpreted for substrate and product signals respectively. Area under the peak calculations for ratios of product isomers were determined using Origin software’s recommended peak integration protocol. Analytical separations of mixtures were carried out on Daicel®

(normal phase) AS chiral column using a 35% iPrOH:65% hexanes and were run over 25 min with a flow rate of 0.5 mL/min in an isocratic mode. Individual retention times for indole-isonitriles are reported along with analytical data for each isomer.

3.4.2.3 LC-HRMSMS AnalysisA

The small molecule metabolites were extracted by adding 100 µL of EtOAc to 100

µL of cell lysate and vortexing the emulsion for 2 minutes. The emulsion was centrifuged for 2 minutes at 600 x g and 3 layers were observed (a lower clear layer, a middle brown layer and an upper clear layer). Compounds 3.3a-h were analyzed as follows: 20 µL of the top layer were removed and analyzed by RP HPLC-ESI-QTOF MS/MS on a Waters

Acuity/Synapt G2 instrument. Five microliters of the extract was injected onto a Waters

2.1x100 mm column packed with 1.8 µm BEH C18 particles. Mobile phase A was 0.1% v/v formic acid in water; mobile phase B was 0.1% v/v formic acid in acetonitrile, and the gradient profile was hold 100% A for 0.5 minutes, linear ramp to 90% B at 12.00 minutes, hold 90% B for 3 minutes, ramp down to 100%A at 15.5 minutes and hold 100% A for 4.5 minutes. The mass spectrometer was operated in positive ESI, sensitivity mode and the data dependent MS/MS survey parameters were: record from m/z 150-650 at 2 Hz with

MS/MS recorded on the top 3 ions exceeding 20000 counts per second in any spectrum.

The masses of protonated molecular ions for compounds 3.3a-h were put on a preferential inclusion list (i.e. if those masses were observed above 2e4 cps, record their MS/MS even if they were not in the top 3 ions). The trap voltage (collision energy) was ramped from

20-50 V with 7.6 x 10-3 mbar of Ar in the collision cell; MS/MS spectra were recorded from m/z 50-650. Compounds 3.3d and 3.3e were not retained very well on the C18 column using the method above. The ethyl acetate was removed from these samples by taking 20 µL of the top layer of the extraction and evaporating it to dryness under a stream

A Data acquisition performed by Jonathan A. Karty

95 of nitrogen and reconstituting the sample in 20 µL of methanol prior to LC-HRMSMS analysis as described above.

3.4.3 TmTrpB1

Recombinant tryptophan synthase from the hyperthermophilic bacterium

Thermotoga maritima has been overexpressed and purified previously, as described in the literature.7-8 The following sections describe culturing of E. coli BL21-DE3-RIPL, overexpression of TmTrpB1, and lysis of the E. coli cells based on those initial investigations.

3.4.3.1 Culturing and Induction

E. coli BL21-DE3-RIPL containing pET28a-TmTrpB1B was cultivated in 6 L LB containing 35 μg mL-1 kanamycin and 35 μg mL-1 chloramphenicol at 37 °C with shaking at 180 rpm to an OD600 of approximately 0.6. The culture was cooled to 20 °C and overexpression of TmTrpB1 was induced with a final concentration of 0.5 mM IPTG.

3.4.3.2 Lysis

After an induction period of 18 hours, the cells were harvested by centrifugation

(2,700 x g, 15 min, 4 °C). Pelleted cells were resuspended in approximately 35 mL lysis buffer (100 mM K2HPO4, 300 mM KCl, 10 mM imidazole, 40 μM PLP, pH 7.5) containing a cocktail of protease inhibitors (Sigma Aldrich, SIGMAFAST™ protease inhibitor cocktail tablets, EDTA-free for use in purification of histidine-tagged proteins) and sonicated (Fisher Scientific) for 6 x (30 second pulse, 30 second pause) at 4 °C. The cell

B E. coli BL21-DE3-RIPL containing pET28a-TmTrpB1 was graciously provided by the Poulter8 group with permission from the Sterner7 group.

96 lysate was clarified by centrifugation (23, 400 x g, 15 min, 4 °C). The supernatant resulting from the centrifugation was heated to 55 °C for 25 min and centrifuged again (23, 400 x g,

15 min, 4 °C).

3.4.3.3 Purification The lysis procedure was followed as described above. Once the cellular debris was removed from the sonicate, imidazole was added to a final concentration of 10 mM.

The sample was incubated with approximately 10 mL of Ni-NTA resin (Qiagen) at 4 °C for 1 hour. The Ni-NTA resin/sample mixture was then poured into a column.

Purification of 6x-His tagged TmTrpB1 was achieved using elution buffer (100 mM

K2HPO4, 300 mM KCl, pH 7.5) with a step gradient of imidazole (25 mM, 50 mM, 100 mM, 250 mM, 500 mM, and 1 M). Eluted column fractions were analyzed for protein content of the appropriate molecular weight by SDS-PAGE analysis. Fractions containing the desired protein (according to molecular weight) were pooled and dialyzed against storage buffer (100 mM K2HPO4, pH 7.5). The protein was portioned into 1 mL aliquots, flash frozen under liquid-nitrogen, and stored at -80 °C.

