Towards the directed evolution of an -aspartate oxidase from L Pseudomonas putida Charlotte Leese PhD University of York Chemistry July 2012 Abstract Amino acid oxidases (AAOs) are enantioselective flavoenzymes that catalyse the oxidation of amino acids into imino acids, which spontaneously hydrolyse in water to form keto acids. AAOs have several potential applications, most notably as biocatalysts in the production of enantiomerically pure amino acids and keto acids, or in enzymatic biosensors. Therefore, there is a demand for a range of AAOs with specific activity against various substrates, encouraging the characterisation of less well understood oxidases. Seven putative oxidases were cloned into the pET-YSBLIC-3C expression vector, expressed in E. coli-DE3 expression strains and assayed for activity against all proteinogenic amino acids. Of these seven targets, the L-amino acid oxidase from Pseudomonas putida (PpLAAO) was found to be highly soluble, had detectable activity against L-aspartate and L-asparagine and had not been investigated in depth previously. The purified PpLAAO protein showed high substrate specificity against L-aspartate and lower activity with substrate inhibition against L-asparagine. Very low activity was also detected against L-glutamate. The purified protein had optimal activity around pH 7.5 and at temperatures between 4°C and 30°C. To investigate the role of residues in the active site area of the PpLAAO protein thirteen active site residues, determined by comparison with the structure of the L-aspartate oxidase from E. coli (L- AspO), were mutated to alanine. Eleven of these mutants were purified and assayed against L- aspartate, L-asparagine and L-glutamate. Results were largely consistent with knowledge of L- AspO. Ingenza Ltd. has an interest in potential applications of L-tyrosine and L-alanine oxidases. Because of this iterative combinatorial active site saturation testing, using the active structure of L-AspO was performed alongside a small scale epPCR mutagenesis in an attempt to introduce activity against L-alanine and L-tyrosine. L-homoserine was also targeted as part of a substrate walking approach towards L-alanine; however no novel activity was detected in any transformants. 1 Contents Abstract 1 Contents 2 Figures and Tables 6 List of Abbreviations 11 Acknowledgments 15 Declaration 15 Chapter 1: Introduction 16 1.1 Uses of enantiomerically pure amino acids and amines 16 1.1.1 Uses of enantiomerically pure L-amino acids 16 1.1.2 Uses of enantiomerically pure D-amino acids 21 1.1.3 Uses of enantiomerically pure amines 23 1.2 Production of enantiomerically pure amino acids 26 1.2.1 Extraction of L-amino acids 26 1.2.2 Chemical synthesis of amino acids 26 1.2.3 Biocatalytic production and racemization of amino acids 29 1.3 Amino acid oxidases 37 1.3.1 Structure and Activity of AAOs 37 1.3.2 Roles of AAOs in Nature 42 1.3.3 Applications of AAOs 44 1.4 Objectives of Project 49 1.4.1 Investigation of target oxidases 49 1.4.2 Evolution of new substrate activity 51 1.4.3 Summary 52 Chapter 2: Materials and Methods 53 2.1 General Section 53 2 2.1.1 Agarose gel electrophoresis 53 2.1.2 SDS-polyacrylamide gel electrophoresis (SDS-PAGE) 53 2.1.3 Transformation of chemically competent E. coli with plasmid 54 2.1.4 Transformation of electrocompetent E. coli with plasmid 55 2.1.5 Production of electrocompetent E. coli cells 56 2.1.6 Polymerase Chain Reaction (PCR) 57 2.2 Generation of target oxidases 63 2.2.1 PCR amplification of four target genes 63 2.2.2 Ligation Independent Cloning (LIC) 64 2.2.3 Restriction analysis of the plasmids 65 2.2.4 Analysis of recombinant genes by nucleotide sequencing 65 2.2.5 Overexpression analysis of target enzymes 66 2.2.6 Overexpression of target enzymes for assay analysis 67 2.2.7 Assay of target enzyme substrates 68 2.2.8 Effects of AIM, L-Glu and L-Arg on solubility of AsAAO and BaAO 69 