Acetylene Hydratase from Pelobacter acetylenicus Functional Studies on a Gas-Processing Tungsten, Iron-Sulfur Enzyme by Site Directed Mutagenesis and Crystallography Dissertation zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften Dr. rer. nat. vorgelegt von Dipl.-Biol. Felix ten Brink Konstanz, April 2010 1. Referent: Prof. Dr. P.M.H. Kroneck 2. Referent: Prof. Dr. O. Einsle Datum der mündlichen Prüfung: 09.04.2010 II Table of contents Table of contents Table of contents ....................................................................................................................I Zusammenfassung ................................................................................................................ V Summary.............................................................................................................................VII 1. Introduction ................................................................................................................... 1 1.1 Molybdenum and tungsten in biological systems ................................................. 1 Chemical properties and bioavailability of molybdenum and tungsten ........................ 1 Molybdenum and tungsten in organisms....................................................................... 2 Molybdenum and tungsten enzymes ............................................................................. 3 Molybdenum cofactor and enzyme classification ......................................................... 4 Heterologous expression of molybdopterin-dependent enzymes.................................. 7 Cofactor biosynthesis in Escherichia coli ..................................................................... 8 Chaperone mediated cofactor delivery........................................................................ 10 1.2 Iron sulfur clusters............................................................................................... 11 Cluster biosynthesis..................................................................................................... 12 1.3 Acetylene............................................................................................................. 13 Reactions of Acetylene................................................................................................ 14 Sources and bio-availability ........................................................................................ 15 1.4 Acetylene Hydratase from Pelobacter acetylenicus............................................ 16 Overall structure .......................................................................................................... 17 Active site.................................................................................................................... 18 Reaction mechanism.................................................................................................... 19 1.5 Scope of the study ............................................................................................... 20 2. Materials and Methods ................................................................................................ 23 2.1 Chemicals and biochemicals ............................................................................... 23 2.2 Acetylene Hydratase from P. acetylenicus.......................................................... 24 Cultivation of P. acetylenicus...................................................................................... 24 Purification .................................................................................................................. 25 Crystallization.............................................................................................................. 26 2.3 Heterologous Expression of AH in E. coli .......................................................... 30 Cloning ........................................................................................................................ 30 I Table of contents Transformation............................................................................................................ 30 Test expressions .......................................................................................................... 31 Addition of chaperone binding sites............................................................................ 32 Site directed mutagenesis............................................................................................ 33 Cultivation and expression of AH variants ................................................................. 34 Purification of AH variants ......................................................................................... 34 Crystallization of NarG-AH........................................................................................ 35 2.4 Further methods................................................................................................... 36 Experiments under exclusion of dioxygen.................................................................. 36 Determination of protein concentration ...................................................................... 36 Enzyme activity........................................................................................................... 37 SDS-PAGE.................................................................................................................. 38 Agarose gel electrophoresis ........................................................................................ 39 Qualitative and quantitative analysis of molybdenum cofactor.................................. 39 UV/Vis spectroscopy................................................................................................... 39 Circular dichroism spectroscopy................................................................................. 40 Metal analysis by ICP-MS .......................................................................................... 40 Sequence alignments................................................................................................... 40 3. Results ......................................................................................................................... 42 3.1 Acetylene Hydratase from P. acetylenicus ......................................................... 42 Cultivation................................................................................................................... 42 Purification.................................................................................................................. 43 Crystallization of acetylene hydratase from P. acetylenicus....................................... 45 3.2 Heterologous expression of AH in E. coli........................................................... 52 Addition of chaperone binding sites............................................................................ 53 Site directed mutagenesis............................................................................................ 54 Cultivation and expression of AH variants ................................................................. 56 Purification of heterologously expressed AH ............................................................. 57 Characterization of AH and variants........................................................................... 60 Molecular parameters of heterologously expressed AH and variants......................... 60 Crystallization of heterologously expressed NarG-AH .............................................. 66 4. Discussion ................................................................................................................... 68 Influence of MPD on activity of AH........................................................................... 68 II Table of contents 4.1 Crystallization of AH(W) and AH(Mo) .............................................................. 69 Incubation of crystals with C2H2 and CO.................................................................... 69 Soaking with acetaldehyde and propargylalcohol....................................................... 72 Crystallization of AH(Mo) .......................................................................................... 73 4.2 Expression of AH in E. coli................................................................................. 74 Molybdenum cofactor insertion and addition of chaperone binding sequences ......... 77 Control of protein folding with circular dichroism spectroscopy................................ 79 Site directed mutagenesis ............................................................................................ 81 Metal content and activity ........................................................................................... 82 Reaction mechanism and substrate binding..................................................................... 84 5. Conclusions ................................................................................................................. 87 5.1 Reactivity in crystallo................................................................................................ 87 5.2 Site directed mutagenesis of amino acid residues at the active site .......................... 87 6. References ................................................................................................................... 89 7. Acknowledgements ....................................................................................................