THE COENZYME M BIOSYNTHETIC PATHWAY IN PROTEOBACTERIUM

XANTHOBACTER AUTOTROPHICUS PY2

by
Sarah Eve Partovi

A dissertation submitted in partial fulfillment of the requirements for the degree

of
Doctor of Philosophy in
Biochemistry

MONTANA STATE UNIVERSITY
Bozeman, Montana

January 2018
©COPYRIGHT by
Sarah Eve Partovi
2018
All Rights Reserved ii
DEDICATION

I dedicate this dissertation to my family, without whom none of this would have been possible. My husband Ky has been a part of the graduate school experience since day one, and I am forever grateful for his support. My wonderful family; Iraj, Homa, Cameron, Shireen, Kevin, Lin, Felix, Toby, Molly, Noise, Dooda, and Baby have all been constant sources of encouragement. iii
ACKNOWLEDGEMENTS

First, I would like to acknowledge Dr. John Peters for his mentorship, scientific insight, and for helping me gain confidence as a scientist even during the most challenging aspects of this work. I also thank Dr. Jennifer DuBois for her insightful discussions and excellent scientific advice, and my other committee members Dr. Brian Bothner and Dr. Matthew Fields for their intellectual contributions throughout the course of the project. Drs. George Gauss and Florence Mus have contributed greatly to my laboratory technique and growth as a scientist, and have always been wonderful resources during my time in the lab. Members of the Peters Lab past and present have all played an important role during my time, including Dr. Oleg Zadvornyy, Dr. Jacob Artz, and future Drs. Gregory Prussia, Natasha Pence, and Alex Alleman. Undergraduate researchers/REU students including Hunter Martinez, Andrew Gutknecht and Leah Connor have worked under my guidance, and I thank them for their dedication to performing laboratory assistance. Dr. Bernd Markus Lange of Washington State University has been instrumental in helping us collect mass spectrometry data, and Dr. Brian Tripet assisted in collecting exciting time-resolved ¹H-NMR data. Many other people in the Department of Chemistry and Biochemistry have contributed through stimulating conversation despite being involved in disciplines other than biochemistry, including Drs. Ky Mickelsen, Colin Miller, Ryan Latterman, David Skowron, Ashley Beckstead, Christine Gobrogge, Anna Michel, Amanda Mattson, and Doreen Brown. Finally, I would like to thank the DOE for their ongoing support. iv
TABLE OF CONTENTS

1. INTRODUCTION ...........................................................................................................1
Introduction to Coenzyme M.........................................................................................1
A role for CoM in methanogenesis..........................................................................2
Introduction to Xanthobacter autotrophicus Py2 ..........................................................5
Methods for identifying CoM as C3 carrier.............................................................6 A role for CoM in bacterial propylene metabolism...............................................11 Unifying structural features in propylene metabolism enzymes............................12 Similarities in CoM utilization between methanogenesis and propylene metabolism ...........................................................................................20
Biosynthesis of CoM in methanogenic archaea ............................................................21 Putative pathway for bacterial CoM biosynthesis ........................................................24 Research Directions ......................................................................................................28 References......................................................................................................................32

2. COENZYME M BIOSYNTHESIS IN BACTERIA INVOLVES PHOSPHATE
ELIMINATION BYA UNIQUE MEMBER OF THE ASPARTASE/FUMARASE SUPERFAMILY............................................................................................................40

Contribution of Authors and Co-Authors ......................................................................40 Manuscript Information .................................................................................................42 Abstract .........................................................................................................................43 Introduction ...................................................................................................................44 Methods .........................................................................................................................48
Growth of Xanthobacter autotrophicus Py2..........................................................48 Amplification of genes for putative CoM biosynthesis .........................................48 Expression and purification of putative CoM biosynthesis gene products............49 Determining sulfite uptake by the XcbB1-catalyzed reaction ...............................50 Measuring inorganic phosphate production by the XcbC1-catalyzed reaction......................................................................................51 Determination of XcbE1 activity with an assay for H₂S formation.......................52 Mass spectrometric analysis of reaction products .................................................52 Q-TOF MS.............................................................................................................53 Time Resolved ¹H-NMR........................................................................................53 Phylogeny and homology modeling ......................................................................54
Results and Discussion ..................................................................................................54
Sequence analyses identify gene familes and suggest possible roles for putative CoM biosynthetic genes...........................................................................54 XcbB1 catalyzes the conversion of phosphoenolpyruvate to phosphosulfolactate ...........................................................................................58 v
TABLE OF CONTENTS CONTINUED

