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 Of Chi-h, a Chi-h from Ostrinia furnacalis , was investigated. Crystal structures of both Of Chi-h and its complex with chitoheptaose ((GlcN) 7 ) reveal that Of Chi-h possesses a long and asymmetric substrate binding cleft, which is a typical characteristics of a processive exo-chitinase. The structural comparison between Of Chi-h and its bacterial homolog Sm ChiA uncovered two phenylalanine-to-tryptophan site variants in Of Chi-h at subsites +2 and possibly −7. The F232W/F396W double mutant endowed Sm ChiA with higher hydrolytic activities toward insoluble substrates, such as insect cuticle, α-chitin, and chitin nanowhisker. An enzymatic assay demonstrated that Of Chi-h outperformed Of ChtI, an insect endo-chitinase, toward the insoluble substrates, but showed lower activity toward the soluble substrate ethylene glycol chitin. Furthermore, Of Chi-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.
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Liu et al. (2017) studied this question.