Analysis of essential carboxylic amino acid residues for catalytic activity of fungal chitosanases by site-directed mutagenesis.
A comparison of amino acid sequences of fungal chitosanases, belonging to family 75 of glycosyl hydrolases, revealed three carboxylic amino acid residues completely conserved among all of the chitosanases. To study the role of these residues in catalysis, they were replaced with other residues by site-directed mutagenesis in the chitosanase gene of Fusarium solani. The mutated genes were expressed in the yeast Saccharomyces cerevisiae and the resulting recombinant chitosanases were used in kinetic analysis. Chitosanases with Asp-175-->Asn and Glu-188-->Gln mutations were essentially inactive, whereas those with Asp-175-->Glu and Glu-188-->Asp mutations retained 25-50% specific activity as compared with the wild-type enzyme. The mutation of Asp-212-->Asn did not decrease specific activity to a large extent. Circular dichroism analysis confirmed that the mutant chitosanases had similar secondary structures to that of the wild-type enzyme. These results indicate that Asp-175 and Glu-188 are essential residues for the catalytic activity of chitosanase. Time-dependent (1)H-NMR analysis for the hydrolysis of D-glucosamine hexamer revealed that a fungal chitosanase is an inverting enzyme producing only the alpha anomeric form of reaction products.
Shimosaka
Makoto
M
Department of Applied Biology, Faculty of Textile Science and Technology, Shinshu University, 3-15-1 Tokida, Ueda, Nagano 386-8567, Japan. mashimo@shinshu-u.ac.jp
Sato
Kazuaki
K
Nishiwaki
Naohide
N
Miyazawa
Takashi
T
Okazaki
Mitsuo
M
D019943:Amino Acid Substitution; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics; D005670:Fusarium / Q000201:enzymology* / Q000235:genetics; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics; D017354:Point Mutation; D017433:Protein Structure, Secondary / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013379:Substrate Specificity
D005656:Fungal Proteins; D006026:Glycoside Hydrolases; C019868:chitosanase