title: Characterization of an exochitinase from Epiphyas postvittana nucleopolyhedrovirus (family Baculoviridae).
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 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 alpha/beta 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 K(m) values of 270+/-60 and 240+/-40 microM against 4MU-(GlcNAc)2 and 20+/-6 and 14+/-7 microM 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 beta-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.
authors.affiliation: Department of Microbiology and Immunology, School of Medical Sciences, University of Otago, PO Box 56, Dunedin, New Zealand.
authors.forename: Vivienne L
authors.initials: VL
authors.lastname: Young
authors.affiliation: Horticulture and Food Research Institute of New Zealand, Palmerston North, New Zealand.
authors.forename: Robert M
authors.initials: RM
authors.lastname: Simpson
authors.affiliation: Department of Microbiology and Immunology, School of Medical Sciences, University of Otago, PO Box 56, Dunedin, New Zealand.
authors.forename: Vernon K
authors.initials: VK
authors.lastname: Ward
mesh_terms: D000117:Acetylglucosamine / Q000302:isolation & purification; D016367:Baculoviridae / Q000201:enzymology*; D020134:Catalytic Domain; D002686:Chitin / Q000378:metabolism; D002688:Chitinases / Q000737:chemistry* / Q000378:metabolism*; D004186:Disaccharidases / Q000302:isolation & purification; D004791:Enzyme Inhibitors; D013058:Mass Spectrometry; D008958:Models, Molecular; D008970:Molecular Weight; D009844:Oligosaccharides / Q000378:metabolism; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D014764:Viral Proteins / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D009844:Oligosaccharides; D011994:Recombinant Proteins; D014764:Viral Proteins; D002686:Chitin; D004186:Disaccharidases; D002688:Chitinases; D000117:Acetylglucosamine