Details

Autor(en) / Beteiligte

• Boucher, Isabelle
• Fukamizo, Tamo
• Honda, Yuji
• Willick, Gordon E.
• Neugebauer, Witold A.
• Brzezinski, Ryszard

Titel

Site-directed Mutagenesis of Evolutionary Conserved Carboxylic Amino Acids in the Chitosanase from Streptomyces sp. N174 Reveals Two Residues Essential for Catalysis (∗)

Ist Teil von

• The Journal of biological chemistry, 1995-12, Vol.270 (52), p.31077-31082

Ort / Verlag

United States: Elsevier Inc

Erscheinungsjahr

1995

Quelle

MEDLINE

Beschreibungen/Notizen

• 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.