Tungsten-acetylene hydratase from Pelobacter acetylenicus and molybdenum-transhydroxylase from Pelobacter acidigallici: Two novel molybdopterin and iron-sulfur containing enzymes Dissertation submitted to Fachbereich Biologie, Universität Konstanz, Germany for the degree of Doctor of Natural Sciences by Dipl.-Biol. Dietmar Josef Abt Konstanz, July 2001 Dissertation der Universität Konstanz Datum der mündlichen Prüfung: 10. September 2001 Referent: Prof. Dr. P.M.H. Kroneck Referent: Prof. Dr. B. Schink Für meine Eltern “... to boldly go where no man has gone before.“ (Star Trek) Table of Contents I Table of Contents Zusammenfassung...................................................................................................................... IV Summary....................................................................................................................................VII 1. Introduction......................................................................................................................1 1.1. Physical and chemical properties of molybdenum and tungsten .......................................1 1.2. The mononuclear molybdenum and tungsten enzymes .....................................................3 1.3. The molybdenum cofactor .................................................................................................6 1.4. Iron-sulfur centers..............................................................................................................7 1.5. Pelobacter acetylenicus acetylene hydratase.....................................................................9 1.5.1. Acetylene metabolism......................................................................................................10 1.5.2. Molecular properties of acetylene hydratase ...................................................................11 1.6. Pelobacter acidigallici transhydroxylase.........................................................................12 1.6.1. Metabolism of gallic acid by Pelobacter acidigallici......................................................12 1.6.2. Molecular properties of transhydroxylase........................................................................13 1.7. Scope of the study............................................................................................................15 2. Materials and Methods..................................................................................................16 2.1. Chemicals and biochemicals............................................................................................16 2.2. Organisms ........................................................................................................................17 2.2.1. Pelobacter acetylenicus ...................................................................................................17 2.2.2. Pelobacter acidigallici.....................................................................................................18 2.3. Cultivation of bacteria......................................................................................................18 2.3.1. Pelobacter acetylenicus ...................................................................................................18 2.3.2. Pelobacter acidigallici.....................................................................................................22 2.4. Glycerol cryo-cultures......................................................................................................23 2.5. Enzyme purification.........................................................................................................23 2.5.1. Acetylene hydratase.........................................................................................................24 2.5.2. Transhydroxylase.............................................................................................................25 2.6. Enzyme activity................................................................................................................26 2.6.1. Acetylene hydratase.........................................................................................................26 2.6.2. Transhydroxylase.............................................................................................................27 2.6.3. Alcohol dehydrogenase....................................................................................................28 2.7. UV/Vis spectroscopy.......................................................................................................28 2.8. EPR spectroscopy............................................................................................................29 2.9. Crystallization ..................................................................................................................29 2.10. Sequencing of the acetylene hydratase gene....................................................................30 2.10.1. N-terminal amino acid sequencing ..................................................................................30 2.10.2. Cyanogen bromide digestion ...........................................................................................31 2.10.3. DNA preparation..............................................................................................................31 2.10.3. Primers for polymerase chain reaction.............................................................................32 2.10.4. PCR techniques................................................................................................................32 2.10.5. Sequencing.......................................................................................................................35 2.10.6. Cloning.............................................................................................................................36 2.10.7. Computer programs and Internet websites.......................................................................37 II Table of Contents 2.10.8. Sequence handling and phylogenetic analysis.................................................................38 2.11. Analytical methods ..........................................................................................................38 2.11.1. ICP-MS ............................................................................................................................38 2.11.2. Protein ..............................................................................................................................39 2.11.3. Polyacrylamide gel electrophoresis.................................................................................39 2.11.4. Agarose gels.....................................................................................................................39 3. Results .............................................................................................................................40 3.1. Acetylene hydratase of Pelobacter acetylenicus .............................................................40 3.1.1. Growth of Pelobacter acetylenicus under various conditions .........................................40 3.1.2. Purification of acetylene hydratase..................................................................................41 3.1.3. Thermostability of acetylene hydratase, Y-ADH, and S-ADH .......................................45 3.1.4. Long-term study on acetylene hydratase activity ............................................................47 3.1.5. Metal content of acetylene hydratase...............................................................................47 3.1.6. UV/Vis spectra of acetylene hydratase ............................................................................48 3.1.7. EPR spectra of acetylene hydratase .................................................................................49 3.1.8. Identification, amplification, and sequencing of the acetylene hydratase gene structure via PCR based techniques................................................................................................53 3.1.9. Crystallization and three-dimensional structure of acetylene hydratase..........................61 3.1.10. Acetylene hydratase activity in Archaeoglobus fulgidus.................................................62 3.2. Transhydroxylase of Pelobacter acidigallici...................................................................63 3.2.1. Growth of Pelobacter acidigallici ...................................................................................63 3.2.2. Purification of transhydroxylase......................................................................................63 3.2.3. UV/Vis spectra of transhydroxylase ................................................................................65 3.2.4. EPR spectra of transhydroxylase .....................................................................................66 3.2.5. Crystallization and three dimensional structure of transhydroxylase ..............................68 3.3. Phylogenetic analysis of acetylene hydratase and transhydroxylase...............................69 3.3.1. The DMSO-reductase family...........................................................................................69 3.3.2. The DMSO-reductase subfamily .....................................................................................73 4. Discussion........................................................................................................................76