SITE-DIRECTED MUTAGENESIS OF EVOLUTIONARY CONSERVED CARBOXYLIC AMINO-ACIDS IN THE CHITOSANASE FROM STREPTOMYCES SP N174 REVEALS 2 RESIDUES ESSENTIAL FOR CATALYSIS

The comparison of four sequences of prokaryotic chitosanases, belongin g to the family 46 of glycosyl hydrolases, revealed a conserved N-term inal module of 50 residues, including five invariant carboxylic residu es. To verify if some of these residues are important for catalytic ac tivity in the chitosanase from Streptomyces sp. N174, these 5 residues were replaced by site-directed mutagenesis. Substitutions of Glu-22 o r Asp-40 with sterically conservative (E22Q, D40N) or functionally con servative (E22D, D40E) residues reduced drastically specific activity and k(cat), while K-m was only slightly changed. The other residues ex amined, Asp-6, Glu-SG, and Asp-37, retained significant activity after mutation. Circular dichroism studies of the mutant chitosanases confi rmed that the observed effects are not due to changes in secondary str ucture. These results suggested that Glu-22 and Asp-40 are directly in volved in the catalytic center of the chitosanase and the other residu es are not essential for catalytic activity.