XcbC1 catalyzes the β-elimination of phosphate from phosphosulfolactate to form sulfoacrylic acid .......................................................61 Modeling XcbC1 active site reactivity ..................................................................64
Conclusions....................................................................................................................66 Acknowledgements........................................................................................................67 References......................................................................................................................69

3. A PYRIDOXAL 5’-PHOSPHATE-DEPENDENT ENZYME MAY PROVIDE
A SOURCE FOR THE COENZYME M THIOL MOIETY.........................................76

Introduction ...................................................................................................................76
PLP-Dependent Enzymes .....................................................................................76 Cysteine Desulfhydrases .......................................................................................80
L-cysteine desulfhydrase ...........................................................................80 D-cysteine desulfhydrase...........................................................................81
Hypothesized role of XcbE1 in CoM biosynthesis ...............................................85
Methods .........................................................................................................................86
Amplification of xcbE1 .........................................................................................86 Expression and purification of XcbE1 ..................................................................86 XcbE1 Phylogenetics.............................................................................................88 Determination of activity with an assay for H₂S formation...................................88 Screening XcbE1 reaction for aldehyde/ketone products......................................88 Measurement of pyruvate production ....................................................................89 Detection of cysteine consumption........................................................................89 Confirming PLP-dependence.................................................................................90 Determination if thiol-specific alkylating agent inhibits XcbE1 activity..............90 XcbE1 Crystal Screens .........................................................................................91 Proposed cosubstrate activity screens....................................................................91
Assays with proposed cosubstrates............................................................91 Synthesis and biological preparation of sulfoacetaldehyde.......................92
Detection of CoM using HPLC-FLD.....................................................................93
Results and Discussion ..................................................................................................94
XcbE1 bioinformatics provides preliminary insight into activity .........................94 Purification of XcbE1 ............................................................................................96 Testing XcbE1 for canonical D-cysteine desulfhydrase activity ..........................98 Screening XcbE1 reactions for proposed cosubstrates ........................................103
Isethionate and vinyl sulfonate ................................................................105 Sulfolactate ..............................................................................................107 Sulfolacetaldehyde...................................................................................110 Preliminary crystallization conditions .....................................................112 vi
TABLE OF CONTENTS CONTINUED

Determining if thiol-specific alkylating agent is an effective XcbE1 inhibitor ....................................................................................113
Proposed XcbE1 scheme .............................................................................................114 Conclusions and Future Directions .............................................................................116 References ...................................................................................................................118

4. AN ASPARTASE/FUMARASE SUPERFAMILY ENZYME MAY CATALYZE
A SECOND REACTION IN COM BIOSYNTHESIS................................................122

Introduction .................................................................................................................122 Methods .......................................................................................................................126
Amplification of XcbD1 ......................................................................................126 Expression and purification of XcbD1 ................................................................127 Detection of AMP and AMP derivatives.............................................................129
UV-Vis.....................................................................................................129 HPLC .......................................................................................................130
Q-TOF MS...........................................................................................................130
Screening proposed cosubstrates using Q-TOF MS ................................130 Using X. autotrophicus Py2 cell lysates to investigate XcbD1 reaction......................................................................131
Time resolved ¹H-NMR ......................................................................................132 Phylogeny and homology modeling ....................................................................132
Results and Discussion ................................................................................................132
Purification of XcbD1..........................................................................................132 Testing XcbD1 for canonical ADL activity.........................................................134 Investigating the XcbD1 reaction with Q-TOF MS and time resolved ¹H-NMR .................................................................................134 X. autotrophicus Py2 cell lysates could provide alternative route to solving the reaction ................................................................................139 Phylogeny and homology modeling of XcbD1 ..................................................141
Conclusions and future directions................................................................................145 References ...................................................................................................................149

5. CONCLUDING REMARKS.......................................................................................151 APPENDICES .................................................................................................................159
APPENDIX A: Supplemental Material to Chapter 1 ..........................................160 APPENDIX B: Supplemental Material to Chapter 2...........................................166

CUMULATIVE REFERENCES CITED ........................................................................173 vii
LIST OF TABLES

