Characterization of an exochitinase from Epiphyas postvittana nucleopolyhedrovirus (family Baculoviridae)

• Published in: Journal of general virology 2005-12, Vol.86 (12), p.3253-3261
• Main Authors: Young, V.L, Simpson, R.M, Ward, V.K
• Format: Article
• Language: English
• Subjects: Acetylglucosamine - isolation & purification; Baculoviridae - enzymology; Baculovirus; Biological and medical sciences; Catalytic Domain; chitin; Chitin - metabolism; chitinase; Chitinases - chemistry; Chitinases - metabolism; Disaccharidases - isolation & purification; enzymatic hydrolysis; enzyme activity; Enzyme Inhibitors; Epiphyas postvittana; exochitinase; Fundamental and applied biological sciences. Psychology; Mass Spectrometry; Microbiology; Miscellaneous; Models, Molecular; Molecular Weight; Nuclear polyhedrosis virus; Nucleopolyhedrovirus; Oligosaccharides - metabolism; Protein Conformation; Protein Structure, Tertiary; Recombinant Proteins - metabolism; Serratia marcescens; Substrate Specificity; Viral Proteins - metabolism; Virology
• ISSN: 0022-1317; 1465-2099
• DOI: 10.1099/vir.0.81262-0

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 Epiphyas postvittana nucleopolyhedrovirus (EppoNPV) against Serratia marcescens chitinase A indicated that the enzyme possesses an N-terminal polycystic kidney 1 (PKD1) domain for chitin-substrate feeding and an α/β 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 Km values of 270 +/- 60 and 240 +/- 40 micromolar against 4MU-(GlcNAc)2 and 20 +/- 6 and 14 +/- 7 micromolar against 4MU-(GlcNAc)3 for native and recombinant versions of the enzyme, respectively. In contrast, digestion and thin-layer chromatography analysis of short-chain (GlcNAc)2-6 chito-oligosaccharides without the fluorescent 4-methylumbelliferone (4MU) moiety produced predominantly (GlcNAc)2, indicating an exochitinase, although low-level endochitinase activity was detected. Digestion of long-chain colloidal β-chitin and analysis by mass spectrometry identified a single 447 Da peak, representing a singly charged (GlcNAc)2 complexed with a sodium adduct ion, confirming the enzyme as an exochitinase with no detectable endochitinolytic activity. Furthermore, (GlcNAc)3-6 substrates, but not (GlcNAc)2, 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.