NAD+ and Metal-ion Dependent Hydrolysis by Family 4 Glycosidases: Structural Insight into Specificity for Phospho-β-d-glucosides

Northwestern University, Evanston, Illinois, United States
Journal of Molecular Biology (Impact Factor: 4.33). 03/2005; 346(2):423-35. DOI: 10.1016/j.jmb.2004.11.058
Source: PubMed


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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    • "Except for AglTm (ASP260 and ARG 263) and AglBs (TYR265), residues belonging to region1 are situated at the interior of the proteins and are not involved in cofactor binding, catalytic activities or specific interactions with the substrates (Lodge et al., 2003; Leisch et al., 2012; Rajan et al, 2004; Varrot et al., 2005; Yip et al., 2004). Also, except for Gly290 of BglTm, residues belonging to region 2 are not involved in catalytic activities, but they are exposed to the solvent (Lodge et al., 2003; Leisch et al., 2012; Rajan et al, 2004; Varrot et al., 2005; Yip et al., 2004). It suggests that these structurally distinct regions may be involved in oligomerization processes or in other specific protein-protein interactions. "
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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; 60(4). · 1.15 Impact Factor
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    • "Significantly , and in contrast with Koshland's double-displacement mechanisms [8] [9] [10] [11], members of GH4 catalyze the hydrolysis of the glycosidic linkage via a novel sequence of oxidation–elimination–addition and reduction reactions [11] [12] [13] [14] [15] [16] [17]. The GH4 Family is also unique in that it includes five groups of enzymes [18], whose substrate specificities correlate remarkably with the presence of a four amino acid motif including the active-site Cysteine residue: 6-phospho-b-glucosidases, CN(V/I)P [5] [9] [11] [13], 6-phos- pho-a-glucosidases, CDMP [4] [12] [19] [20], a-glucosidases, CHEI [6] [7] [21], a-galactosidases, CH(S/G)V [3,22–24] and a-glucuronidases , CHGx [25]. In an earlier phylogenetic analysis of 201 GH4 enzymes , we noted a group of proteins of unknown catalytic activity with the motif CHEV (see Fig. 1A, Ref. [18]). "
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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; 587(6). DOI:10.1016/j.febslet.2013.02.004 · 3.17 Impact Factor
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    • "The four-residue sequence G(L/I)NH is conserved in all GH4 enzymes, and structural analyses of phospho-a-glucosidase (GlvA) and phospho-b-glucosidase (BglT) show that the His residue of this motif, as well as Cys in the Cys motif, are ''both'' coordinately linked to the catalytically essential Mn 2þ ion. The loss of these metal-binding residues clearly makes glycoside hydrolysis by the GH4 mechanism impossible (Rajan et al. 2004; Yip et al. 2004; Varrot et al. 2005). Although the A. laidlawii protein is phylogenetically solidly within the 6-phospho-b-glucosidase clade, our recent cloning and expression studies have shown that this protein is not only devoid of phospho-b-glucosidase activity, but it also exhibits no detectable activity to pNP-b-glucopyranoside , a-glucopyranoside, a-galactopyranoside, or a-mannopyranoside (J. "
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    ABSTRACT: 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.
    Molecular Biology and Evolution 08/2009; 26(11):2487-97. DOI:10.1093/molbev/msp162 · 9.11 Impact Factor
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