Sequence comparisons between beta-glucosidases of the same family show that residues Glu183 and Glu397 are highly conserved. Both residues are positioned at the end of a pocket located at the C terminus of the barrel and have been assigned the respective roles of proton donor and nucleophile on the basis of inhibitor-binding and mutagenesis experiments. These roles are consistent with the environments of the two residues. The pocket itself is typical of a sugar-binding site as it contains a number of charged, aromatic and polar groups. In support of this role, we present crystallographic data on a possible product complex between CBG and glucose, resulting from co-crystallization of the native enzyme with its natural substrate, linamarin.
Bile salt hydrolase (BSH) is an enzyme produced by the intestinal microflora that catalyzes the deconjugation of glycine-or taurine-linked bile salts. The crystal structure of BSH reported here from Bifidobacterium longum reveals that it is a member of N-terminal nucleophil hydrolase structural superfamily possessing the characteristic ␣␣ tetra-lamellar tertiary structure arrangement. Site-directed mutagenesis of the catalytic nucleophil residue, however, shows that it has no role in zymogen processing into its corresponding active form. Substrate specificity was studied using Michaelis-Menten and inhibition kinetics and fluorescence spectroscopy. These data were compared with the specificity profile of BSH from Clostridium perfrigens and pencillin V acylase from Bacillus sphaericus, for both of which the three-dimensional structures are available. Comparative analysis shows a gradation in activity toward common substrates, throwing light on a possible common route toward the evolution of pencillin V acylase and BSH.
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