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Structural studies of the catalytic mechanism of bacillus circulans xylanase Sidhu, Gary

Abstract

The goals of the work described in this thesis were to gain further insight into the structural aspects of the catalytic mechanism of the retaining β-1,4-xylanase from Bacillus circulans. This included the study of both the wild type and certain variants of this enzyme as well as glycosyl-enzyme catalytic intermediates. The 1.6 A resolution recombinant wild type enzyme structure determined in this thesis is very similar to the previously published 1.49 A resolution structure, especially with respect to the residues in the active site. In fact, differences between the two models are restricted to the conformations of mobile surface residues. The structure of Asn35Asp BCX, determined to a resolution of 1.55 A, confirms that the hydrogen bond between residue 35 and the acid/base catalyst, Glu172, is maintained despite the asparagine to aspartate substitution. This hydrogen bond is expected to monopolize the proton on the side chain carboxyl group of Glu172 at pH values above the pKa of Asp35 in a manner that will disrupt catalysis and thereby modify the pH optimum of the enzyme. In the 1.5 A resolution structure of the catalytically inactive Tyr69Phe variant, it is observed, rather surprisingly, that the conformations of residues hydrogen bonded to the phenolic oxygen of Tyr69 in the wild type enzyme, including the catalytic nucleophile, Glu78, change little. These results disprove an earlier hypothesis suggesting that the role of Tyr69 in this enzyme is to correctly position the catalytic nucleophile for attack of the substrate. The structures of the wild type and the Asn35Asp glycosyl-enzyme intermediates were determined to a resolution of 1.8 A. The 2-fluoro-xylose residue bound in the -1 subsite adopts a 2.5B (boat) conformation, allowing atoms C5, O5, C1, and C2 of the sugar to achieve coplanarity as required at the oxocarbenium ion-like transition states of the double-displacement catalytic mechanism. Comparison of the glycosyl-enzyme intermediates to a mutant of this same enzyme non-covalently complexed with xylotetraose reveals a number of differences beyond the distortion of the sugar moiety. Most notably, a bifurcated hydrogen bond is formed in the glycosyl-enzyme intermediates involving the OH atom of Tyr69, the endocyclic oxygen atom (O5) of the xylose residue in the -1 subsite, and atom OE2 of the catalytic nucleophile, Glu78. Since mutation of Tyr69 to phenylalanine produces an inactive enzyme, it is suggested that the interactions involving the phenolic oxygen of Tyr69, O5 of the proximal saccharide, and Glu78 OE2 are important for the catalytic mechanism of this enzyme and, through charge redistribution, serve to stabilize the oxocarbenium-like ion of the transition state. Overall, studies of the covalent glycosyl-enzyme intermediates of this xylanase provide insights into specificity, as contacts with O5 of the proximal saccharide exclude the hydroxymethyl group of glucose-based substrates, and the mechanism of catalysis, including aspects of stereoelectronic theory as applied to glycoside hydrolysis. [Scientific formulae used in this abstract could not be reproduced.]

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