Structure, Catalysis, and Inhibition of OfChi-h, the Lepidoptera-exclusive Insect Chitinase

Abstract

Edited by Gerald W. HartChitinase-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 endowed SmChiA with higher hydrolytic activities toward insoluble substrates, such as insect cuticle, ␣-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.

Identification and RNAi‐based function analysis of chitinase family genes in diamondback moth, Plutella xylostella

“…Although other chitinase genes, such as Cht1 , Cht9 , Cht‐h and IDGF 4 were also reported to have important roles during the molting process in some insect species, no observed effects were found in P. xylostella in this research. In addition, no specific functions of orthologues of PxCht2 , PxCht3 , PxCht8 and PxCht25 in P. xylostella or other insects have been reported to date .…”

“…Recently, chitinases have been considered potential targets for new insecticide screening and RNA interference (RNAi)‐based management because of their critical roles in insect development. Some small molecules have been reported to be effective inhibitors against chitinases, such as the pseudotrisaccharide allosamidin and the cyclopentapeptide argifin, and these inhibitors were considered potential insecticides or fungicides. In addition, some insect chitinase genes were identified as ideal silencing targets and resulted in increased mortality or delayed development, which facilitated the development of a promising RNAi‐based method for the pest control .…”

Functional analysis of eight chitinase genes in rice stem borer and their potential application in pest control

“…It can be used to control SSB (Zibaee and Malagoli, 2014), thrips (Arthurs et al, 2013), whiteflies (Buitenhuis et al, 2016) and pine moths (Fan et al, 2013). Therefore, the joint use of fungal insecticides and chitinase inhibitors against the orthologues of CsCht10 could also potentially be applied to other lepidopteran insect pests (Sakuda et al, 1987;Hirose et al, 2010;Chen et al, 2014;Liu et al, 2017). As CsCh10 was highly expressed in the midgut and during the pupal stage, it can be deduced that CsCht10 might be involved in defence by degrading the chitin of exogenous pathogens.…”

Chitinase: diversity, limitations, and trends in engineering for suitable applications

“…Their chitinases are produced during ecdysis for the shedding of old cuticle. Recently, studies on the catalysis and structural characterization of Lepidoptera chitinase, Of ChtI from Ostrinia furnacalis demonstrated that the enzyme was indispensable to moulting [ 53 , 54 ].…”