Structure and function of domains constructing bacterial chitinase

点击翻译按钮获取中文摘要

The aim of this study is to unravel the mechanisms underlying degradation of crystalline chitin by Bacillus circulans WL-12 chitinase A1. The structures and properties of the domains constructing chitinse A1 were studied in detail as follows.(1) Structure and catalytic mechanism of the catalytic domain (CatD) of chitinase A1. Three-dimensional structure of CatD was determined by X-ray crystallography. CatD consists of (β/α)8-TIM-barrel attached on top with two small β- subdomain which provide deep cleft for substrate binding. Complexed structures of CatD with inhibitors and (GlcNAc)ィイD26ィエD2 revealed that chitinase A1 has seven subsites, from (-5) to (+2), and degrades substrate from its reducing end. In addition, from the structure of distorted GlcNAc residue at subsite -1, the experiments using affinity-label reagent, and site-directed mutagenesis of possible nucleophile residue, it was concluded that substrate-assisted catalysis is the catalytic mechanism of chitinase A1.(2) Molecul … More ar directionality of chitin degradation by chitinase A1. Detailed study of (GlcNAc)ィイD26ィエD2 hydrolysis suggested that this enzyme has six subsites, from (-2) to (+4), in the catalytic site and hydrolyzes second linkage from non-reducing end most frequently. On the other hand, microdiffraction electron micrography carried out on the β-chitin microfibrils treated with chitinase A1 strongly suggested that degradation of microfibril occurs from reducing ends. From these results, it was suggested that the directionality of hydrolysis is different between oligosaccharides and crystalline chitin.(3) Structure and properties of chitin-binding domain (ChBD) and type III-like domain (FnlII domain) of chitinase A1. High level expression system of ChBD in E. coli was constructed and its structure and properties were studied. Binding of ChBDィイD2ChiA1ィエD2 was highly specific to insoluble chitin and did not interact with soluble substrates. ChBDィイD2ChiA1ィエD2 lacks linearly exposed three aromatic residues. In addition, it was shown that the ChBD is not absolutely required for hydrolysis of crystalline β-chitin but greatly enhances the efficiency of degradation.On the other hand, purified FnIIIィイD2R2ィエD2 domain did not show any significant binding activity to insoluble chitin. Less