3.4.3.4 Enzymatic Assay with Purified TmTrpB1

The assay with TmTrpB1 was carried out as previously described8 with minor modifications, including amount of enzyme, buffer composition (this protocol did not utilize DMSO) and reaction time. In addition, the enzyme was not removed from the reaction mixture prior to silica gel flash chromatography in this method. 2-methylindole

(1.20 mmol) and L-serine (2.38 mmol) were added to 25 mL of assay buffer (100 mM

K2HPO4, pH 7.5, 180 mM KCl, 120 µM PLP) containing approximately 5 mg of purified

TmTrpB1. The reaction was covered in a blanket of nitrogen, sealed, and incubated at 80

°C for approximately 48 hours. Progress of the reaction was monitored by TLC analysis using 20% H2O in acetonitrile. The reaction was cooled to room temperature and purified by silica gel flash chromatography using a gradient of 0-20% H2O in acetonitrile. Identity of the purified product was confirmed by 1H and 13C NMR.

3.5 References

1. Scheuer, P. J., Isocyanides and cyanides as natural products. Acc. Chem. Res. 1992,

25 (10), 433-439.

2. Simpson, J. S.; Garson, M. J., Biosynthetic pathways to isocyanides and isothiocyanates; precursor incorporation studies on terpene metabolites in the tropical marine sponges Amphimedon terpenensis and Axinyssa n.sp. Org. Biomol. Chem. 2004, 2

(6), 939-948.

3. Wainman, Y. A.; Neves, A. A.; Stairs, S.; Stockmann, H.; Ireland-Zecchini, H.;

Brindle, K. M.; Leeper, F. J., Dual-sugar imaging using isonitrile and azido-based click chemistries. Org. Biomol. Chem. 2013, 11 (42), 7297-7300.

4. Wilson, R. M.; Stockdill, J. L.; Wu, X.; Li, X.; Vadola, P. A.; Park, P. K.; Wang,

P.; Danishefsky, S. J., A Fascinating Journey into History: Exploration of the World of

Isonitriles En Route to Complex Amides. Angewandte Chemie International Edition 2012,

51 (12), 2834-2848.

5. (a) Hillwig, M. L.; Fuhrman, H. A.; Ittiamornkul, K.; Sevco, T. J.; Kwak, D. H.;

Liu, X., Identification and Characterization of a Welwitindolinone Alkaloid Biosynthetic

Gene Cluster in the Stigonematalean Cyanobacterium Hapalosiphon welwitschii.

ChemBioChem 2014, 15 (5), 665-669; (b) Hillwig, M. L.; Liu, X., A new family of iron- dependent halogenases acts on freestanding substrates. Nat. Chem. Biol. 2014, 10 (11),

921-923.

6. Hoppe, I.; Schöllkopf, U., Synthesis and Biological Activities of the Antibiotic B

371 and its Analogs. Liebigs Annalen der Chemie 1984, 1984 (3), 600-607.

7. Hettwer, S.; Sterner, R., A novel tryptophan synthase beta-subunit from the hyperthermophile Thermotoga maritima. Quaternary structure, steady-state kinetics, and putative physiological role. J. Biol. Chem. 2002, 277 (10), 8194-201.

8. Rudolf, J. D.; Wang, H.; Poulter, C. D., Multisite Prenylation of 4-Substituted

Tryptophans by Dimethylallyltryptophan Synthase. J. Am. Chem. Soc. 2013, 135 (5), 1895-

1902.

9. Micallef, M. L.; Sharma, D.; Bunn, B. M.; Gerwick, L.; Viswanathan, R.; Moffitt,

M. C., Comparative analysis of hapalindole, ambiguine and welwitindolinone gene clusters and reconstitution of indole-isonitrile biosynthesis from cyanobacteria. BMC Microbiol.

2014, 14 (213), 1-18.

10. Hillwig, M. L.; Zhu, Q.; Liu, X., Biosynthesis of Ambiguine Indole Alkaloids in

Cyanobacterium Fischerella ambigua. ACS Chem. Biol. 2014, 9 (2), 372-377.

11. Clarke-Pearson, M. F.; Brady, S. F., Paerucumarin, a new metabolite produced by the pvc gene cluster from Pseudomonas aeruginosa. J. Bacteriol. 2008, 190 (20), 6927-30.

12. (a) Drake, E. J.; Gulick, A. M., Three-dimensional Structures of Pseudomonas aeruginosa PvcA and PvcB, Two Proteins Involved in the Synthesis of 2-Isocyano-6,7- dihydroxycoumarin. J. Mol. Biol. 2008, 384 (1), 193-205; (b) Gulick, A. M.; Drake, E. J.,

3E59: Crystal structure of the PvcA (PA2254) protein from Pseudomonas aeruginosa.

Worldwide Protein Data Bank, 2008; (c) Gulick, A. M.; Drake, E. J., 3EAT: Crystal structure of the PvcB (PA2255) protein from Pseudomonas aeruginosa. Worldwide

Protein Data Bank, 2008.

100

13. Zhu, J.; Lippa, G. M.; Gulick, A. M.; Tipton, P. A., Examining Reaction Specificity in PvcB, a Source of Diversity in Isonitrile-Containing Natural Products. Biochemistry

2015, 54 (16), 2659-2669.

