Characterization of an exochitinase from Epiphyas postvittana nucleopolyhedrovirus (family Baculoviridae).
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.
Young
Vivienne L
VL
Department of Microbiology and Immunology, School of Medical Sciences, University of Otago, PO Box 56, Dunedin, New Zealand.
Simpson
Robert M
RM
Horticulture and Food Research Institute of New Zealand, Palmerston North, New Zealand.
Ward
Vernon K
VK
Department of Microbiology and Immunology, School of Medical Sciences, University of Otago, PO Box 56, Dunedin, New Zealand.
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
D004791:Enzyme Inhibitors; D009844:Oligosaccharides; D011994:Recombinant Proteins; D014764:Viral Proteins; D002686:Chitin; D004186:Disaccharidases; D002688:Chitinases; D000117:Acetylglucosamine