NAD+ and Metal-ion Dependent Hydrolysis by Family 4 Glycosidases: Structural Insight into Specificity for Phospho-β-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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- "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. "
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; · 1.39 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. "
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 · 14.31 Impact Factor
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- "Gene identifier Gene name Original annotation Current annotation Family Crystal Structure Reference(s) TM0076 xyl3 Xylosidase b-Xylosidase GH3 Xue & Shao (2004) TM0209 pfk 6-PFK phosphofructokinase 6-PFK phosphofructokinase, ATP-dependent Ding et al. (2001) TM0281 a-L-Arabinofuranosidase a-L-Arabinofuranosidase GH51 Miyazaki (2005) TM0289 pfp 6-Phosphofructokinase, pyrophosphate-dependent 6-Phosphofructokinase, pyrophosphate-dependent Ding et al. (2001) TM0306 a-L-Fucosidase, putative a-L-Fucosidase GH29 1HL8, 1HL9, 1ODU Tarling et al. (2003), Sulzenbacher et al. (2004) TM0434 agu4A a-Glucosidase a-Glucuronidase GH4 Suresh et al. (2002) TM0437 pelB Exopolygalacturonase Exopolygalacturonase GH28 Kluskens et al. (2005) TM0504 Hypothetical protein Putative signaling peptide Johnson et al. (2005b) TM0539 Tryptophan synthase, b subunit Indole rescue protein Hettwer & Sterner (2002) TM0653 miaB Conserved hypothetical protein t-RNA methylthiotransferase Pierrel et al. (2003) TM0752 agu4B a-Glucosidase a-Glucuronidase GH4 1VJTA Suresh et al. (2003) TM0841 S-Layer like array protein Fatty-acid binding protein DegV/ COG1307 Schulze-Gahmen et al. (2003) TM0875 Hypothetical protein YggU-like protein YggU-like 1O22 Bakolitsa et al. (2004) TM0913 mazG mazG protein Pyrophosphatase, nucleotide triphosphate pyrophosphorylase, MazG Zhang et al. (2003) TM1062 gusB b-Glucuronidase b-Glucuronidase GH2 Salleh et al. (2006) TM1068 a-Glucosidase a-Glucuronidase (499% id/466 aa with TM0434) GH4 Suresh et al. (2002) TM1192 gal36A a-Galactosidase a-Galactosidase GH36 1ZY9 Liebl et al. (1998b) TM1193 lacZ b-Galactosidase b-Galactosidase GH2 Kim et al. (2004) TM1201 Arabinogalactan endo-1,4-b- galactosidase, putative Endo-b-1,4-galactanase GH53 Yang et al. (2006) TM1267 hydG thiH protein, putative Putative hydrogenase maturation protein Rubach et al. (2005) TM1269 hydE Biotin synthetase, putative Putative hydrogenase maturation protein Rubach et al. (2005) TM1281 bglT 6-P-b-Glucosidase 6-P-b-Glucosidase GH 4 Varrot et al. (2005), Yip et al. (2004) TM1371 iscU "
ABSTRACT: High-throughput sequencing of microbial genomes has allowed the application of functional genomics methods to species lacking well-developed genetic systems. For the model hyperthermophile Thermotoga maritima, microarrays have been used in comparative genomic hybridization studies to investigate diversity among Thermotoga species. Transcriptional data have assisted in prediction of pathways for carbohydrate utilization, iron-sulfur cluster synthesis and repair, expolysaccharide formation, and quorum sensing. Structural genomics efforts aimed at the T. maritima proteome have yielded hundreds of high-resolution datasets and predicted functions for uncharacterized proteins. The information gained from genomics studies will be particularly useful for developing new biotechnology applications for T. maritima enzymes.FEMS Microbiology Reviews 12/2006; 30(6):872-905. DOI:10.1111/j.1574-6976.2006.00039.x · 13.81 Impact Factor