14. Zhang, Z.; Ren, J.; Stammers, D. K.; Baldwin, J. E.; Harlos, K.; Schofield, C. J.,

Structural origins of the selectivity of the trifunctional oxygenase clavaminic acid synthase.

Nat. Struct. Biol. 2000, 7 (2), 127-133.

15. Alkhalaf, Lona M.; Ryan, Katherine S., Biosynthetic Manipulation of Tryptophan in Bacteria: Pathways and Mechanisms. Chem. Biol. 2015, 22 (3), 317-328.

16. Barry, S. M.; Challis, G. L., Mechanism and Catalytic Diversity of Rieske Non-

Heme Iron-Dependent Oxygenases. ACS Catalysis 2013, 3 (10), 2362-2370.

17. Krebs, C.; Galonić Fujimori, D.; Walsh, C. T.; Bollinger, J. M., Non-Heme Fe(IV)–

Oxo Intermediates. Acc. Chem. Res. 2007, 40 (7), 484-492.

101

Chapter 4: Thesis Summary and Future Directions

4.1 Thesis Summary

4.1.1 Part I: Heterologous Expression and in vitro Reconstitution of Isonitrile Synthase (WelI1) and Fe(II)-α-Ketoglutarate Dependent Oxygenase (WelI3)

Cyanobacteria have proven to be a significant source of bioactive compounds1 with potential therapeutic applications.2 Connecting genes to molecules3 is an approach used in today’s genome driven biosynthetic investigations to access unique structural scaffolds and new biocatalysts. Gene clusters have been associated with several cyanobacterial natural products;4 however, the biosynthesis of the antibacterial,5 anticancer,6 anti-MDR,7 isonitrile/isothiocyanate containing hapalindole-type alkaloids remained underexplored until more recently.

Chapter 2 described the synthetic biology approach utilized to characterize two enzymes involved in hapalindole-type alkaloid biosynthesis,8 Figure 4.1. A putative biosynthetic gene cluster was identified to be involved in welwitindolinone (wel)

trans indole-isonitrile cis 4.5 trans 4.5 4. 4.3

4.2 4.4

Figure 4.1 Biosynthetic route to the first stable intermediate en route to the hapalindole-type alkaloids, indole isonitrile (4.5), catalyzed by WelI1 and WelI3 from the wel cluster of W. intricata (HT-29-1). WelI1 utilizes L-tryptophan (4.1) and ribose-5-phosphate (4.2) to yield an unstable intermediate (4.3). This intermediate (4.3) is then oxidatively decarboxylated by WelI3, an oxygenase that requires Fe(II) and α-ketoglutarate.

102 biosynthesis in W. intricata (HT-29-1) and two genes, welI1 and welI3, were hypothesized to encode for biocatalysts responsible for producing the first stable intermediate towards the hapalindole-type alkaloids, indole isonitrile (4.5). Bioinformatic analysis revealed homology of WelI1 and WelI3 to an isonitrile synthase and an oxidative decarboxylase, respectively. The two proteins were heterologously expressed and utilized in assays as E. coli cell lysates. Reconstitution of the biosynthetic steps catalyzed by WelI1 and WelI3 led to the production of indole isonitrile; which confirmed the roles of WelI1 and WelI3, an isonitrile synthase and an Fe2+/α-ketoglutarate-dependent oxygenase, respectively.

Products from enzymatic assays were verified through GC-MS, LC-MS, and HPLC analyses with comparison to synthetically prepared authentic standards.

Interestingly, the enzymatic steps catalyzed by WelI1 and WelI3 were found to produce both the cis (cis-4.5) and trans (trans-4.5) isomers of indole isonitrile. This is in contrast to other isonitrile biosynthetic enzymes characterized within the hapalindole-type alkaloid-producing strains, AmbI1/AmbI3 (F. ambigua),9 and WelI1/WelI3 (H. welwitschii),10 which produce only the cis isomer of indole isonitrile. Based on these results, it is proposed that biosynthetic clusters for some of the hapalindole-type alkaloids produce both isomers while others do not.

103

4.1.2 Part II: Substrate Tolerance of Isonitrile Synthase and Fe(II)-α-Ketoglutarate Dependent Dioxygenase affords Unnatural Variants of Cyanobacterial Hapalindole Pathway Intermediate

Isonitrile containing natural products continue to enrich the treasure trove of natural products, leading to interesting bioactivities. Isonitriles have shown promise towards imaging11 and are known for their antimalarial12 activities. Access to novel isonitrile- containing natural products could lead to promising therapeutic applications. A synthetic biology approach to novel natural products can complement traditional synthetic methods;13 in addition to being a more environmentally friendly, time and cost effective method compared to synthetic approaches.

