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Pdul Is an Evolutionary Distinct Phosphotransacylase Involved in B12-Dependent 1,2-Propanediol Degradation by Salmonella Enteric

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Pdul Is an Evolutionary Distinct Phosphotransacylase Involved in B12-Dependent 1,2-Propanediol Degradation by Salmonella Enteric Iowa State University Capstones, Theses and Graduate Theses and Dissertations Dissertations 2009 PduL is an evolutionary distinct phosphotransacylase involved in B12-dependent 1,2-propanediol degradation by Salmonella enterica serovar Typhimurium LT2 and is associated with the propanediol utilization microcompartments Yu Liu Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/etd Part of the Biochemistry, Biophysics, and Structural Biology Commons Recommended Citation Liu, Yu, "PduL is an evolutionary distinct phosphotransacylase involved in B12-dependent 1,2-propanediol degradation by Salmonella enterica serovar Typhimurium LT2 and is associated with the propanediol utilization microcompartments" (2009). Graduate Theses and Dissertations. 10513. https://lib.dr.iastate.edu/etd/10513 This Thesis is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. PduL is an evolutionary distinct phosphotransacylase involved in B12-dependent 1,2-propanediol degradation by Salmonella enterica serovar Typhimurium LT2 and is associated with the propanediol utilization microcompartments by Yu Liu A thesis submitted to the graduate faculty in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Major: Immunobiology Program of Study Committee: Thomas Bobik, Major Professor Louisa Tabatabai Qijing Zhang Kenneth J. Koehler Iowa State University Ames, Iowa 2009 Copyright © Yu Liu, 2009. All rights reserved. ii TABLE OF CONTENTS LIST OF FIGURES vi LIST OF TABLES vii LIST OF ABBREVIATIONS viii ABSTRACT x INTRODUCTION 1 Cobalamin B12 1 Structure of AdoCbl 1 B12–Dependent Reactions in Enteric Bacteria 2 Propanediol Dehydratase 2 Ethanolamine Ammonia Lyase 2 Glycerol Dehydratase 2 Methionine Synthetase 2 B12 Synthesis in S.typhimurium and Cob Operon 3 1,2-Propanediol 3 Pathway for 1,2-PD Degradation 4 Metabolism of 1,2-PD under Anaerobic and Aerobic Conditions 4 The pdu Locus 5 Regulation of pdu Operon 6 Bacteria Microcompartment 9 Carboxysome 9 Composition of Carboxysome 10 α- and β-Carboxysomes 10 iii Carboxysome Genomics 11 Carboxysomes are Icosahedral 11 Molecular Conduits for Small Molecules Span the Carboxysome Shell 12 Propanediol Utilization Microcompartment 13 Model for the Pdu Microcompartment 14 Proteomics of the Pdu Microcompartments 16 The Pathway of 1,2-Propanediol Degradation 17 The Genetics of 1,2-Propanediol Degradation 18 Assembly of Microcompartments 19 Mitigation of Aldehyde Toxicity and Diffusive Loss 19 Homologues of Carboxysome Proteins 20 Structure of the Pdu Microcompartment 21 Research Overview 22 MATERIALS AND METHODS 24 Chemicals and Reagents 24 Bacterial Strains, Media, and Growth Conditions 24 General Molecular Methods 25 Protein Methods 26 P22 Transduction 26 Construction of plasmids for production of PduL and PduL-His8 26 Growth of the PduL-His8 Production Strain 27 Purification of PduL-His8 27 iv Preparation of Cell Extracts of S. Enterica 28 PTAC Assays 28 Construction of a Nonpolar pduL Deletion 29 Aerobic Growth Curves 30 Anaerobic Growth Curves 30 DNA Sequencing and Analysis 31 High-Pressure Liquid Chromatography (HPLC) 31 HPLC Electrospray Ionization Mass Spectrometry (HPLC-ESI-MS) 31 PTAC Assays for Shell Encoding Gene Mutants 32 Pdu Microcompartments Purification 32 Antibody Preparation 33 Western Blot 33 Two-Hybrid Screen 34 RESULTS 35 The pta Gene is Nonessential for 1,2-PD Degradation 35 Strains with pduL Mutations Produce Less Propionic Acid on MacConkey–1,2-Propanediol Indicator Medium 36 Enzyme Assays Indicate that pduL Encodes a PTAC Enzyme 36 pduL Mutants are Impaired for Aerobic Growth on 1,2-PD 38 pduL Mutants are Unable to Obtain Energy from 1,2-PD under Fermentative Conditions 39 The Observed Phenotypes of a pduL Mutant are Complemented by a pduL Minimal Clone 40 v PduL Substitutes for Pta in Vivo During Acetate Utilization 41 Propionyl-CoA is a Product of the PduL Reaction 42 Production of PduL-His8 Protein 44 Purification and Kinetic Characterization of PduL-His8 45 Localization of PduL 48 Preparation of PduL-Specific Antibody 49 Western Blot Analysis of Purified Pdu microcompartments 50 Interactions between PduL and Other Pdu microcompartments Proteins 51 DISCUSSION 54 CONCLUSIONS AND FUTURE DIRECTIONS 59 Genetic and Biochemical Evidence of PduL in 1,2-PD Degradation 59 PduL is a Component of Pdu microcompartments and Probably Associate with pdu Proteins 60 Future Experimentation 61 ACKNOWLEDGMENTS 62 REFERENCES 63 vi LIST OF FIGURES 1. Regulation of the cob/pdu regulon of Salmonella enterica. 