The Pennsylvania State University The Graduate School Department of Chemistry CYSTEINE BIOSYNTHESIS AND SULFUR FIXATION IN THE ARCHAEA A Thesis in Chemistry by Birthe Borup 2001 Birthe Borup Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy May 2001 We approve the thesis of Birthe Borup. Date of Signature James G. Ferry Professor of Anaerobic Microbiology Thesis Advisor Co-Chair of Committee Juliette T. J. Lecomte Associate Professor of Chemistry Co-Chair of Committee Philip C. Bevilacqua Assistant Professor of Chemistry Joseph M. Bollinger Assistant Professor of Biochemistry and Molecular Biology Andrew G. Ewing Professor of Chemistry Head of the Department of Chemistry iii ABSTRACT Cysteine plays a critical role in the structure, stability and catalytic function of many proteins in all domains of life. Cysteine is also the major source of sulfur for the synthesis of sulfur-containing compounds in organisms of the Bacteria and Eukarya domains. Two routes for cysteine biosynthesis in nature have been documented, pathways I and II. Plants and members of the Bacteria domain synthesize cysteine and fix sulfur via pathway I. Fungi fix sulfide and synthesize cysteine using pathway I or II. Although the genomes of seven members of the archaea have been sequenced to date, the data offer little understanding of cysteine biosynthesis in this domain. Presented here is the purification and characterization of the first archaeal enzyme catalyzing sulfur fixation and cysteine biosynthesis, O-acetylserine sulfhydrylase, from the methanoarchaeon Methanosarcina thermophila. Evidence was obtained for the involvement of O-acetylserine sulfhydrylase in cysteine biosynthesis by pathway I through biochemical, physiological and genetic means. O-acetylserine sulfhydrylase from M. thermophila exhibits positive co-operativity indicative of involvement in a biosynthetic pathway. O-acetylserine sulfhydrylase expression is elevated under growth conditions where the organism must synthesize cysteine, compared to expression levels where cysteine is provided in the media. The two genes involved in pathway I are transcribed in the same direction and possibly form an operon in M. thermophila. Activities of enzymes of pathway II are not detected in cell free extract, indicating that the only other pathway known for the synthesis of cysteine is not operable in M. thermophila under the conditions tested. Evidence is presented implicating an additional function for O-acetylserine sulfhydrylase. Over 90% of cysteine desulfurase activity in cell extracts of cells grown with cysteine as the sole sulfur source is due to O-acetylserine sulfhydrylase, suggesting the possibility that O-acetylserine sulfhydrylase provides the primary source of sulfide for biosynthesis. Patterns of expression levels in the presence and absence of sulfide, growth patterns with trimethylamine, inhibition by ammonia of the desulfurase activity, iv and changes in sulfide concentration of the media during growth support this interpretation. Experimental advances were made to obtain genetic knock-out mutants in M. thermophila deficient in the genes of pathway I to determine if an additional, novel pathway exists for cysteine biosynthesis in the archaea, and to test the hypothesis for the role of O-acetylserine sulfhydrylase in the desulfuration of cysteine to supply sulfur for biosynthesis. In addition a novel assay was developed for sulfide detection in cell extracts that is less prone to high background absorbances than published methods. v TABLE OF CONTENTS LIST OF FIGURES......................................................................................................vii LIST OF TABLES.......................................................................................................x ACKNOWLEDGEMENTS.........................................................................................xi FORWARD..................................................................................................................xii Chapter 1 Biochemistry and physiology of the archaea...............................................1 Archaea – an overview .........................................................................................1 Methanogens.........................................................................................................4 Pathways........................................................................................................6 Energetics ......................................................................................................8 Amino Acid Biosynthesis..............................................................................9 Protein Biosynthesis ......................................................................................10 Methanosarcina thermophila ........................................................................11 References.............................................................................................................12 Chapter 2 Physiological functions and biosynthesis of cysteine..................................15 Functions of L-Cysteine........................................................................................15 Sulfur containing cofactor biosynthesis................................................................15 Iron-sulfur cluster formation.................................................................................18 Sulfur assimilation................................................................................................24 Methionine synthesis and degradation..................................................................26 Cysteine biosynthesis............................................................................................26 Enzymes involved in cysteine biosynthesis..........................................................28 Serine acetyltransferase (SAT) ......................................................................28 O-acetylserine sulfhydrylase (OASS)............................................................29 Cystathionine β-synthase...............................................................................36 Cystathionine γ-lyase.....................................................................................37 References.............................................................................................................40 Chapter 3 O-acetylserine sulfhydrylase from Methanosarcina thermophila...............53 vi Abstract.................................................................................................................53 Introduction...........................................................................................................54 Materials and Methods .........................................................................................55 Results...................................................................................................................59 Discussion.............................................................................................................71 Acknowledgments ................................................................................................72 References.............................................................................................................73 Chapter 4 Cysteine biosynthesis in the Archaea: Methanosarcina thermophila utilizes O-acetylserine sulfhydrylase for the synthesis of cysteine.......................79 Abstract.................................................................................................................79 Introduction...........................................................................................................79 Materials and methods..........................................................................................81 Results...................................................................................................................83 Discussion.............................................................................................................87 Acknowledgments ................................................................................................89 References.............................................................................................................90 Chapter 5 Experimental approaches towards defining an alternative role of O- acetylserine sulfhydrylase in addition to cysteine biosynthesis ............................92 Abstract.................................................................................................................92 Introduction...........................................................................................................93 Materials and Methods .........................................................................................95 Results...................................................................................................................98 Discussion.............................................................................................................109 Acknowledgements...............................................................................................118 References.............................................................................................................119 Chapter 6 Conclusion...................................................................................................122 Appendix A Protocols for assays