Chapter 3 described an investigation probing the substrate promiscuity of WelI1 and WelI3, indole-isonitrile (4.3) producing biocatalysts whose characterizations8 were described in Chapter 2. These enzymes work in tandem to synthesize cis and trans indole- isonitrile encoded within the hapalindole-type alkaloid, welwitindolinone (wel), biosynthetic pathway. WelI1 and WelI3 were reconstituted in vitro, and their substrate versatility was evaluated utilizing seven tryptophan derivatives, including one derivative

(C2-methyl L-tryptophan) biosynthesized by a third biocatalyst, the tryptophan synthase

TmTrpB114 (Figure 4.2). All seven tryptophan derivatives yielded corresponding cis and trans indole-isonitrile analogs with all assays favoring the cis isomer, but in varying degrees. The results were characterized by HPLC for turnover and analysis of cis to trans product ratios. Product identities were established by LC-HRMS and MS/MS. Through bioinformatic analysis, it was rationalized that the smaller amino acid residues within the active site of WelI3 (compared to those of its homolog PaPvcB) were responsible for the flexible substrate tolerance observed in this investigation for WelI3. Additionally, a

104 plausible mechanism for WelI3 was hypothesized, again based on PaPvcB. The relatively unstable tryptophan-isonitrile intermediate (4.1) is proposed to be hydroxylated in situ at the Cposition by WelI3 and simultaneously decarboxylated and desaturated. However, the fact that both WelI1 and WelI3 display substrate promiscuity leaves room for exciting investigations in the future.

4. 4.3 cis 4.3 1 Figure 4.2 This work employs sequential biocatalysis of TmTrpB1, WelI1, and WelI3 in order to generate analogs of indole isonitrile. All three biocatalysts were heterologously expressed utilizing T7 inducible plasmids and purified to homogeneity for enzymatic assays. TmTrpB1 catalyzes the formation of tryptophan analogs (4.1), which can be utilized by WelI1 to generate an isonitrile-containing intermediate (4.3) that is oxidatively decarboxylated by WelI3 to yield analogs of indole isonitrile (4.3).

105

4.2 Future Directions

The work described within this thesis sparks curiosity in several ways, paving the way for future investigations on hapalindole-type alkaloid biosynthesis and unnatural analog generation of this family of compounds. The biocatalytic functions of WelI1 and WelI3 from W. intricata (HT-29-1) were established within this thesis; however, many mechanistic questions remain unanswered for these biocatalysts. Future investigations that would benefit the continuation of this exploration include assessing the turnover efficiency of both biocatalysts (both with the natural substrate and substrate analogs), mutagenesis of proposed active site amino acid residues, and crystallography studies.

Kinetic investigations conducted on WelI1 and WelI3 would allow for a more efficient enzymatic assay set up. Concentrations of substrates and required cofactors could be optimized, in addition to assay time constraints to maximize the yield of desired product.

With turnover data for the native substrate, substrate analogs could be tested for turnover efficiency as well. Some natural product analogs may be biosynthesized more proficiently than others, which could be a potential benefit if said analogs were prospective candidates for therapeutics.

In order to draw conclusions on the mechanisms of these biocatalysts, mutagenesis studies and crystallography investigations would be beneficial. Substituting amino acid residues proposed to play a role within the active site would confirm or refute their involvement. At present, any models of WelI1 and WelI3 are homology-based; therefore, crystallography data would allow for an improved, more accurate structure of the enzymes.

Additionally, crystallography studies conducted with bound substrates or substrate

106 cofactors would provide additional mechanistic information. These investigations could also clarify how both isomers of indole-isonitrile are produced by the WelI1/I3 system.

Lastly, since the WelI1/I3 system has already been shown to work under cell lysate conditions, the TmTrpB1 catalyzed reaction could be attempted at the lysate stage as well.

Side-stepping the purification of all three proteins in order to produce natural product analogs would only cause this biosynthetic methodology to become even more time and cost effective.

107

4.3 References

1. (a) Burja, A. M.; Banaigs, B.; Abou-Mansour, E.; Grant Burgess, J.; Wright, P. C.,

Marine cyanobacteria—a prolific source of natural products. Tetrahedron 2001, 57 (46),

9347-9377; (b) Welker, M.; Dittmann, E.; von Döhren, H., Chapter Two - Cyanobacteria as a Source of Natural Products. In Methods in Enzymology, David, A. H., Ed. Academic

Press: 2012; Vol. Volume 517, pp 23-46.

2. (a) Salvador-Reyes, L. A.; Luesch, H., Biological targets and mechanisms of action of natural products from marine cyanobacteria. Nat. Prod. Rep. 2015, 32 (3), 478-503; (b)

Sainis, I.; Fokas, D.; Vareli, K.; Tzakos, A. G.; Kounnis, V.; Briasoulis, E., Cyanobacterial cyclopeptides as lead compounds to novel targeted cancer drugs. Mar. Drugs 2010, 8 (3),

629-57.

3. (a) Walsh, C. T.; Fischbach, M. A., Natural Products Version 2.0: Connecting

Genes to Molecules. J. Am. Chem. Soc. 2010, 132 (8), 2469-2493; (b) Ploux, O.; Méjean,

A., Genomics of the Biosynthesis of Natural Products: From Genes to Metabolites. In

Outstanding Marine Molecules, La Barre, S.; Kornprobst, J.-M., Eds. Wiley-VCH Verlag

GmbH & Co. KGaA: 2014; pp 473-488.

4. (a) Méjean, A.; Ploux, O., A Genomic View of Secondary Metabolite Production in Cyanobacteria. In Advances in Botanical Research, Elsevier Ltd.: 2013; Vol. 65, pp 189-

234; (b) Ramaswamy, A. V.; Flatt, P. M.; Edwards, D. J.; Simmons, T. L.; Han, B.;

Gerwich, W. H., The Secondary Metabolites and Biosynthetic Gene Clusters of Marine

Cyanobacteria. Applications in Biotechnology. Front. Mar. Biotechnol. 2006, 175-224;

108 cyanobacteria: A treasure trove of unique enzymes. Beilstein J. Org. Chem. 2011, 7, 1622-

1635.

