Characterization of two important histidine residues in the active site of xylanase A from Streptomyces lividans, a family 10 glycanase.
The active site of xylanase A (XlnA) from Streptomyces lividans contains three histidine residues, two of which (H81 and H207) are almost completely conserved in family 10 glycanases. The structural analysis of the enzyme shows that H81 and H207 are part of an important hydrogen bond network in the vicinity of the two catalytic residues (E128 and E236). In order to investigate the role of these two histidine residues for the structure/function of XlnA, three mutant enzymes were produced at each position, namely, H81R/S/Y and H207E/K/R. The specific activity of these mutant enzymes is reduced by more than 95%, revealing the importance of these two residues for the catalytic function of XlnA. The kinetic parameters of the three more active enzymes were determined, of which mutation H207K increased the K(M) 3-fold. The k(cat) of the mutant enzymes is reduced proportionally to the specific activity. Furthermore, the pKa values of the two catalytic residues are decreased in all six mutations, demonstrating a role for H81 and H207 in the hydrogen bond network responsible for maintaining the ionization state of the two catalytic residues. In most cases, the unfolding of mutated XlnA in guanidine hydrochloride (Gdn-HCl) showed that the concentration required to denature 50% of the XlnA decreased, thus demonstrating the importance of those two residues for the stability of the enzyme. Moreover, the m value [m = d(deltaG)/d[Gdn-HCl]] for the unfolding of XlnA in Gdn-HCl is increased for each of the six mutations, suggesting that the mutant proteins have less residual structure in the denatured state than does the wild-type enzyme.
Roberge
M
M
Centre de recherche en microbiologie appliquée, Institut Armand-Frappier, Université du Québec, Laval, Canada.
Shareck
F
F
Morosoli
R
R
Kluepfel
D
D
Dupont
C
C
D000595:Amino Acid Sequence; D001665:Binding Sites; D043364:Endo-1,4-beta Xylanases; D006026:Glycoside Hydrolases / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry*; D006358:Hot Temperature; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D013302:Streptomyces / Q000201:enzymology*; D014995:Xylosidases / Q000737:chemistry* / Q000235:genetics
D006639:Histidine; D006026:Glycoside Hydrolases; D014995:Xylosidases; C020668:glycanase; D043364:Endo-1,4-beta Xylanases