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Indexed by:期刊论文

Date of Publication:2017-02-10

Journal:JOURNAL OF BIOLOGICAL CHEMISTRY

Included Journals:SCIE、EI、PubMed、Scopus

Volume:292

Issue:6

Page Number:2080-2088

ISSN No.:0021-9258

Key Words:chitin,chitinase,crystal structure,inhibitor,insect,molting

Abstract:Chitinase-h (Chi-h) is of special interest among insect chitinases due to its exclusive distribution in lepidopteran insects and high sequence identity with bacterial and baculovirus homologs. Here OfChi-h, a Chi-h from Ostrinia furnacalis, was investigated. Crystal structures of both OfChi-h and its complex with chitoheptaose ((GlcN) 7) reveal that OfChi-h possesses a long and asymmetric substrate binding cleft, which is a typical characteristics of a processive exo-chitinase. The structural comparison between OfChi-h and its bacterial homolog SmChiA uncovered two phenylalanine-to-tryptophan site variants in OfChi-h at subsites +2 and possibly -7. The F232W/F396W double mutant endowedSmChiA with higher hydrolytic activities toward insoluble substrates, such as insect cuticle, alpha-chitin, and chitin nanowhisker. An enzymatic assay demonstrated that OfChi-h outperformed OfChtI, an insect endochitinase, toward the insoluble substrates, but showed lower activity toward the soluble substrate ethylene glycol chitin. Furthermore, OfChi-h was found to be inhibited by N, N',N"-trimethylglucosamine- N, N',N",N"'-tetraacetylchitotetraose (TMG(GlcNAc)(4)), a substrate analog which can be degraded into TMG-(GlcNAc)(1-2). Injection of TMG-(GlcNAc)(4) into 5th-instar O. furnacalis larvae led to severe defects in pupation. This work provides insights into a molting-indispensable insect chitinase that is phylogenetically closer to bacterial chitinases than insect chitinases.