5. Moore, R. E.; Cheuk, C.; Yang, X. Q. G.; Patterson, G. M. L.; Bonjouklian, R.;

Smitka, T. A.; Mynderse, J. S.; Foster, R. S.; Jones, N. D., Hapalindoles, antibacterial and antimycotic alkaloids from the cyanophyte Hapalosiphon fontinalis. J. Org. Chem. 1987,

52 (6), 1036-1043.

6. Bhat, V.; Dave, A.; MacKay, J. A.; Rawal, V. H., Chapter Two - The Chemistry of

Hapalindoles, Fischerindoles, Ambiguines, and Welwitindolinones. In The Alkaloids:

Chemistry and Biology, Hans-Joachim, K., Ed. Academic Press: 2014; Vol. Volume 73, pp 65-160.

7. Stratmann, K.; Moore, R. E.; Bonjouklian, R.; Deeter, J. B.; Patterson, G. M. L.;

Shaffer, S.; Smith, C. D.; Smitka, T. A., Welwitindolinones, Unusual Alkaloids from the

Blue-Green Algae Hapalosiphon welwitschii and Westiella intricata. Relationship to

Fischerindoles and Hapalinodoles. J. Am. Chem. Soc. 1994, 116 (22), 9935-9942.

8. Micallef, M. L.; Sharma, D.; Bunn, B. M.; Gerwick, L.; Viswanathan, R.; Moffitt,

M. C., Comparative analysis of hapalindole, ambiguine and welwitindolinone gene clusters and reconstitution of indole-isonitrile biosynthesis from cyanobacteria. BMC Microbiol.

2014, 14 (213), 1-18.

9. Hillwig, M. L.; Zhu, Q.; Liu, X., Biosynthesis of Ambiguine Indole Alkaloids in

Cyanobacterium Fischerella ambigua. ACS Chem. Biol. 2014, 9 (2), 372-377.

10. Hillwig, M. L.; Fuhrman, H. A.; Ittiamornkul, K.; Sevco, T. J.; Kwak, D. H.; Liu,

X., Identification and Characterization of a Welwitindolinone Alkaloid Biosynthetic Gene

109

Cluster in the Stigonematalean Cyanobacterium Hapalosiphon welwitschii. ChemBioChem

2014, 15 (5), 665-669.

11. Wainman, Y. A.; Neves, A. A.; Stairs, S.; Stockmann, H.; Ireland-Zecchini, H.;

Brindle, K. M.; Leeper, F. J., Dual-sugar imaging using isonitrile and azido-based click chemistries. Org. Biomol. Chem. 2013, 11 (42), 7297-7300.

12. (a) König, G. M.; Wright, A. D.; Angerhofer, C. K., Novel Potent Antimalarial

Diterpene Isocyanates, Isothiocyanates, and Isonitriles from the Tropical Marine Sponge

Cymbastela hooperi. J. Org. Chem. 1996, 61 (10), 3259-3267; (b) Fattorusso, E.;

Taglialatela-Scafati, O., Marine antimalarials. Mar. Drugs 2009, 7 (2), 130-52.

13. Keasling, J. D.; Mendoza, A.; Baran, P. S., Synthesis: A constructive debate.

Nature 2012, 492 (7428), 188-189.

14. (a) Hettwer, S.; Sterner, R., A novel tryptophan synthase beta-subunit from the hyperthermophile Thermotoga maritima. Quaternary structure, steady-state kinetics, and putative physiological role. J. Biol. Chem. 2002, 277 (10), 8194-201; (b) Rudolf, J. D.;

Wang, H.; Poulter, C. D., Multisite Prenylation of 4-Substituted Tryptophans by

Dimethylallyltryptophan Synthase. J. Am. Chem. Soc. 2013, 135 (5), 1895-1902.

110

Appendix 1 Bioinformatic Analysis of WelI1 and WelI3 Appendix 1.1 Illustration of gene clusters

gene gene

amb amb

hpi hpi

wel

gene cluster from from cluster gene

wel wel

gene cluster from from cluster gene

wel

UTEX 1903. UTEX

UH strain IC strain UH

) biosynthetic gene clusters. clusters. gene biosynthetic )

wel

sp. PCC 9339 (JGI IMG/ER: 2516653082). 2516653082). IMG/ER: (JGI 9339 PCC sp.

sp. PCC 9431 (JGI IMG/ER: 2512875027). 2512875027). IMG/ER: (JGI 9431 PCC sp.

Fischerella ambigua Fischerella

Fischerella

Hapalosiphon welwitschii Hapalosiphon

Fischerella

) and welwitindolinone ( welwitindolinone and )

amb

gene cluster from from cluster gene

amb

gene cluster from from cluster gene

), ambiguine ( ),ambiguine

1 (JGI IMG/ER: 2548876995). IMG/ER: (JGI 1

hpi

wel

hpi

wel wel

SAG 1427 SAG

UTEX 1903. UTEX

UTEXB1830.

sp. ATCC 43239. ATCC sp.

