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Genetic and biochemical characterization of YrkF, a novel two-domain sulfurtransferase in Bacillus subtilis Jeremy Hunt Thesis submitted to the faculty of Virginia Polytechnic Institute and State University in partial fulfillment of the requirements for the degree of Master of Science In Biochemistry Timothy J. Larson, Chair Dennis R. Dean Brenda J. Winkel August 11, 2004 Blacksburg, Virginia Keywords: YrkF, rhodanese, Ccd1, persulfide sulfur, disulfide cross-linking Genetic and biochemical characterization of YrkF, a novel two-domain sulfurtransferase in Bacillus subtilis Jeremy Hunt Timothy J. Larson, Chair Department of Biochemistry ABSTRACT Sulfur-containing compounds such as thiamin, biotin, molybdopterin, lipoic acid, and [Fe-S] clusters are essential for life. Sulfurtransferases are present in eukaryotes, eubacteria, and archaea and are believed to play important roles in mobilizing sulfur necessary for biosynthesis of these compounds and for normal cellular functions. The rhodanese homology domain is a ubiquitous structural module containing a characteristic active site cysteine residue. Some proteins containing a rhodanese domain display thiosulfate:cyanide sulfurtransferase activity in vitro. However, the physiological functions of rhodaneses remain largely unknown. YrkF, the first rhodanese to be characterized from Bacillus subtilis, is a unique protein containing two domains, an N-terminal Ccd1 domain and a C-terminal rhodanese domain. Ccd1 (conserved cysteine domain 1) is a ubiquitous structural module characterized by a Cys-Pro-X-Pro sequence motif. Thus, YrkF contains two cysteine residues (Cys15 and Cys149), one in each domain. Biochemical, genetic, and bioinformatic approaches were used in order to characterize YrkF. First, YrkF was overexpressed and assayed for rhodanese activity to show that the protein is a functional rhodanese. A variant protein, YrkFC15A, containing a cysteine to alanine substitution in the Ccd1 domain was created to determine if the Ccd1 cysteine is essential for rhodanese activity. The variant protein was overexpressed and rhodanese assays showed that YrkFC15A is also a functional rhodanese. Inherent structural and catalytic differences were observed when comparing YrkF and YrkFC15A, which may reflect the importance of the Ccd1 cysteine residue to normal enzymatic function and structural stability. Initial kinetic studies identified differences in activity between YrkF and YrkFC15A. Cross-linking experiments showed a propensity for the formation of inter- and intramolecular disulfide bonds between the two cysteine residues and indicated that Cys15 and Cys149 are located near one another in the 3- dimensional structure of the protein. Analysis of the proteins by mass spectrometry suggested YrkF contains a stable persulfide sulfur, whereas YrkFC15A showed no evidence of a stable persulfide sulfur and was prone to oxidation and other active site modifications. A homology model of YrkF was created using structures of a rhodanese homolog and a Ccd1 homolog as templates. The model was used to predict the structure of YrkF based on the results of the cross-linking experiments. A strain containing a yrkF chromosomal deletion could be constructed, indicating YrkF is not essential for survival. Phenotypic analysis of the yrkF mutant revealed that YrkF is not needed for biosynthesis of sulfur-containing cofactors (thiamin, biotin, molybdopterin, or lipoic acid) or amino acids. The characterization of YrkF could lead to the discovery of novel physiological roles for rhodaneses and may give insight into possible roles for the Ccd1 module. iii TABLE OF CONTENTS ABSTRACT........................................................................................................................ ii TABLE OF CONTENTS................................................................................................... iv LIST OF FIGURES .........................................................................................................viii LIST OF TABLES............................................................................................................. xi ACKNOWLEDGMENTS ................................................................................................ xii CHAPTER ONE ................................................................................................................. 1 Introduction..................................................................................................................... 1 1.1. Chemistry of sulfur ............................................................................................. 1 1.2. Sulfane sulfur...................................................................................................... 2 1.2.1. Biological significance................................................................................. 2 1.2.2. S0 carrier proteins......................................................................................... 5 1.2.3. Cysteine desulfurase .................................................................................... 6 1.3. Sulfurtransferases................................................................................................ 9 1.3.1. Thiosulfate:cyanide sulfurtransferase (rhodanese) ...................................... 9 1.3.2. Mercaptopyruvate sulfurtransferase (MST)............................................... 16 1.4. Biosynthesis of sulfur-containing compounds.................................................. 18 1.4.1. [Fe-S] clusters ............................................................................................ 18 1.4.2. Thiamin...................................................................................................... 24 1.4.3. Molybdopterin............................................................................................ 26 1.4.4. Thionucleosides in tRNA........................................................................... 28 1.4.5. Biotin.......................................................................................................... 31 1.4.6. Lipoic acid ................................................................................................. 35 iv 1.5. Physiological characterization of rhodaneses ................................................... 37 1.6. YrkF: A unique two-domain rhodanese............................................................ 39 1.7. Present work...................................................................................................... 40 CHAPTER TWO .............................................................................................................. 42 Materials and Methods.................................................................................................. 42 2.1. Materials ........................................................................................................... 42 2.2. Bacterial strains and plasmids........................................................................... 42 2.3. Media and growth conditions............................................................................ 45 2.4. General Methods............................................................................................... 46 2.4.1. Polymerase Chain Reaction, DNA electrophoresis, and ligation reactions46 2.4.2. Preparation of competent E. coli cells and transformation........................ 46 2.4.3. Transformation of B. subtilis ..................................................................... 47 2.4.4. Determining protein concentration ............................................................ 47 2.5. Construction of overexpression vectors pVK2B and pVK3............................. 48 2.6. Oligonucleotide-directed mutagenesis.............................................................. 48 2.7. Construction of plasmid pFerm1 ...................................................................... 51 2.8. Deletion of the chromosomal yrkF gene........................................................... 51 2.9. Overexpression and purification of YrkF and YrkFC15A................................... 51 2.10. Polyacrylamide gel electrophoresis ................................................................ 53 2.11. Assay of rhodanese activity ............................................................................ 53 2.12. Disulfide cross-linking.................................................................................... 56 2.12.1. Cross-linking with dibromobimane (bBBr)............................................. 56 2.12.2. Treatment with Cu(1,10-phenanthroline)2SO4 (CuPhen) ........................ 57 v 2.13. Mass Spectrometry.......................................................................................... 58 2.14. Homology modeling of YrkF.......................................................................... 60 2.14.1. Sequence alignments of templates........................................................... 60 2.14.2. Constructing a model for YrkF................................................................ 60 2.14.3. Energy Minimization of the YrkF model................................................. 61 2.15. Molecular dynamics (MD) simulations of homology model.......................... 62 2.16. Limited Proteolysis of YrkF ........................................................................... 62 CHAPTER THREE .......................................................................................................... 64 Results..........................................................................................................................
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