On the phosphorylase activity of GH3 enzymes: A beta-N-acetylglucosaminidase from Herbaspirillum seropedicae SmR1 and a glucosidase from Saccharopolyspora erythraea

摘要

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-GIcNAcp (K-m = 0.24 mM, k(cat) = 1.2 s(-1), k(cat)/K-m = 5.0 mM(-1)s(-1)) than pNP-beta-D-Glcp (K-m = 33 mM, k(cat) = 33 x 10(-3) s(-1), k(cat)/K-m = 9 x 10(-4) mM(-1)s(-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-GIcNAc 1P and beta-DGlc 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. (C) 2016 Elsevier Ltd. All rights reserved.