UH strain HT strain UH

Illustration of the hapalindole ( hapalindole the of Illustration

Fischerella ambig Fischerella muscicola Fischerella

Figure A1. Figure from cluster from cluster welwitschii Hapalosiphon intricata Westiella from cluster

111

Appendix 1.2 Translation of welI1 and welI3 using ExPaSy’s translate tool

Appendix 1.2.1 Translation of welI1

MKLKMISEKILRDIFQYRRLLSDTEPCAKEPCSMCLAPHIPKIQSFIESNEPIHFILP AFPAKSPNPQKVLGPMPDMGERVALQFLQNLCNHISQIYAPGAKITICSDGRVFT DLVAITDENVSLYRQGIGRLLNEINADAIDTFCLENVFTGMSFDQMRKTLVEQYA QPIESIQERVNSEDKHRQFFNGIYHLLFDDYLVLYPDKSREQIEIECNVRAYEVIQR SNAWTTLVGQHFPQSLRLSIHPQDYHSNKIGIHMIKTSDQWGTPWHNAPLFNGQ EFLLMKRKHIEDMGASLVWHNDYPSHYILSKQVSPALITLDSKS

Appendix 1.2.2 Translation of welI3

MVSTSVEQSTQFSVKSLIPFGALLEANEDCSDIQQLSIEQLCQLTWEHRLIVLRGF SLLEREELSTYCQRWGELLVWNFGTVLDLIVHQNPENYLFTNGNVPFHWDGAFA EAVPRFLFFQCLKAPEAGSGGESLFCDTVRILQNLSPQQREIWQKTEINYKSEKVA HYGGEITKSLVTKHPITGLSTLRFAEPLNDASVHLNPLYVEVCNLPTEEQNPFLNE LIENLYLPQNCFAHEWQEGDFLIADNHALLHGRNPFLFNSQRHLQRVHIL

112

Appendix 1.3 BLASTP results for WelI1 and WelI3

Table A1.1 BLASTP results for WelI1

Name of Protein Accession Number Length E Max value identity

Pyoverdine biosynthesis YP_481843 322 3e-105 46% [Frankia sp. CcI3]

Isonitrile synthase AAZ39275 393 3e-99 43% [uncultured organism]

IsnA

Pyoverdine biosynthesis ZP_02383059 282 3e-89 49% protein PvcA

[Burkholderia ubonensis Bu]

Spore wall maturation CBX80661 334 2e-87 44% protein DIT1 [Erwinia

amylovora ATCC BAA-2158]

Hypothetical protein YP_122579 349 3e-83 40% lpp0236 [Legionella

pneumophila str. Paris]

113

Table A1.2 BLASTP results for WelI3

Name of protein Accession Number Length E Max value identity

Pyoverdine biosynthesis YP_481844 270 9e-91 46% protein [Frankia sp. CcI3]

Oxygenase [uncultured AAZ39276 271 3e-74 45% organism-- IsnB

Taurine catabolism ZP_18172455 264 2e-69 40% dioxygenase, TauD/TfdA family [Acinetobacter baumannii WC-141]

Hypothetical protein ZP_17182149 291 5e-67 40% HMPREF1171_00181 NZ_AGWR01000000 [Aeromonas hydrophila SSU]

PvcB [Legionella ZP_06188335 278 6e-67 41% longbeachae D-4968]

Hypothetical protein NP_930051 281 5e-65 38% plu2817 [Photorhabdus luminescens subsp. laumondii TTO1]

Hypothetical protein YP_125606 278 3e-64 40% lpl0237 [Legionella pneumophila str. Lens]

114

Appendix 2 cis and trans Indole-isonitrile Synthetic Standard Characterization Appendix 2.1 NMR Spectra

400 MHz, CDCl3

Figure A2.1 1H NMR for cis indole-isonitrile.

100 MHz, CDCl3

Figure A2.2 13C NMR for cis indole-isonitrile.

115

400 MHz, CDCl3

Figure A2.3 1H NMR for trans indole-isonitrile.

100 MHz, CDCl3

Figure A2.4 13C NMR for trans indole-isonitrile.

116

Appendix 2.2 HRMS Spectra

HRMS Expected: 168.0687

Figure A2.5 HRMS for a sample containing a mixture of cis and trans isomers of indole isonitrile.

117

Appendix 3 Analysis of WelI1/3 Enzymatic Assays with E. coli cell lysates

Appendix 3.1 Analysis of WelI1/3 Enzymatic Assay by LC-MS

Expected MW: 168.07

Figure A3.1 LC-MS of WelI1/3 enzymatic assay with cell lysates.

118

Appendix 4 GC-MS Spectra of Synthesized cis and trans indole isonitrile standards

Figure A4.1 GC-trace for trans indole-isonitrile.

119

Figure A4.2 MS spectrum of peak from GC spectrum with Rt of 7.91 min as shown in Figure A4.1.

120

Figure A4.3 GC-trace for cis indole-isonitrile.

121

Figure A4.4 MS spectrum of peak from GC spectrum with Rt of 7.51 min as shown in Figure A4.3

122

Appendix 5 NMR spectra of 2-methyl-L-tryptophan

Figure A5.1 1H NMR spectrum of 2-methyl-L-tryptophan. 1H NMR spectrum obtained using DMSO and D2O as solvents at 500 MHz.