2.2.9 Effects of oxygen availability on solubility of AsAAO and BaAO 70 2.2.10 Solubility screen of BaAO 71 2.3 Protein Purification and Characterisation 73 2.3.1 Overexpression of PpLAAO for protein purification 73 2.3.2 Purification of PpLAAO protein 74 2.3.3 Liquid phase assay of activity of wild type PpLAAO against substrates 76 2.3.4 Liquid phase assay of wild type PpLAAO against L-aspartate over pH 78 2.3.5 Liquid phase assay of WT PpLAAO against L-aspartate over temperature 78 2.3.6 Overexpression and purification of PpLAAO for crystallisation 79 2.3.7 Crystallisation screening of PpLAAO 79 2.3.8 Native gel and Dynamic Light Scattering analysis of wild type PpLAAO 80 2.3.9 Analysis of effects of different buffers on wild type PpLAAO 81 2.3.10 Effects of cold purification and G. HCl on PpLAAO quaternary form 83 2.4 Generation of active site residue mutants of PpLAAO 85 2.5 Purification and characterisation of active site PpLAAO mutants 87 2.5.1 Overexpression of active site PpLAAO mutant proteins for purification 87 2.5.2 Purification of active site PpLAAO mutant protein 87 2.5.3 Assay of active site PpLAAO mutant proteins against target substrates 88 2.6 Generation of SDSM CASTing libraries 90 2.6.1 Building of SDSM CASTing library A 90 2.6.2 Building of SDSM CASTing library D 93 2.6.3 Attempts to build SDSM libraries B, C & E using PfUTurbo Polymerase 94 3 2.6.4 PNDTE library SDSM reaction and transformation 95 2.6.5 Attempts to build SDSM libraries B, C and E using Phusion Polymerase 95 2.6.6 Attempts to build SDSM libraries B and C using KOD HotStart Polymerase 97 2.7 Random mutagenesis of wild-type PpLAAO gene sequence 99 2.8 Screening of epPCR and SDSM CASTing libraries 100 2.8.1 Solid Phase HRP assay of epPCR and SDSM CASTing libraries A and D 100 2.8.2 Analysis of potential hits from CASTing library A 102 2.8.3 Analysis of potential Screening Hits from CASTing library D 103 2.8.4 Analysis of three error-prone PCR mutants 104 Chapter 3: Cloning, Expression and Assay of Targets 106 3.1 Cloning of targets into pET-YSBLIC3C plasmid 109 3.2 Expression and solubility of targets 112 3.3 Activity assay of target oxidases 120 3.4 Attempts to increase solubility of AsAAO and BaAO 127 3.5 Conclusions 132 Chapter 4: Characterisation of PpLAAO protein 136 4.1 Purification of PpLAAO protein 136 4.2 Characterisation of PpLAAO protein 138 4.3 Attempts to crystallise PpLAAO protein 143 4.4 Conclusions 147 Chapter 5: Site Directed Mutagenesis of PpLAAO protein 150 5.1 Comparison of PpLAAO and E. coli L-AspO 150 5.2 Generation of active site mutants of PpLAAO 153 5.3 Purification of PpLAAO active site mutants 157 5.4 Activity assay of active site mutants 165 5.5 Discussion 170 Chapter 6: CASTing and epPCR of PpLAAO 175 6.1 Generation of SDSM CASTing libraries 177 6.2 Generation of PpLAAO epPCR library 193 4 6.3 Screening of mutagenesis libraries 195 6.3.1 Screening of PpLAAO CASTing library A 195 6.3.2 Screening of PpLAAO CASTing library D 197 6.3.3 Screening of PpLAAO error-prone PCR mutants 206 6.4 Conclusions 208 Chapter 7: General Discussion 214 Appendix A: C. glutamicum as an expression strain 217 A.1 Introduction 217 A.1.1 Physiology of C. glutamicum 217 A.1.2 History of C. glutamicum in the biochemical industry 217 A.1.3 Use of C. glutamicum in protein production and biotransformations 218 A.1.4 Use of C. glutamicum for expression of previous gene targets 218 A.2 Results 219 A.2.1 Attempts to clone AsAAO, BaAO & NfOR into pEK-Ex2 shuttle vector 219 A.2.2 Transformation and expression of pEKEx2-NfOR plasmid 219 A.3 Discussion 221 A.4 Materials and Methods 222 A.4.1 Transformation of electrocompetent E. coli with plasmid 222 A.4.2 Preparation of pEK-EX2 for cloning 223 A.4.3 Preparation of gene inserts for cloning into pEKEx2 plasmid 223 A.4.4 pEKEx2 ligation 225 A.4.5 Preparation of competent C. glutamicum cells 226 A.4.6 Transformation of C. glutamicum