title: On the phosphorylase activity of GH3 enzymes: A β-N-acetylglucosaminidase from Herbaspirillum seropedicae SmR1 and a glucosidase from Saccharopolyspora erythraea.
abstract: 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 × 10-3s-1, kcat/Km= 9 × 10-4mM-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.
authors.affiliation: College of Pharmacy, Dalhousie University, 5968 College Street, P.O. Box 15000, Halifax, Nova Scotia B3H 4R2, Canada; Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Centro Politécnico, CEP 81-531-990, P.O. Box 19046, Curitiba, Paraná, Brazil.
authors.forename: Diogo R B
authors.initials: DR
authors.lastname: Ducatti
authors.affiliation: Department of Chemistry, Dalhousie University, 6274 Coburg Road, P.O. Box 15000, Halifax, Nova Scotia B3H 4R2, Canada.
authors.forename: Madison A
authors.initials: MA
authors.lastname: Carroll
authors.affiliation: College of Pharmacy, Dalhousie University, 5968 College Street, P.O. Box 15000, Halifax, Nova Scotia B3H 4R2, Canada; Department of Chemistry, Dalhousie University, 6274 Coburg Road, P.O. Box 15000, Halifax, Nova Scotia B3H 4R2, Canada. Electronic address: david.jakeman@dal.ca.
authors.forename: David L
authors.initials: DL
authors.lastname: Jakeman
mesh_terms: D000118:Acetylglucosaminidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D003001:Cloning, Molecular; D005959:Glucosidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D042803:Herbaspirillum / Q000201:enzymology* / Q000235:genetics; D006868:Hydrolysis; D010766:Phosphorylation; D016930:Saccharopolyspora / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D005959:Glucosidases; D000118:Acetylglucosaminidase
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)