The chitosanase from Streptomyces sp. N174: Site-directed mutagenesis studies
Structural stability


Studies on the three tryptophan residues in N174 chitosanase (Trp-28; Trp-101 and Trp-227) showed that none of them participated directly in the substrate binding, but rather stabilized the protein structure by making various interactions with hydrophobic side chains (42). Mutations of these residues destabilize the protein structure, which results in lower specific activity of the mutant enzymes, while affinity to chitosan substrate is almost unaffected.

Other three residues that seem to be important for structural stability have been identified recently (57). These are Asp-145, Arg-190, and Arg-205. Chitosanases that have been mutated in R205 position (R205A, R205Y, R205H) were found to have less than 0.3% of the activity of the wild type enzyme and to possess thermal stabilities 4-5 kcal/mol lower than that of wild type protein. It is interesting to note that these three residues are strictly conserved in all the chitosanases belonging to family 46 and so far sequenced. See the alignment by clicking here.

The examination of the x-ray crystal structure reveals that Arg-205 is localized very closely to the catalytic residue Glu-22 and is involved in a network of interactions with Asp-145 and Arg-190. Similar networks were identified in the crystal structures of the Bacillus circulans MH-K1 chitosanase, T4 lysozyme and barley chitinase (57).




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This page was created by Ryszard Brzezinski and Andrzej Neugebauer.
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Last updated: 2001/03/16