Structure and mechanism of chitin deacetylase from the fungal pathogen Colletotrichum lindemuthianum

被引:137
|
作者
Blair, David E.
Hekmat, Omid
Schuttelkopf, Alexander W.
Shrestha, Binesh
Tokuyasu, Ken
Withers, Stephen G.
van Aalten, Daan M. F. [1 ]
机构
[1] Univ Dundee, Sch Life Sci, Div Biol Chem & Mol Microbiol, Dundee DD1 5EH, Scotland
[2] Univ British Columbia, Prot Engn Network Ctr Excellence Canada, Dept Chem, Vancouver, BC V6T 1Z1, Canada
[3] Univ Paris 11, Inst Genet & Microbiol, F-91405 Orsay, France
[4] Natl Food Res Inst, Carbohydrate Lab, Food Mat Div, Tsukuba, Ibaraki 3058642, Japan
基金
英国惠康基金;
关键词
D O I
10.1021/bi0606694
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The fungal pathogen Colletotrichum lindemuthianum secretes an endo-chitin de-N-acetylase (ClCDA) to modify exposed hyphal chitin during penetration and infection of plants. Although a significant amount of biochemical data is available on fungal chitin de-N-acetylases, no structural data exist. Here we describe the 1.8 angstrom crystal structure of a ClCDA product complex and the analysis of the reaction mechanism using Hammett linear free energy relationships, subsite probing, and atomic absorption spectroscopy studies. The structural data in combination with biochemical data reveal that ClCDA consists of a single domain encompassing a mononuclear metalloenzyme which employs a conserved His-HisAsp zinc-binding triad closely associated with the conserved catalytic base (aspartic acid) and acid (histidine) to carry out acid/base catalysis. The data presented here indicate that ClCDA possesses a highly conserved substrate-binding groove, with subtle alterations that influence substrate specificity and subsite affinity. Strikingly, the structure also shows that the hexahistidine purification tag appears to form a tight interaction with the active site groove. The enzyme requires occupancy of at least the 0 and +1 subsites by (GlcNAc) 2 for activity and proceeds through a tetrahedral oxyanion intermediate.
引用
收藏
页码:9416 / 9426
页数:11
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