Novel Catalytic Mechanism of Glycoside Hydrolysis Based on the Structure of an NAD+/Mn2+-Dependent Phospho-α-Glucosidase from Bacillus subtilis

Molecular Pharmacology and Biological Chemistry, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA.
Structure (Impact Factor: 5.62). 10/2004; 12(9):1619-29. DOI: 10.1016/j.str.2004.06.020
Source: PubMed


GlvA, a 6-phospho-alpha-glucosidase from Bacillus subtilis, catalyzes the hydrolysis of maltose-6'-phosphate and belongs to glycoside hydrolase family GH4. GH4 enzymes are unique in their requirement for NAD(H) and a divalent metal for activity. We have determined the crystal structure of GlvA in complex with its ligands to 2.05 A resolution. Analyses of the active site architecture, in conjunction with mechanistic studies and precedent from the nucleotide diphosphate hexose dehydratases and other systems, suggest a novel mechanism of glycoside hydrolysis by GlvA that involves both the NAD(H) and the metal.

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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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    • "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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    • "A feature of almost all classical CAZy families is that, since sequence dictates structure, and structure determines function, the catalytic mechanism is conserved within a sequence-based family (Henrissat and Davies, 1997). Exceptions to this rule are rare and unusual: GH4 and GH109 enzymes are not classical hydrolases, but instead use NAD + in a transient reduction/oxidation reaction with leaving group elimination (Rajan et al., 2004), and GH23 is a family with both inverting and retaining hexosaminidases, but the catalytic mechanism of neither is understood, and may involve substrate participation in catalysis. The α-glucosidase encoded by the susB gene belongs to family GH97 (Hughes et al., 2003) of this Carbohydrate Active enZymes (CAZy) classification (, the catalytic mechanism of which was unknown prior to this work, but had been predicted to be retaining in an insightful bioinformatics analysis (Naumoff, 2005). "
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    ABSTRACT: Enzymatic cleavage of the glycosidic bond yields products in which the anomeric configuration is either retained or inverted. Each mechanism reflects the dispositions of the enzyme functional groups; a facet of which is essentially conserved in 113 glycoside hydrolase (GH) families. We show that family GH97 has diverged significantly, as it contains both inverting and retaining alpha-glycosidases. This reflects evolution of the active center; a glutamate acts as a general base in inverting members, exemplified by Bacteroides thetaiotaomicron alpha-glucosidase BtGH97a, whereas an aspartate likely acts as a nucleophile in retaining members. The structure of BtGH97a and its complexes with inhibitors, coupled to kinetic analysis of active-site variants, reveals an unusual calcium ion dependence. 1H NMR analysis shows an inversion mechanism for BtGH97a, whereas another GH97 enzyme from B. thetaiotaomicron, BtGH97b, functions as a retaining alpha-galactosidase.
    Chemistry & Biology 11/2008; 15(10):1058-67. DOI:10.1016/j.chembiol.2008.09.005 · 6.65 Impact Factor
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