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