8 2. Electron micrograph of bacterial microcompartments. 14 3. Model for 1,2-propanediol degradation by S. enterica. 15 4. Purified Pdu microcompartments. 17 5. Growth of a pduL null mutant on 1,2-PD. 40 6. Ectopic expression of a pduL minimal clone corrects the growth defect of a pta mutant on acetate minimal medium. 42 7. Propionyl-CoA is produced by the PduL reaction. 44 8. SDS-PAGE analysis of PduL-His8 purification. 47 9. Western analysis with anti-PduL polyclonal antibody preparations. 49 10. Western blot analysis of purified Pdu microcompartments. 50 11. Construction of bait and target recombinant plasmids and the schematic of the BacterioMatch II two-hybrid system dual reporter construct. 52 vii LIST OF TABLES 1. Bacterial strains used in this study 25 2. Phosphotransacylase activity in cell extracts from selected strains of S. enterica 38 3. Kinetic constants for purified recombinant PduL-His8 47 4. Phosphotransacylase activity in pdu mutants 48 5A. Two-hybrid test for pBT/pduL and each of other Pdu proteins 53 5B. Two-hybrid test for pTRG/pduL and each of other Pdu proteins 53 viii LIST OF ABBREVIATIONS 1,2-PD 1,2 propanediol 3-AT 3-amino-1,2,4-triazole 3-PGA 3-phosphoglycerate AdeCbl adenosylcobalamin AEBSF 4-(2-Aminoethyl)benzenesulfonylfluoride HCl Amp ampicillin ATP adenosine triphosphate B-PER II bacterial protein extraction reagent II CA carbonic anhydrase CCM CO2-concentrating mechanism CH3Cbl methylcobalamin CNCbl cyanocobalamin Cob cobalamin CoA coenzyme A DDH diol dehydratase Dmb dimethylbenzimidazole E. Coli Escherichia coli EDTA ethylenediaminetetraacetic acid HCl hydrogen chloride His8 8 C-terminal histidine residues HPLC high pressure liquid chromatography HPLC-ESI-MS HPLC electrospray ionization mass spectrometry ix IgG immunoglobulin G AP alkaline phosphatase KanR kanamycin resistance IPTG isopropyl-β-D-thiogalactopyranoside LB luria-bertani/Lennox MALDI-TOF MS matrix assisted laser desorption ionization-time-of-flight mass spectrometry Mcp microcompartment NCE no-carbon-E PAGE polyacrylamide gel electrophoresis PCR polymerase chain reaction pI isoelectric point pdu propanediol utilization PSI-BLAST protein specific iterative basic local alighment search tool Pta phosphotransacetylase PTAC phosphotransacylase PVDF polyvinylidene fluoride RuBisco ribulose bis-phosphate carboxylase monooxygenase RuBP ribulose bisphosphate SDS sodium dodecyl sulfate S. enterica Salmonella enterica s.n. supernatant Tn transposon x ABSTRACT Salmonella enterica degrades 1,2-propanediol (1,2-PD) in a coenzyme B12- dependent manner. Prior enzymatic assays of crude cell extracts indicated that a phosphotransacylase (PTAC) was needed for this process, but the enzyme involved was not identified. Here we show that the pduL gene encodes an evolutionarily distinct PTAC used for 1,2-PD degradation. Growth tests showed that pduL mutants were unable to ferment 1,2-PD and were also impaired for aerobic growth on this compound. Enzyme assays showed that cell extracts from a pduL mutant lacked measurable PTAC activity in a background that also carried a pta mutation (the pta gene was previously shown to encode a PTAC enzyme). Ectopic expression of pduL corrected the growth defects of pta mutant. PduL fused to 8 C-terminal histidine residues (PduL-his8) was purified -1 -1 and its kinetic constants determined: Vmax = 51.7±7.6 µmol min mg ; and Km 2- 2- for propionyl-PO4 and acetyl-PO4 = 0.61 and 0.97 mM, respectively. Sequence analyses showed that PduL is unrelated in amino acid sequence to known PTAC enzymes and that PduL homologues are distributed among at least 49 bacterial species, but are absent from the Archaea and Eukarya. Immunoblots showed that PduL was a component of propanediol utilization microcompartment. PduL enzyme assay and two-hybrid analysis indicated PduL was associated with several candidate partner proteins including PduJK, PduB’, PduM, PduN and PduW expressed from pdu operon. 1 INTRODUCTION Cobalamin B12 Vitamin B12 which is also known as cyanocobalamin (CNCbl) is a precursor of two coenzymes, adenosylcobalamin (AdoCbl) and methylcobalamin (CH3Cbl). These coenzymes are required cofactors for a variety of enzymes found in bacteria and higher animals. CH3Cbl-dependent enzymes catalyze a variety of methyl transfer reactions and AdoCbl-dependent enzymes mediate difficult rearrangements and reductions via free radical mechanisms. Two human enzymes that require B12 are known and these enzymes are vital for good health. Among the Bacteria and Archaea, about 15 different B12-dependent enzymes have been reported and several have important biotechnology applications. Structure of AdoCbl AdoCbl has a molecular weight of 1580, and at least 25 enzymes are
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