cells with pEK-Ex2-NfOR plasmid 227 A.4.7 Gene overexpression of transformed C. glutamicum cells 228 Appendix B: Alignment of Known and Target Oxidases 229 Bibliography 231 5 Figures and Tables Fig. 1.1: Conversion of D-methionine into L-methionine by animal and adult humans Fig. 1.2: Commercial methods for the synthesis of aspartame Fig. 1.3: L-homophenylalanine-ethyl ester and ACE inhibitor derivatives Fig. 1.4: Synthesis of β-lactam antibiotics from synthetic D-amino acids and 6-APA Fig. 1.5: Possible reaction scheme for the production of sweetener Alitame Fig. 1.6: Comparison of pyrethroid insecticide Fluvalinate and D-valine Fig. 1.7: Racemate resolution of (R,S)-thiazolidinone carboxylic acid Fig. 1.8: Example of enantioselective ketone deprotonation using a chiral lithium amide Fig. 1.9: Example of enantioselective epoxide rearrangement using a chiral lithium amide Fig. 1.10: Example of production of a (S)-4-(1-phenylethylamino)quinazoline Fig. 1.11: (S)-1-methoxy-2-aminopropane and its derivative herbicide FRONTIER X2® Fig. 1.12: Strecker synthesis method of producing racemic amino acids Fig. 1.13: Amidocarbonylation method for production of racemic N-acyl α-amino acids Fig. 1.14: Methionine synthesis method developed by Degussa Fig. 1.15: Asymmetric Strecker synthesis of an α-aminonitrile Fig. 1.16: Production scheme of D-phenylalanine by fermentation in E. coli Fig. 1.17: Resolution of an amino acid to its D-form using an L-amino acid oxidase Fig. 1.18: Synthesis of L-aspartate from fumarate using L-aspartate ammonia lyase Fig. 1.19: Synthesis of L-phenylalanine using L-phenylalanine ammonia lyase Fig. 1.20: Synthesis of L-alanine from L-aspartate using L-aspartate β-decarboxylase Fig. 1.21: Degussa’s method for production of L-tert-leucine using leucine dehydrogenase Fig. 1.22: Great Lakes Chemicals’ production method for L- and D-2-aminobutyric acid Fig. 1.23: Method for production of amino acids by racemization of hydantoins Fig. 1.24: Fermentation route for the production of D-phenylglycine Fig. 1.25: Production of L-cysteine from D,L-ATC Fig. 1.26: Resolution of D,L-amino acids using acylases Fig. 1.27: Reaction scheme for an L-amino acid oxidase Fig. 1.28: Possible reaction mechanisms for the oxidation by amino acid oxidases Fig. 1.29: Superimposition of active site area of MAO-N and RoLAAO Fig. 1.30: LigPlot+ comaprison of MAO-N-D5, RoLAAO and MAO-B Fig. 1.31: Mechanism of oxidation of L-aspartate by the E. coli L-aspO Fig. 1.32: Synthesis of Quinolinic acid by L-aspartate oxidase and quinolinate synthase Fig. 1.33: Conversion of cephalosporin C into 7-ACA using D-AAO and a glutaryl hydrolase 6 Fig. 2.1: 96 well plate HRP-assay layout Fig. 2.2: Standard Bradford calibration Fig. 2.3: Layout of well solutions in 24-well crystallisation screen of PpLAAO protein. Fig. 2.4: Example of solid phase HRP-based oxidase assay Fig. 3.1: Sequence alignment seven targets selected to be cloned into pET-YSBLIC3C Fig. 3.2: Cloning region of pET28a and pET-YSBLIC-3C vectors Fig. 3.3: Agarose gel of amplified target oxidase gene sequences Fig. 3.4: Agarose gel of restriction endonuclease check of cloned targets Fig. 3.5: SDS-PAGE of induced cells transformed with non recombinant pET-YSBLIC3C Fig. 3.6: SDS-PAGE of induced cells transformed with pET-YSBLIC3C-AsAAO Fig. 3.7: SDS-PAGE of induced cells transformed with pET-YSBLIC3C-BaAO Fig. 3.8: SDS-PAGE of induced cells transformed with pET-YSBLIC3C-BsGO Fig. 3.9: SDS-PAGE of induced cells transformed with pET-YSBLIC3C-NfOR Fig. 3.10: SDS-PAGE of induced cells transformed with pET-YSBLIC3C-PpLAAO Fig. 3.11: SDS-PAGE of induced cells transformed with pET-YSBLIC3C-RoLAAO Fig. 3.12: SDS-PAGE of induced cells transformed with pET-YSBLIC3C-SLAAO Fig. 3.13: Example of 96-well HRP-based activity assay Fig. 3.14: Activity screen of induced cells transformed with pET-YSBLIC3C plasmid Fig. 3.15: Activity screen of induced