Final Report Summary - XYLANASES (Xylanases as models for understanding enzymatic catalysis) In this project the fellow (Dr Martin A. Fascione, MAF) has adopted a multidisciplinary approach to determine how subtle changes to TS configurations are transmitted from remote sites in enzymes. With a particular focus on the training MAF in a range of new chemical biology techniques including: molecular biology (protein cloning, expression and purification); protein semisynthesis (intein-mediated protein semisynthesis, incorporation of unnatural amino acids); enzyme kinetics; protein NMR, all during the outgoing phase with Professor Stephen G. Withers, FRS at the University of British Columbia, Canada (UBC); protein X-ray crystallography, during the reintegration phase with Professor Gideon J. Davies FRS at the University of York, UK.The primary goal of the project was to explore how remote parts of a protein contribute to catalysis. Glycosidases are particularly appropriate model systems for such studies since they are ubiquitous, amenable to kinetic, structural and mutagenic studies and well organised into sequence defined glycoside hydrolase (GH) families.4 The xylanases from families GH10 and GH11, including Bacillus circulans xylanase (Bcx, GH11, 20 kDa) have been established by the groups of Professor Stephen G. Withers and Professor Gideon J. Davies as among the best mechanistically and structurally characterised enzymes, making them ideal systems with which to probe the roles specific residues play in catalysis by using a combination of mutagenesis, protein semisynthesis, enzyme kinetics, protein NMR and X-ray crystallography. The individual objectives initially highlighted to help achieve this goal were:Objective 1: Identify a set of 2-3 remote mutations that each yield stable mutants with substantially altered activity for expression and purification (UBC).Objective 2: Perform detailed kinetic studies on mutants.Objective 3: Use protein NMR to characterise mutants.Objective 4: Investigate protein semisynthesis strategies using unnatural amino acids and circularly permutated Bcx constructs. Objective 5: Perform detailed kinetic and protein NMR studies on cpBcx mutants containing unnatural amino acids (UBC/York).Objective 6: Use X-ray studies to determine 3D structures of selected mutants (York).During the course of the project to date MAF has focussed his research on the xylanase from Bacillus circulans (Bcx). Initial training in molecular biology/cloning and protein expression was provided within the Withers lab, as well as on attendance at the one-week long AMBL Molecular Biology Workshop held by the Michael Smith Laboratories at UBC. These new skills were developed in the cloning and expression of a number of Bcx mutants which were then screened for putative glycosynthase activity. Following training in molecular cloning MAF expressed and purified wild type Bcx for enzyme kinetic characterisation, with in-house Withers lab training. The activity of Bcx was characterised using michaelis-menten kinetics with a range of xylobioside substrates, including 3,4-dinitrophenyl xylobioside (DNPX2) and p-nitrophenyl xylobioside (PNPX2), a pH profile of activity was also measured using DNPX2. Inactivation and reactivation kinetics were also measured using covalent inactivator 2-fluoro-2,5-dinitrophenyl xylobioside (2F-DNPX2). Following studies on wild type Bcx, two tyrosine residues in Bcx were then targeted for mutation. Plasmids containing TAG amber stop codon mutations were constructed for the purpose of incorporating unnatural amino acids into Bcx using in vivo unnatural amino acid mutagenesis. The plasmid of interest was co-transformed into BL21 cells alongside pEVOL plasmids containing two copies of an evolved orthogonal tyrosyl tRNA synthetase and tRNA from M. jannaschii. These plamdis were kind gifts from the Stubbe group at MIT, and facilitated the site specific incorporation of either 3-fluoro tyrosine or 3-nitro tyrosine to afford Bcx mutants. These mutants were also characterised by a full range of enzyme kinetic experiments, including pH profiles, inactivation and reactivation kinetics. Selected mutants were also characterised by 19F-protein NMR, with training and collaboration from the McIntosh group at UBC. Although attempted titrations of the catalytic glutamic acid residues using 19F-NMR afforded ambiguous results to date, it was possible to follow the inactivation and reactivation of the Bcx mutants in real time, using 2F-DNPX2. The main results achieved so far in the project therefore include the training of MAF in protein cloning and expression, enzyme kinetics and protein NMR, alongside the first application of in vivo site-specific unnatural amino acid mutagenesis on a carbohydrate active enzyme (CAZyme). This methodology facilitated the study of two tyrosines in Bcx. The incorporation of 3F-Tyr and 3-NO2 Tyr into these positions allowed the modulation of the pKa of the phenol groups in these residues, as well as the pKas of the active site nucleophile E78 and the acid/base E172. Although the mutants ranged from between 55 fold to 8 fold less active than wt Bcx, it was noted that the one mutation had a greater effect on the rate of inactivation (25 fold slower than wt), while the other mutation had a greater effect on the rate of reactivation and no significant effect on the rate of inactivation. These results were also confirmed by 19F-protein NMR using 2F-DNPX2 as an inactivator. The nature of the effect resulting from mutation of these Tyr residues will be more transparent following structural studies using the X-ray crystallography facilities available during the return phase of the project in York, UK. It is anticipated that crystallisation studies on mutants in the presence and absence of inactivator 2F-DNPX2 will provide insight into its modulation of inactivation, which is considered a reporter of the first part of the enzyme mechanism- formation of the covalent intermediate. The major objective of period 2 of the project was to complete the kinetic characterisation of Bcx mutants, and establish robust protein expression conditons which yielded enough protein for x-ray crystallography studies. Studies were also ongoing towards the semi synthesis of Bcx using circularly permutated mutant scaffolds, using constructs generated in period 1, however protein expression trials were unsuccessful in increasing the levels of ciruclarly permutated protein production. Anticipating difficulties with x-ray crystallography trials with only small amounts of protein, the project was refocussed upon expression, crystallisations and characteristion of the Bcx analogues containing unnatural amino acids. We also produced mutant constructs of previously crystallised sialisdase proteins, for expression of unnatural amino acid containing sialidases. Extensive protein expression trials with these two proteins established optimum conditions for unnatural amino acid incorporation into carbohydrate active enzymes, and therefore the highest protein yields for subsequent x-ray crystallography trials. Although Bcx has been crystallised on many occasions previously, to date extensive crystallisation trials have yet to produce crystals of unnatural amino acid containing proteins that diffract to afford structures at appropriate resolution. It is anticipated that further crystal trials will afford reproducible crystallisation conditions that will faciliate the acquisition of structural data which will help to clarify the kinetic affects of the incorporation of unnatural amino acids at key residues, and conclude the project.