Biochemistry-Electrochemistry Research Unit and School of Chemistry, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima, Thailand; Center of Excellence in Advanced Functional Materials, Suranaree University of Technology, Nakhon Ratchasima, Thailand.
Biochemistry-Electrochemistry Research Unit and School of Chemistry, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima, Thailand.
Int J Biol Macromol. 2016 Dec;93(Pt A):1111-1117. doi: 10.1016/j.ijbiomac.2016.09.066. Epub 2016 Sep 22.
Vibrio harveyi chitinase A (VhChiA) is a GH-18 glycosyl hydrolase with a structure containing three distinct domains: i) the N-terminal chitin-binding domain; ii) the (α/β) TIM barrel catalytic domain; and iii) the α+β insertion domain. In this study, we cloned the gene fragment encoding the chitin-binding domain of VhChiA, termed ChBD. The recombinant ChBD was heterologously expressed in E. coli BL21 strain Tuner(DE3)pLacI host cells, and purified to homogeneity. CD measurements suggested that ChBD contained β-sheets as major structural components and fluorescence spectroscopy showed that the protein domain was folded correctly, and suitable for functional characterization. Chitin binding assays showed that ChBD bound to both α- and β-chitins, with the greatest affinity for β-colloidal chitin, but barely bound to polymeric chitosan. These results identified the tandem N-acetamido functionality on chitin chains as the specific sites of enzyme-substrate interactions. The binding affinity of the isolated domain was significantly lower than that of intact VhChiA, suggesting that the catalytic domain works synergistically with the chitin-binding domain to guide the polymeric substrate into the substrate binding cleft. These data confirm the physiological role of the chitin-binding domain of the marine bacterial GH-18 chitinase A in chitin-chitinase interactions.
哈维弧菌几丁质酶 A（VhChiA）是一种 GH-18 糖苷水解酶，其结构包含三个不同的结构域：i）N 端几丁质结合域；ii）（α/β）TIM 桶催化结构域；和 iii）α+β插入结构域。在这项研究中，我们克隆了编码 VhChiA 几丁质结合域的基因片段，称为 ChBD。重组 ChBD 在大肠杆菌 BL21 菌株 Tuner(DE3)pLacI 宿主细胞中异源表达，并进行了均质纯化。CD 测量表明 ChBD 含有 β-折叠作为主要结构成分，荧光光谱表明该蛋白结构域折叠正确，适合功能表征。几丁质结合实验表明 ChBD 结合 α-和 β-几丁质，对 β-胶体几丁质具有最大亲和力，但对聚合壳聚糖几乎没有亲和力。这些结果确定了几丁质链上的串联 N-乙酰氨基功能是酶-底物相互作用的特异性位点。分离的结构域的结合亲和力明显低于完整的 VhChiA，这表明催化结构域与几丁质结合结构域协同作用，将聚合物底物引导到底物结合裂隙中。这些数据证实了海洋细菌 GH-18 几丁质酶 A 的几丁质结合结构域在几丁质-几丁质酶相互作用中的生理作用。
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