title: Asparagine-127 of xylanase A from Streptomyces lividans, a key residue in glycosyl hydrolases of superfamily 4/7: kinetic evidence for its involvement in stabilization of the catalytic intermediate.
abstract: Site-directed mutagenesis of asparagine-127 (N127) of xylanase A (XlnA) from Streptomyces lividans, belonging to family 10 and superfamily 4/7 of glycosyl hydrolases, was chosen to study the role of this conserved residue. The isosteric mutation N127D introduced did not affect the fold of XlnA as revealed by circular dichroism. Comparison of the kinetic constants of N127D and wild-type XlnA revealed a 70-fold decrease in the specificity constant (kcat/K(M)) towards birchwood xylan, which is attributed solely to the difference in the kcat value and indicates a role of N127 in stabilization of the catalytic intermediate. N127 also plays a role in maintaining the ionization states of the two catalytic residues, as shown by the modified pH profile of XlnA-N127D. Characterization of XlnA-N127D and the analysis of the three-dimensional structure of XlnA converge towards a stabilization role for N127 in the catalytic site of XlnA.
authors.affiliation: Centre de Recherche en Microbiologie Appliquée, Institut Armand-Frappier, Université du Québec, Laval, Canada.
authors.forename: M
authors.initials: M
authors.lastname: Roberge
authors.forename: C
authors.initials: C
authors.lastname: Dupont
authors.forename: R
authors.initials: R
authors.lastname: Morosoli
authors.forename: F
authors.initials: F
authors.lastname: Shareck
authors.forename: D
authors.initials: D
authors.lastname: Kluepfel
mesh_terms: D001216:Asparagine / Q000378:metabolism*; D002384:Catalysis; D055598:Chemical Phenomena; D002621:Chemistry; D002942:Circular Dichroism; D043364:Endo-1,4-beta Xylanases; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011551:Pseudomonas fluorescens / Q000201:enzymology; D013302:Streptomyces / Q000201:enzymology*; D014995:Xylosidases / Q000235:genetics / Q000378:metabolism*; D001617:beta-Glucosidase / Q000378:metabolism
chemical_list: D001216:Asparagine; D014995:Xylosidases; D001617:beta-Glucosidase; D043364:Endo-1,4-beta Xylanases; C090001:XynB xylanase