• Table
• Page

4.1. Summarized Q-TOF MS reaction sets with XcbD1 ....................................136 4.2. Summarized ¹H-NMR experiments with XcbD1 .........................................138 4.3. Preliminary X. autotrophicus Py2 cell lysate Q-TOF MS results.................140 4.4. Active site residues of canonical ADL vs XcbD1 homology model............145
A.S.1. All known copies of putative CoM biosynthetic operon ..............................161 A.S.2. Expression vectors discussed throughout thesis ...........................................162 A.S.3. The 20 genes upstream and downstream from CoM operon........................163 A.S.4. List of genes/enzymes for methanoarchaeal CoM biosynthesis...................165 B.S.1. Primers used for amplifying xcbB1, xcbC1, and xcbE1................................167 viii
LIST OF FIGURES

• Figure
• Page

1.1. Structure of CoM ..............................................................................................1 1.2. Propylene metabolism pathway........................................................................3 1.3. MCR in anaerobic oxidation of methane..........................................................4 1.4. Progression of propylene metabolism...............................................................7 1.5. Spectral identification of 2-hydroxypropyl-CoM.............................................8 1.6. Spectral identification of 2-ketopropyl-CoM....................................................9 1.7. Spectral identification of ¹³C-acetoacetate......................................................10 1.8. Propylene metabolism in X. autotrophicus Py2..............................................12 1.9. Alkene monooxygenase..................................................................................13
1.10. Proposed mechanism and CoM-coordination for EaCoMT ...........................15 1.11. Comparison of sulfonate binding sites for R- and S-HPCDH.........................16 1.12. Comparison of substrate binding sites for R- and S-HPCDH.........................17 1.13. Mechanism for 2-KPCC catalyzed reaction ...................................................18 1.14. Sulfonate binding site in 2-KPCC...................................................................19 1.15. Methanogenic CoM biosynthetic pathways....................................................22 1.16. Gene cluster that encodes proteins involved in propylene metabolism..........25 1.17 Gene neighborhoods for putative CoM operon in alkene metabolizers ..........28
2.1. Plasmid region containing genes for CoM biosynthesis.................................46 2.2. Phylogenetic tree of AFS enzymes including XcbC and XcbD .....................57 2.3. Data supporting XcbB1 reaction.....................................................................60 ix
LIST OF FIGURES CONTINUED

• Figure
• Page

2.4. Data supporting XcbC1 reaction.....................................................................63 2.5. Homology model of XcbC1 and proposed mechanism ..................................65 3.1. PLP-dependent reaction with bound substrate................................................77 3.2. PLP quinonoid intermediate ...........................................................................78 3.3. Scheme for D-cysteine desulfhydrase and ACCD enzymes...........................82 3.4. Catalytic mechanism of D-cysteine desulfhydrases .......................................83 3.5. Amino acid alignment of XcbE1 with ACCD and
D-cysteine desulfhydrase...............................................................................94

3.6. XcbE1 phylogenetic tree.................................................................................95 3.7. Amino acid alignment of XcbE1 with cysteine desulfurases .........................96 3.8. Eluted XcbE1..................................................................................................97 3.9. UV-Vis scan of purified XcbE1......................................................................97
3.10. H₂S formation by XcbE1 when supplied with L- or D-cysteine ....................99 3.11. XcbE1 pH optima .........................................................................................100 3.12. Confirmation of aldehyde/ketone product in XcbE1 reaction ......................101 3.13. XcbE1 kinetics of pyruvate formation for D-and L-cysteine .......................102 3.14. Truncated mechanism for XcbE1 .................................................................103 3.15. CoM derivatives tested as cosubstrates.........................................................104 3.16. XcbE1 H₂S formation when supplemented with isethionate or vinyl sulfonate...............................................................................................106 x
LIST OF FIGURES CONTINUED

• Figure
• Page

3.17. XcbE1 H₂S formation when supplemented with sulfolactate.......................107 3.18. XcbE1 plus sulfolactate HPLD-FLD chromatogram....................................109 3.19. XcbE1 H₂S formation when supplemented with sulfoacetaldehyde ............110 3.20. XcbE1 crystals from three crystallization trials............................................113 3.21. Levels of H₂S when XcbE1 incubated with NEM........................................114 3.22. Proposed scheme for bacterial CoM biosynthesis ........................................115
4.1. Summary of first two solved steps for bacterial CoM biosynthesis .............122 4.2. Phylogenetic tree showing XcbD relationship among AFS enzymes ..........123 4.3. Canonical ADL reaction ...............................................................................124 4.4. Scheme for XcbD1 catalyzed addition of AMP across sulfoacrylic double bond...............................................................................126

