Young Vivienne L, Simpson Robert M, Ward Vernon K
Department of Microbiology and Immunology, School of Medical Sciences, University of Otago, PO Box 56, Dunedin, New Zealand.
Horticulture and Food Research Institute of New Zealand, Palmerston North, New Zealand.
J Gen Virol. 2005 Dec;86(Pt 12):3253-3261. 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 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.
杆状病毒几丁质酶和其他18家族糖水解酶在针对荧光几丁寡糖进行检测时，已显示同时具有外切几丁质酶和内切几丁质酶活性。苹果蠹蛾核多角体病毒（EppoNPV）的几丁质酶与粘质沙雷氏菌几丁质酶A的同源性建模表明，该酶具有一个用于几丁质底物摄取的N端多囊肾1（PKD1）结构域和一个18家族糖水解酶特有的α/β TIM桶状催化结构域。EppoNPV几丁质酶与其他杆状病毒几丁质酶有许多共同特征，包括高氨基酸同一性、N端分泌信号和功能性C端内质网保留序列。EppoNPV几丁质酶对荧光几丁寡糖表现出外切和内切几丁质分解活性，该酶天然型和重组型对4MU-(GlcNAc)2的K(m)值分别为270±60和240±40 μM，对4MU-(GlcNAc)3的K(m)值分别为20±6和14±7 μM。相比之下，对不含荧光4-甲基伞形酮（4MU）部分的短链(GlcNAc)(2-6)几丁寡糖进行消化和薄层色谱分析，主要产生(GlcNAc)2，表明其为外切几丁质酶，尽管检测到了低水平的内切几丁质酶活性。对长链胶体β-几丁质进行消化并通过质谱分析，鉴定出一个单一的447 Da峰，代表与钠加合物离子络合的单电荷(GlcNAc)2，证实该酶为无内切几丁质分解活性的外切几丁质酶。此外，(GlcNAc)(3-6)底物而非(GlcNAc)2可作为EppoNPV几丁质酶的抑制剂。短链底物不太可能与PKD1底物摄取机制的芳香族残基相互作用，因此可能无法准确反映这些酶对天然底物的活性。基于这些结果，杆状病毒EppoNPV的几丁质酶是一种外切几丁质酶。
