Site-directed mutagenesis of evolutionary conserved carboxylic amino acids in the chitosanase from Streptomyces sp. N174 reveals two residues essential for catalysis.
The comparison of four sequences of prokaryotic chitosanases, belonging to the family 46 of glycosyl hydrolases, revealed a conserved N-terminal module of 50 residues, including five invariant carboxylic residues. To verify if some of these residues are important for catalytic activity in the chitosanase from Streptomyces sp. N174, these 5 residues were replaced by site-directed mutagenesis. Substitutions of Glu-22 or Asp-40 with sterically conservative (E22Q, D40N) or functionally conservative (E22D, D40E) residues reduced drastically specific activity and kcat, while Km was only slightly changed. The other residues examined, Asp-6, Glu-36, and Asp-37, retained significant activity after mutation. Circular dichroism studies of the mutant chitosanases confirmed that the observed effects are not due to changes in secondary structure. These results suggested that Glu-22 and Asp-40 are directly involved in the catalytic center of the chitosanase and the other residues are not essential for catalytic activity.
Département de Biologie, Faculté des Sciences, Université de Sherbrooke, Québec, Canada.
I
I
Boucher
T
T
Fukamizo
Y
Y
Honda
G E
GE
Willick
W A
WA
Neugebauer
R
R
Brzezinski
D000595:Amino Acid Sequence; D001483:Base Sequence; D005075:Biological Evolution; D002264:Carboxylic Acids / Q000378:metabolism; D002384:Catalysis; D002942:Circular Dichroism; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004926:Escherichia coli / Q000235:genetics; D006026:Glycoside Hydrolases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009844:Oligosaccharides / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013302:Streptomyces / Q000201:enzymology*
D002264:Carboxylic Acids; D009844:Oligosaccharides; C050523:chitohexaose; D006026:Glycoside Hydrolases; C019868:chitosanase