The relationship between structure and specificity in the plant nitrilases

..................................................................................... 5 List of abbreviations...................................................................... 6 0.1 Introduction........................................................................... 10 0.1.1 Nitrilases..................................................................... 10 0.1.2 Industrial considerations................................................... 10 0.1.3 Structural biology of nitrilases............................................ 11 0.1.4 Physiological role of nitrilases............................................ 13 0.1.5 Aims.......................................................................... 15 0.2 Materials and methods............................................................... 16 0.2.1 Homology modelling....................................................... 16 0.2.2 Molecular biology........................................................... 16 0.2.2.1 Overlap extension PCR.......................................... 16 0.2.2.2 Site-directed mutagenesis....................................... 17 0.2.2.3 Transformation and sequencing................................. 19 0.2.3 Protein expression and purification for activity assays.................. 20 0.2.4 Protein expression and purification for electron microscopy........... 21 0.2.5 SDS-PAGE.................................................................... 22 0.2.6 Bradford assay................................................................ 22 0.2.7 Specific activity measurement............................................. 23 0.2.8 Photometric detection of free ammonia................................... 24 0.2.9 Negative-stain electron microscopy........................................ 25 0.2.10 Helical image processing.................................................. 25 0.2.11 Helical reconstruction...................................................... 25 0.2.12 Single-particle image processing......................................... 27 0.2.13 Single-particle reconstruction............................................. 27 0.2.14 Resolution estimation...................................................... 28 0.2.15 Data visualisation.......................................................... 28 0.2.16 Mutant list................................................................... 28 Un ive rsi ty of Ca pe To wn 
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 SECTION 1: Substrate specificity: modifying preferred substrate size and chemistry 1.1 Introduction.......................................................... 29 1.1.1 Interface formation............................................. 29 1.1.2 Substrate specificity........................................... 30 1.1.3 Aims............................................................. 32 1.1.4 Brief summary of findings.................................... 33 1.2 Results................................................................. 34 1.2.1 Interface disruption............................................ 34 1.2.2 The position of the substrate within the binding pocket.. 35 1.2.3 The role of quaternary structure.............................. 36 1.2.4 Nitrilase helical reconstruction............................... 36 1.2.5 Substrate size vs. helical twist................................ 39 1.2.6 Exchanging the helical twist.................................. 39 1.2.7 Interface formation and specificity.......................... 46 1.2.8 Active-site pocket mutations.................................. 47 1.2.9 Nitrilase 4 β-cyanoalanine binding........................... 53 1.3 Discussion............................................................. 56 1.3.1 Substrate specificity........................................... 56 1.3.2 Methodology................................................... 57 1.3.2.1 Helical reconstruction.................................. 57 1.3.2.2 Specific activity measurement........................ 57 1.3.3 Active site pocket specificity determinants................ 58 1.3.4 Interface disruption and inactivation........................ 59 SECTION 2: Amide/ acid ratio: reaction mechanism and modifying product ratio 2.1 Introduction.......................................................... 61 2.1.1 Mechanism...................................................... 61 2.1.2 Electronic effects............................................... 62 2.1.3 Amide:acid ratio of nitrilase enzymes...................... 63 2.1.4 Aims............................................................. 64 2.1.5 Brief summary of findings.................................... 64 Un ive rsi ty of Ca pe To wn 
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 2.2 Results................................................................. 66 2.2.1 Nitrilase active site............................................. 66 2.2.2 The unbound enzyme state.................................... 66 2.2.3 Tetrahedral intermediate state................................ 67 2.2.4 The nitrilase reaction model.................................. 67 2.2.5 Determinants of nitrilase amide:acid product ratio........ 72 2.2.6 S. alba NIT1c and S. arvensis NIT1c: effect of tyrosine. 72 2.2.7 Amide: acid ratio in nitrilase 4: effect of arg/lys.......... 74 2.3 Discussion............................................................. 76 2.3.1 Mechanism modelling......................................... 76 2.3.2 Determinants of nitrilase activity............................ 77 2.3.3 Nitrilase mechanism........................................... 78 2.3.4 Nitrilase mechanism: amide formation..................... 79 Conclusions and future work............................................................ 82 References.................................................................................... 85 Appendix..................................................................................... 96