4.5. SDS-PAGE of purified XcbD1.....................................................................133 4.6. Sequence coverage map of XcbD1 in-gel trypsin digest..............................134 4.7. ¹H-NMR spectrum showing sulfoacrylic acid peaks....................................138 4.8. Cysteine plus sulfoacrylic acid adduct..........................................................141 4.9. Alignment of XcbD1 with canonical ADL and pCMLE..............................143
4.10. XcbD1 homology model superimposed over canonical ADL......................144
5.1. Proposed pathway for bacterial CoM biosynthesis.......................................152 5.2. Eluted XcbA2................................................................................................155
B.S.1. Levels of H₂S formed with XcbE1 incubated with D-and L-cysteine..........168 xi
LIST OF FIGURES CONTINUED

• Figure
• Page

B.S.2. Controls for ¹H-NMR experiments...............................................................169 B.S.3. Scheme for canonical phosphatase reaction and predicted
¹H-NMR for sulfoacrylic acid.......................................................................171

B.S.4. Amino acid alignment of AFS members and XcbC1 ...................................172 xii
LIST OF SCHEMES

Scheme
2.1. PEP- and L-phosphoserine-dependent pathways for
Page methanoarchaeal CoM biosynthesis ...............................................................45
2.2. Proposed pathway for bacterial CoM biosynthesis.........................................55 xiii
GLOSSARY

CoM – coenzyme M PEP - phosphoenolpyruvate PLP- pyridoxal 5’-phosphate AMO – alkene monooxygenase ECP – epoxide carboxylation pathway AMP – adenosine monophosphate NADH – nicotinamide adenine dinucleotide EaCoMT – epoxyalkane: coenzyme M transferase R-HPCDH – (R)-hydroxypropyl-CoM-dehydrogenase S-HPCDH – (S)- hydroxypropyl-CoM-dehydrogenase 2-KPCC – 2-ketopropyl-CoM-oxidoreductase/carboxylase 2-KPC – 2-ketopropyl-CoM XcbB1 – Product of xcbB1, homologous to phosphosulfolactate synthase (ComA) XcbC1 – Product of xcbC1, homologous to argininosuccinate lyases XcbD1 – Product of xcbD1, homologous to adenylosuccinate lyases XcbE1 – Product of xcbE1, homologous to D-cysteine desulfhdyrases ACCD – 1-aminocyclopropane-1-carboxylate deaminase AFS – aspartase/fumarase superfamily ASL – argininosuccinate lyase ADL – adenylosuccinate lyase xiv
ABSTRACT

The metabolically versatile bacterium Xanthobacter autotrophicus Py2 has been the focus of many studies within the field of bioenergy sciences, as it contains two unique CO₂ fixing enzymes, and can utilize unconventional substrates such as propylene and acetone as the sole supplemented carbon source while fixing CO₂ in the process. Unexpectedly, coenzyme M (CoM) was found to play a crucial role as a C₃ carrier in the pathway for propylene metabolism in the late 1990s. Previously, CoM was thought to be present solely as a C₁ carrier in methanogenic archaea for nearly 30 years. Though CoM biosynthesis has been characterized in methanogenic archaea, bacterial CoM biosynthesis remained uncharacterized. In X. autotrophicus Py2, four putative CoM biosynthetic enzymes encoded by xcbB1, C1, D1, and E1 have been identified through informatics and proteomic approaches. XcbB1 is homologous to the archaeal ComA which catalyzes the addition of sulfite to phosphoenolpyruvate, and forms the initial intermediate, phosphosulfolactate, in one of the methanogen CoM biosynthetic pathways. The remaining genes do not encode homologues of any of the previously characterized enzymes in methanogen CoM biosynthesis, suggesting bacteria have a unique pathway. The production of phosphosulfolactate by ComA homolog XcbB1 was verified, indicating that bacterial CoM biosynthesis is initiated in an analogous fashion to the PEP- dependent methanogenic archaeal CoM biosynthesis pathway. XcbC1 and D1 are