123

Figure A5.2 13C NMR spectrum of 2-methyl-L-tryptophan. 13C NMR spectrum obtained using DMSO and D2O as solvents at 125 MHz.

124

Appendix 6 Analysis of WelI1/3 Enzymatic Assay Results with L-tryptophan and tryptophan analogs Appendix 6.1 HPLC Analysis

Figure A6.1 HPLC traces for all 8 enzymatic assay extracts.

125

Appendix 6.2 LC-MS and MS/MS Analysis of WelI1/3 Enzymatic Assay Results with L-tryptophan and tryptophan analogs

Appendix 6.2.1 Assay with L-tryptophan

WelI WelI 1 Ribose 5-phosphate α-ketoglutarate3 Ammonium iron (II)

sulfate + Expected [M + H] : 169.0766

B Experimental mass: 169.0758 Δppm = 0.0008

Figure A6.2 A) Analysis of ethyl acetate extract of WelI1-I3 enzymatic assay with L-tryptophan. Biosynthetic reaction involving WelI1-I3 leading to cis indole isonitrile. B) EIC for reaction mixture and HRMS data for peak at 3.83 min. C) MS-MS data for molecular ion identified in B.

126

Expected Mass: Expected Mass: 142.0651 169.0766 Δppm = 0.0002 Δppm = 0.0006 Expected Mass: 116.0495 Δppm = 0.0043

Figure A6.3 MS-MS data for molecular ion identified in Figure A6.2B with fragment ion annotation for fingerprinting identity of cis indole isonitrile.

127

Appendix 6.2.2 Assay with 2-methyl-L-tryptophan

WelI1 WelI3

Ribose 5-phosphate α-ketoglutarate Ammonium iron (II) sulfate + Expected [M + H] : 183.0922 B

Experimental mass: 183.0910 Δppm = 0.0012

Figure A6.4 A) Analysis of ethyl acetate extract of WelI1-I3 enzymatic assay with 2-methyl-L- tryptophan. Biosynthetic reaction involving WelI1-I3 leading to 2-methyl indole isonitrile. B) EIC for reaction mixture and HRMS data for peak at 5.17min. C) MS-MS data for molecular ion identified in B.

128

Expected Mass: 168.0682 Δppm = 0.9830 Expected Mass: 183.0917 Δppm = 0.0087

Expected Mass: 154.0651 Δppm = 0.0022

Figure A6.5 MS-MS data for molecular ion identified in Figure A6.4B with fragment ion annotation for fingerprinting identity of 2-methyl indole isonitrile.

129

Appendix 6.2.3 Assay with 1-methyl-L-tryptophan

WelI1 WelI3 Ribose 5-phosphate α-ketoglutarate Ammonium iron (II) sulfate + Expected [M + H] : B 183.0922

Experimental mass: 183.0921 Δppm =0.0001

Figure A6.6 A) Analysis of ethyl acetate extract of WelI1-I3 enzymatic assay with 1-methyl-L- tryptophan. Biosynthetic reaction involving WelI1-I3 leading to 1-methyl indole isonitrile. B) EIC for reaction mixture and HRMS data for peak at 4.00 min. C) MS-MS data for molecular ion identified in B.

130

Figure A6.7 MS-MS data for molecular ion identified in Figure A6.6B with fragment ion annotation for fingerprinting identity of 1-methyl indole isonitrile.

131

Appendix 6.2.4 Assay with 4-fluoro-DL-tryptophan

α-ketoglutarate

B Experimental mass: 187.0668 Δppm = 0.0004

Figure A6.8 A) Analysis of ethyl acetate extract of WelI1-I3 enzymatic assay with 4-fluoro- DL-tryptophan. Biosynthetic reaction involving WelI1-I3 leading to 4-fluoro indole isonitrile. B) EIC for reaction mixture and HRMS data for peak at 5.23 min. C) MS-MS data for molecular ion identified in B.

132

Expected Mass: 187.0672 Expected Mass: Δppm = 0.0011 158.0401 Δppm = 0.0367

Figure A6.9 MS-MS data for molecular ion identified in Figure A6.8B with fragment ion annotation for fingerprinting identity of 4-fluoro indole isonitrile.

133

Appendix 6.2.5 Assay with 5-methyl-DL-tryptophan

WelI1 WelI3 Ribose 5-phosphate α-ketoglutarate Ammonium iron (II) sulfate + Expected [M + H] : 183.0922

B Experimental mass: 183.0916 Δppm = 0.0006

Figure A6.10 A) Analysis of ethyl acetate extract of WelI1-I3 enzymatic assay with 5-methyl-DL- tryptophan. Biosynthetic reaction involving WelI1-I3 leading to 5-methyl indole isonitrile. B) EIC for reaction mixture and HRMS data for peak at 4.45 min. C) MS-MS data for molecular ion identified in B.

134

Expected Mass: 168.0682 Δppm = 0.0021

Expected Mass: Expected Mass: 183.0917 141.0573 Δppm = 0.0011 Δppm = 0.0016

Expected Mass: 140.0495 Expected Mass: Δppm = 0.0042 154.0651 Δppm = 0.0037

Figure A6.11 MS-MS data for molecular ion identified in Figure A6.10B with fragment ion annotation for fingerprinting identity of 5-methyl indole isonitrile.