pET-YSBLIC3C-AsAAO cells Fig. 3.16: Activity screen of induced cells transformed with pET-YSBLIC3C-BaAO plasmid Fig. 3.17: Activity screen of induced cells transformed with pET-YSBLIC3C-BsGo plasmid Fig. 3.18: Activity screen of induced cells transformed with pET-YSBLIC3C-NfOR plasmid Fig. 3.19: Activity screen of induced pET-YSBLIC3C-PpLAAO cells Fig. 3.20: Activity screen of induced pET-YSBLIC3C-SLAAO cells Fig. 3.21: SDS-PAGE of solubilisation attempts of BL21-pET-YSBLIC3C-AsAAO protein Fig. 3.22: SDS-PAGE of solubilisation attempts of BL21-pET-YSBLIC3C-BaAO protein Fig. 3.23: SDS-PAGE of effects of O on BL21-pET-YSBLIC3C-AsAAO/BaAO protein 2 Fig. 3.24: SDS-PAGE of Lindwall solubility screen of BL21-pET-YSBLIC3C-BaAO protein Fig. 3.25: SDS-PAGE of scaled up BL21-pET-YSBLIC3C-BaAO protein production Fig. 4.1: SDS-PAGE of protein purified from E. coli BL21-pET-YSBLIC3C-PpLAAO cells Fig. 4.2: Elution peak of PpLAAO through 120 mL HiLoad 16/60 Superdex 75 column Fig. 4.3: Initial rates of purified PpLAAO against varying concentration of L-aspartate Fig. 4.4: Initial rates of purified PpLAAO against varying concentration of L-asparagine Fig. 4.5: Initial rates of purified PpLAAO against varying concentration of L-glutamate Fig. 4.6: Activity assay of purified PpLAAO against L-aspartate over reaction buffer pH 7 Fig. 4.7: Activity assay of purified PpLAAO against L-aspartate over protein temperature Fig. 4.8: 7.5% Native gel separation of purified wild type PpLAAO Fig. 4.9: 7.5% native gel separation of wild-type PpLAAO protein in various buffers Fig. 5.1: Sequence alignment of the L-aspartate oxidases from P. putida and E. coli Fig. 5.2: Active site residues of the E. coli L-AspO structure that were targeted for mutation Fig. 5.3: SDS-PAGE of protein from induced cells transformed with Q242A1-10 plasmids Fig. 5.4: SDS-PAGE of protein from induced H244A1, P245A1-3 and E260A1-2 cells Fig. 5.5: SDS-PAGE of protein from L257A1-3, R290A1-3, Y352A1-2 and S391A1-2 cells Fig. 5.6: SDS-PAGE of protein from induced cells transformed with T259A1-10 plasmids Fig. 5.7: SDS-PAGE of protein from induced V293A1-5 and S389A1-5 cells Fig. 5.8: SDS-PAGE of protein from induced H351A1-2 and R386A1-2 cells Fig. 5.9: SDS-PAGE of attempted purification of PpLAAO-E260A protein Fig. 5.10: SDS-PAGE of purified BL21-pET-YSBLIC3C-PpLAAO-Q242A protein Fig. 5.11: SDS-PAGE of purified BL21-pET-YSBLIC3C-PpLAAO-H244A protein Fig. 5.12: SDS-PAGE of purified BL21-pET-YSBLIC3C-PpLAAO-P245A protein Fig. 5.13: SDS-PAGE of purified BL21-pET-YSBLIC3C-PpLAAO-L257A protein Fig. 5.14: SDS-PAGE of purified BL21-pET-YSBLIC3C-PpLAAO-T259A protein Fig. 5.15: SDS-PAGE of purified BL21-pET-YSBLIC3C-PpLAAO-R290A protein Fig. 5.16: SDS-PAGE of purified BL21-pET-YSBLIC3C-PpLAAO-H351A protein Fig. 5.17: SDS-PAGE of purified BL21-pET-YSBLIC3C-PpLAAO-Y352A protein Fig. 5.18: SDS-PAGE of purified BL21-pET-YSBLIC3C-PpLAAO-R386A protein Fig. 5.19: SDS-PAGE of purified BL21-pET-YSBLIC3C-PpLAAO-S389A protein Fig. 5.20: SDS-PAGE of purified BL21-pET-YSBLIC3C-PpLAAO-S391A protein Fig. 5.21: Kinetics plots of activity PpLAAO-based proteins against L-aspartate Fig. 5.22: Unfitted kinetics data of PpLAAO-based proteins against L-aspartate Fig. 5.23: Unfitted kinetics data of PpLAAO-based proteins against L-asparagine Fig. 5.24: Kinetics plots of activity PpLAAO-based proteins against L-glutamate Fig. 6.1: Substrate walking from L-aspartate to L-alanine using L-homoserine Fig. 6.2: Active site of E. coli L-aspartate oxidase showing library CASTing groups Fig. 6.3: Agarose gel of PCR amplification of purified pET-YSBLIC-3C-PNDTA plasmids Fig. 6.4: Multiple sequence alignment of Wild Type PpLAAO and PNDTA mutants Fig. 6.5: SDS-PAGE of induced cells transformed with pET-YSBLIC-3C-PNDTAA-D Fig. 6.6: Agarose gel of SDSM reactions using PNDTA primer set Fig. 6.7: SDS-PAGE of induced cells transformed pET- YSBLIC-3C-PNDTA70A-I 8