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.
- Angewandte Chemie 01/2006; 118(37):6325-6328.
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ABSTRACT: Co(2+) binding to the nicotinamide adenine dinucleotide (NAD(+)) molecule in water solution was studied by electron paramagnetic resonance (EPR) and electron spin echo at low temperatures. Cobalt is coordinated by NAD(+) when the metal is in excess only, but even in such conditions, the Co/NAD(+) complexes coexist with Co(H2O)6 complexes. EPR spin-Hamiltonian parameters of the Co/NAD(+) complex at 6 K are g z = 2.01, g x = 2.38, g y = 3.06, A z = 94 × 10(-4) cm(-1), A x = 33 × 10(-4) cm(-1) and A y = 71 × 10(-4) cm(-1). They indicate the low-spin Co(2+) configuration with S = 1/2. Electron spin echo envelope modulation spectroscopy with Fourier transform of the modulated spin echo decay shows a strong coordination by nitrogen atoms and excludes the coordination by phosphate and/or amide groups. Thus, Co(2+) ion is coordinated in pseudo-tetrahedral geometry by four nitrogen atoms of adenine rings of two NAD(+) molecules.Applied Magnetic Resonance 07/2013; 44(7):817-826. · 0.83 Impact Factor
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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; · 1.19 Impact Factor