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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

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. "
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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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    • "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 "
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