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  • Title: Site-directed mutagenesis of evolutionary conserved carboxylic amino acids in the chitosanase from Streptomyces sp. N174 reveals two residues essential for catalysis.
    Author: Boucher I, Fukamizo T, Honda Y, Willick GE, Neugebauer WA, Brzezinski R.
    Journal: J Biol Chem; 1995 Dec 29; 270(52):31077-82. PubMed ID: 8537367.
    Abstract:
    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.
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