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Query: UNIPROT:P06889 (Mol)
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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.
J Mol Biol 2005 Feb 18
PMID:NAD+ and metal-ion dependent hydrolysis by family 4 glycosidases: structural insight into specificity for phospho-beta-D-glucosides. 1567 May 94

Glycosyl hydrolase Family 4 (GH4) is exceptional among the 114 families in this enzyme superfamily. Members of GH4 exhibit unusual cofactor requirements for activity, and an essential cysteine residue is present at the active site. Of greatest significance is the fact that members of GH4 employ a unique catalytic mechanism for cleavage of the glycosidic bond. By phylogenetic analysis, and from available substrate specificities, we have assigned a majority of the enzymes of GH4 to five subgroups. Our classification revealed an unexpected relationship between substrate specificity and the presence, in each subgroup, of a motif of four amino acids that includes the active-site Cys residue: alpha-glucosidase, CHE(I/V); alpha-galactosidase, CHSV; alpha-glucuronidase, CHGx; 6-phospho-alpha-glucosidase, CDMP; and 6-phospho-beta-glucosidase, CN(V/I)P. The question arises: Does the presence of a particular motif sufficiently predict the catalytic function of an unassigned GH4 protein? To test this hypothesis, we have purified and characterized the alpha-glucoside-specific GH4 enzyme (PalH) from the phytopathogen, Erwinia rhapontici. The CHEI motif in this protein has been changed by site-directed mutagenesis, and the effects upon substrate specificity have been determined. The change to CHSV caused the loss of all alpha-glucosidase activity, but the mutant protein exhibited none of the anticipated alpha-galactosidase activity. The Cys-containing motif may be suggestive of enzyme specificity, but phylogenetic placement is required for confidence in that specificity. The Acholeplasma laidlawii GH4 protein is phylogenetically a phospho-beta-glucosidase but has a unique SSSP motif. Lacking the initial Cys in that motif it cannot hydrolyze glycosides by the normal GH4 mechanism because the Cys is required to position the metal ion for hydrolysis, nor can it use the more common single or double-displacement mechanism of Koshland. Several considerations suggest that the protein has acquired a new function as the consequence of positive selection. This study emphasizes the importance of automatic annotation systems that by integrating phylogenetic analysis, functional motifs, and bioinformatics data, may lead to innovative experiments that further our understanding of biological systems.
Mol Biol Evol 2009 Nov
PMID:Evolution and biochemistry of family 4 glycosidases: implications for assigning enzyme function in sequence annotations. 1962 89