1887

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Volume 86, Issue 12

Characterization of an exochitinase from (family ) Free

Abstract

Baculovirus chitinases and other family 18 glycohydrolases have been shown to possess both exo- and endochitinase activities when assayed against fluorescent chito-oligosaccharides. Homology modelling of the chitinase of (EppoNPV) against chitinase A indicated that the enzyme possesses an N-terminal polycystic kidney 1 (PKD1) domain for chitin-substrate feeding and an TIM barrel catalytic domain characteristic of a family 18 glycohydrolase. EppoNPV chitinase has many features in common with other baculovirus chitinases, including high amino acid identity, an N-terminal secretion signal and a functional C-terminal endoplasmic reticulum-retention sequence. EppoNPV chitinase displayed exo- and endochitinolytic activity against fluorescent chito-oligosaccharides, with values of 270±60 and 240±40 μM against 4MU-(GlcNAc) and 20±6 and 14±7 μM against 4MU-(GlcNAc) for native and recombinant versions of the enzyme, respectively. In contrast, digestion and thin-layer chromatography analysis of short-chain (GlcNAc) chito-oligosaccharides without the fluorescent 4-methylumbelliferone (4MU) moiety produced predominantly (GlcNAc), indicating an exochitinase, although low-level endochitinase activity was detected. Digestion of long-chain colloidal -chitin and analysis by mass spectrometry identified a single 447 Da peak, representing a singly charged (GlcNAc) complexed with a sodium adduct ion, confirming the enzyme as an exochitinase with no detectable endochitinolytic activity. Furthermore, (GlcNAc) substrates, but not (GlcNAc), acted as inhibitors of EppoNPV chitinase. Short-chain substrates are unlikely to interact with the aromatic residues of the PKD1 substrate-feeding mechanism and hence may not accurately reflect the activity of these enzymes against native substrates. Based upon these results, the chitinase of the baculovirus EppoNPV is an exochitinase.

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2005-12-01
2024-04-26
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Characterization of an exochitinase from Epiphyas postvittana nucleopolyhedrovirus (family Baculoviridae)
J. Gen. Virol. 86, 3253 (2005); https://doi.org/10.1099/vir.0.81262-0
/content/journal/jgv/10.1099/vir.0.81262-0
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