NAD(+) and metal-ion dependent hydrolysis by family 4 glycosidases: Structural insight into specificity for phospho-beta-D-glucosides
ABSTRACT The import of disaccharides by many bacteria is achieved through their simultaneous translocation and phosphorylation by the phosphoenolpyruvate-dependent phosphotransferase system (PEP-PTS). The imported phospho-disaccharides are, in some cases, subsequently hydrolyzed by members of the unusual glycoside hydrolase family GH4. The GH4 enzymes, occasionally found also in bacteria such as Thermotoga maritima that do not utilise a PEP-PTS system, require both NAD(+) and Mn(2+) for catalysis. A further curiosity of this family is that closely related enzymes may show specificity for either alpha-d- or beta-d-glycosides. Here, we present, for the first time, the three-dimensional structure (using single-wavelength anomalous dispersion methods, harnessing extensive non-crystallographic symmetry) of the 6-phospho-beta-glycosidase, BglT, from T.maritima in native and complexed (NAD(+) and Glc6P) forms. Comparison of the active-center structure with that of the 6-phospho-alpha-glucosidase GlvA from Bacillus subtilis reveals a striking degree of structural similarity that, in light of previous kinetic isotope effect data, allows the postulation of a common reaction mechanism for both alpha and beta-glycosidases. Given that the "chemistry" occurs primarily on the glycone sugar and features no nucleophilic attack on the intact disaccharide substrate, modulation of anomeric specificity for alpha and beta-linkages is accommodated through comparatively minor structural changes.
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ABSTRACT: Structural bioinformatics approaches applied to the alpha- and beta-glycosidases from the GH4 enzyme family reveal that, despite low sequence identity, these enzymes possess quite similar global structural characteristics reflecting a common reaction mechanism. Locally, there are a few distinctive structural characteristics of GH4 alpha- and beta-glycosidases, namely, surface cavities with different geometric characteristics and two regions with highly dissimilar structural organizations and distinct physicochemical properties in the alpha- and beta-glucosidases from Thermotoga maritima. We suggest that these structurally dissimilar regions may be involved in specific protein-protein interactions and this hypothesis is sustained by the predicted distinct functional partners of the investigated proteins. Also, we predict that alpha- and beta-glycosidases from the GH4 enzyme family interact with difenoconazole, a fungicide, but there are different features of these interactions especially concerning the identified structurally distinct regions of the investigated proteins.Acta biochimica Polonica 12/2013; · 1.39 Impact Factor
Angewandte Chemie 09/2006; 118(37):6325-6328. DOI:10.1002/ange.200601421
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ABSTRACT: A recombinant glycoside hydrolase family 109 α-N-acetylgalactosaminidase from the pathogenic bacteria E. meningosepticum catalyses the hydrolysis of aryl 2-acetamido-2-deoxy-α-D-galactopyranosides. The sensitivities to leaving group abilities (βlg values) on V and V/K are −0.08 ± 0.06 and −0.31 ± 0.12, respectively. These results are consistent with an E2 elimination following hydride transfer from C3.Medicinal Chemistry Communication 07/2014; 5(8). DOI:10.1039/C4MD00104D · 2.63 Impact Factor