Mutation of Trp137 to glutamate completely removes transglycosyl activity associated with the Aspergillus fumigatus AfChiB1.
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Lu Y, Yang H, Hu H, Wang Y, Rao Z, Jin C
Mutation of Trp137 to glutamate completely removes transglycosyl activity associated with the Aspergillus fumigatus AfChiB1.
Glycoconj J. 2009 Jul;26(5):525-34. doi: 10.1007/s10719-008-9203-z. Epub 2008 Oct 31.
- PubMed ID
- 18975073 [ View in PubMed]
- Abstract
Family 18 chitinases hydrolyze chitin through a substrate-assisted catalytic mechanism and are to a variable extent able to catalyze transglycosylation reactions. Previously Aspergillus fumigatus AfChiB1 was found to be able to catalyze transglycosylation reactions. Structural analysis reveals that AfChiB1 consists of an eight-stranded beta/alpha-barrel. Like other members of the family 18 hydrolases, AfChiB1 has conserved substrate binding site and catalytic acid, while a suitable nucleophile is missing. In this study, Trp137, Asp246, and Met243, which are close to the glycosidic cleavage site, were mutated to glutamate individually. As a result, the W137E remained its hydrolytic activity and was completely devoid of transglycosyl activity, while the D246E reduced its chitinolytic activity and increased its transglycosyl activity. And the M243E showed a remarkable reduction of chitinolytic activity and complete loss of transglycosyl activity. These results suggested that the transglycosyl reaction catalyzed by the AfChiB1 is due to lacking of nucleophile.