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: 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: The catalytic activity of the Family 4 glycosidase LplD protein, whose active site motif is CHEV, is unknown despite its crystal structure having been determined in 2008. Here we identify that activity as being an α-galacturonidase whose natural substrate is probably α-1,4-di-galacturonate (GalUA(2)). Phylogenetic analysis shows that LplD belongs to a monophyletic clade of CHEV Family 4 enzymes, of which four other members are also shown to be galacturonidases. Family GH 4 enzymes catalyze the cleavage of the glycosidic bond, via a non-canonical redox-assisted mechanism that contrasts with Koshland's double-displacement mechanism.FEBS letters 02/2013; · 3.54 Impact Factor
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ABSTRACT: In lactic acid bacteria and other bacteria, carbohydrate uptake is mostly governed by phosphoenolpyruvate-dependent phosphotransferase systems (PTSs). PTS-dependent translocation through the cell membrane is coupled with phosphorylation of the incoming sugar. After translocation through the bacterial membrane, the β-glycosidic bond in 6'-P-β-glucoside is cleaved, releasing 6-P-β-glucose and the respective aglycon. This reaction is catalyzed by 6-P-β-glucosidases, which belong to two glycoside hydrolase (GH) families: GH1 and GH4. Here, the high-resolution crystal structures of GH1 6-P-β-glucosidases from Lactobacillus plantarum (LpPbg1) and Streptococcus mutans (SmBgl) and their complexes with ligands are reported. Both enzymes show hydrolytic activity towards 6'-P-β-glucosides. The LpPbg1 structure has been determined in an apo form as well as in a complex with phosphate and a glucose molecule corresponding to the aglycon molecule. The S. mutans homolog contains a sulfate ion in the phosphate-dedicated subcavity. SmBgl was also crystallized in the presence of the reaction product 6-P-β-glucose. For a mutated variant of the S. mutans enzyme (E375Q), the structure of a 6'-P-salicin complex has also been determined. The presence of natural ligands enabled the definition of the structural elements that are responsible for substrate recognition during catalysis.Acta Crystallographica Section D Biological Crystallography 03/2013; 69(Pt 3):451-63. · 12.67 Impact Factor