A phosphorolytic activity has been reported for beta-N-acetylglucosaminidases from glycoside hydrolase family 3 (GH3) giving an interesting explanation for an unusual histidine as catalytic acid/base residue and suggesting that members from this family may be phosphorylases [J. Biol. Chem. 2015, 290, 4887]. Here, we describe the characterization of Hsero1941, a GH3 beta-N-acetylglucosaminidase from the endophytic nitrogen-fixing bacterium Herbaspirillum seropedicae SmR1. The enzyme has significantly higher activity against pNP-beta-D-GlcNAcp (Km = 0.24 mM, kcat = 1.2 s-1, kcat/Km = 5.0 mM-1s-1) than pNP-beta-D-Glcp (Km = 33 mM, kcat = 3.3 × 10-3 s-1, kcat/Km = 9 × 10-4 mM-1s-1). The presence of phosphate failed to significantly modify the kinetic parameters of the reaction. The enzyme showed a broad aglycone site specificity, being able to hydrolyze sugar phosphates beta-D-GlcNAc 1P and beta-D-Glc 1P, albeit at a fraction of the rate of hydrolysis of aryl glycosides. GH3 beta-glucosidase EryBI, that does not have a histidine as the general acid/base residue, also hydrolyzed beta-D-Glc 1P, at comparable rates to Hsero1941. These data indicate that Hsero1941 functions primarily as a hydrolase and that phosphorolytic activity is likely adventitious. The prevalence of histidine as a general acid/base residue is not predictive, nor correlative, with GH3 beta-N-acetylglucosaminidases having phosphorolytic activity.
Keywords: 2,4-Dinitrophenyl 2-fluoro-2-deoxy-beta-D-glucopyranoside (PubChem CID:445227); 2-acetamido-2-deoxy-α-D-glucopyranosyl fluoride (PubChem CID:53906163); 4-nitrophenyl 2-acetamido-2-deoxy-β-D-glucopyranoside (PubChem CID:102416); 4-nitrophenyl β-D-glucopyranoside (PubChem CID:92930); Chemical compounds; GH3 N-acetylglucosaminidases; Glycosidase; Glycoside hydrolase; Herbaspirillum seropedicae SmR1; Phospho-sugar; β-D-glucopyranosyl phosphate (PubChem CID:122250).
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