135

Appendix 6.2.6 Assay with 6-methyl-DL-tryptophan

WelI1 WelI3 Ribose 5-phosphate α-ketoglutarate

Ammonium iron (II) + sulfate Expected [M + H] : 183.0922

Experimental mass: 183.0923 Δppm = 0.0001

Figure A6.12 A) Analysis of ethyl acetate extract of WelI1-I3 enzymatic assay with 6-methyl-DL- tryptophan. Biosynthetic reaction involving WelI1-I3 leading to 6-methyl indole isonitrile. B) EIC for reaction mixture and HRMS data for peak at 4.40 min. C) MS-MS data for molecular ion identified in B.

136

Expected Mass: Expected Mass: 168.0682 183.0917 Δppm = 0.0015 Δppm = 0.0005

Expected Mass: 141.0573 Δppm = 0.0022 Expected Mass: 154.0651 Δppm = 0.0016

Figure A6.13 MS-MS data for molecular ion identified in Figure A6.12B with fragment ion annotation for fingerprinting identity of 6-methyl indole isonitrile.

137

Appendix 6.2.7 Assay with 5-methoxy-L-tryptophan

WelI1 WelI3 Ribose 5-phosphate α-ketoglutarate Ammonium iron (II) sulfate + Expected [M + H] : 199.0871 B

Experimental mass: 199.0887 Δppm = 0.0016

Figure A6.14 A) Analysis of ethyl acetate extract of WelI1-I3 enzymatic assay with 5-methoxy-L- tryptophan. Biosynthetic reaction involving WelI1-I3 leading to 5-methoxy indole isonitrile. B) EIC for reaction mixture and HRMS data for peak at 3.95 min. C) MS-MS data for molecular ion identified in B.

138

Figure A6.15 MS-MS data for molecular ion identified in Figure A6.14B with fragment ion annotation for fingerprinting identity of 5-methoxy indole isonitrile.

139

Appendix 6.2. 8 Assay with 5-hydroxy-L-tryptophan

WelI1 WelI3 Ribose 5-phosphate α-ketoglutarate Ammonium iron (II) sulfate

+ B Expected [M + H] : 185.0709

Experimental mass: 185.0730 Δppm =0.0021

Figure A6.16 A) Analysis of ethyl acetate extract of WelI1-I3 enzymatic assay with 5-hydroxy-L- tryptophan. Biosynthetic reaction involving WelI1-I3 leading to 5-hydroxy indole isonitrile. B) EIC for reaction mixture and HRMS data for peak at 4.31 min. C) MS-MS data for molecular ion identified in B.

140

Expected Mass: 167.0609 Δppm = 0.0002

Expected Mass: 185.0709 Δppm = 0.0002

Figure A6.17 MS-MS data for molecular ion identified in Figure A6.16B with fragment ion annotation for fingerprinting identity of 5-hydroxy indole isonitrile.

141

Appendix 7 WelI3 homology modeling, docking and multiple alignments

Appendix 7.1 Method for generation of homology model for WelI3:

The translated sequence of welI3 gene (see above) was submitted as a FASTA sequence to Phyre suite4 and was searched in an unbiased manner. The job Id for this search was: http://www.sbg.bio.ic.ac.uk/phyre2/phyre2_output/dd3a5c8cd33b177a/aligs/c3eatX_.1.al ig.html The topmost hit was the structure of Pseudomonas aeruginosa PvcB, with its earlier

X-ray structure (PDBID: C3eat). This was a 2.50 Å structure. The % confidence of this top hit was 100%. The % identity shared between WelI3 and PaPvcB is 36%. However, pairwise alignment (see Figure S21) revealed conversation of all 9 residues important for substrate binding, Fe binding, and cofactor and substrate channeling. Next, the PDB structure of the model of WelI3 was superpositioned with an initial threshold of 3.5 Å.

Typically, highly similar models have a TM-score > 0.7, same fold > 0.5 and different folds

< 0.5. Under this mode, the TM-score for WelI3 superimposed on PaPvcB was 0.0.

Therefore, the PDB downloaded for WelI3 after superpositioning was then taken as the model for docking substrates such as 9.

142

Appendix 8 References to Appendices

1. Micallef, M. L.; Sharma, D.; Bunn, B. M.; Gerwick, L.; Viswanathan, R.; Moffitt,

M. C., Comparative analysis of hapalindole, ambiguine and welwitindolinone gene clusters and reconstitution of indole-isonitrile biosynthesis from cyanobacteria. BMC

Microbiol. 2014, 14 (213), 1-18.

2. Hillwig, M. L.; Zhu, Q.; Liu, X., Biosynthesis of Ambiguine Indole Alkaloids in

Cyanobacterium Fischerella ambigua. ACS Chem. Biol. 2014, 9 (2), 372-377.

3. Hillwig, M. L.; Fuhrman, H. A.; Ittiamornkul, K.; Sevco, T. J.; Kwak, D. H.; Liu,

X., Identification and Characterization of a Welwitindolinone Alkaloid Biosynthetic

Gene Cluster in the Stigonematalean Cyanobacterium Hapalosiphon welwitschii.

ChemBioChem 2014, 15 (5), 665-669.

4. Kelley, L. A.; Sternberg, M. J. E., Protein structure prediction on the Web: a case study using the Phyre server. Nat. Protocols 2009, 4 (3), 363-371.

143

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