title: Mutation of Trp137 to glutamate completely removes transglycosyl activity associated with the Aspergillus fumigatus AfChiB1.
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.
authors.affiliation: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Lü
authors.forename: Haitao
authors.initials: H
authors.lastname: Yang
authors.forename: Hongyan
authors.initials: H
authors.lastname: Hu
authors.forename: Ying
authors.initials: Y
authors.lastname: Wang
authors.forename: Zihe
authors.initials: Z
authors.lastname: Rao
authors.forename: Cheng
authors.initials: C
authors.lastname: Jin
mesh_terms: D001232:Aspergillus fumigatus / Q000201:enzymology* / Q000235:genetics; D002688:Chitinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D009154:Mutation; D017433:Protein Structure, Secondary
chemical_list: D005656:Fungal Proteins; D002688:Chitinases