Tertiary structure and carbohydrate recognition by the chitin-binding domain of a hyperthermophilic chitinase from Pyrococcus furiosus
Tertiary structure and carbohydrate recognition by the chitin-binding domain of a hyperthermophilic chitinase from Pyrococcus furiosus
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DOI：
10.1016/j.jmb.2008.06.006
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发表时间：
2008-09-05
影响因子：
5.6
通讯作者：
Uegaki, Koichi
中科院分区：
文献类型：
--
作者：
Nakamura, Tsutomu;Mine, Shouhei;Uegaki, Koichi
几丁质酶是一种能有效水解α和β结晶几丁质的超嗜热糖苷酶;本文研究的几丁质酶是从激烈火球菌的基因PF 1233和PF 1234中改造而来的。该几丁质酶具有独特的结构特征，包含两个催化结构域（AD 1和AD 2）和两个几丁质结合结构域（ChBD; ChBD 1和ChBD 2）。携带AD 2和ChBD 2的部分酶也有效地水解结晶几丁质。我们确定了ChBD 2的NMR和晶体结构，这显著增强了催化结构域的活性。NMR和晶体结构之间没有显着差异。ChBD 2的整体结构由两个四链β-片层组成，由典型的β-夹心结构组成，与其他碳水化合物结合模块2家族蛋白相似，尽管序列相似性较低。通过NMR鉴定的几丁质结合表面是平坦的，并且包含三个溶剂暴露的Trp残基（Trp 274、Trp 308和Trp 326）的条带，其两侧是酸性残基（Glu 279和Asp 281）。这些酸性残基形成带负电荷的斑块，是ChBD 2的特征。突变分析表明，疏水相互作用是占主导地位的结晶甲壳素的识别和酸性残基负责较高的底物特异性的ChBD 2甲壳素相比，纤维素。这些结果提供了第一个结构的超热稳定的ChBD和产生新的见解蛋白质-碳水化合物识别的机制。这对开发生物质的技术很重要。(C)2008爱思唯尔有限公司版权所有。
A chitinase is a hyperthermophilic glycosidase that effectively hydrolyzes both alpha and beta crystalline chitins; that studied here was engineered from the genes PF1233 and PF1234 of Pyrococcus furiosus. This chitinase has unique structural features and contains two catalytic domains (AD1 and AD2) and two chitin-binding domains (ChBDs; ChBD1 and ChBD2). A partial enzyme carrying AD2 and ChBD2 also effectively hydrolyzes crystalline chitin. We determined the NMR and crystal structures of ChBD2, which significantly enhances the activity of the catalytic domain. There was no significant difference between the NMR and crystal structures. The overall structure of ChBD2, which consists of two four-stranded beta-sheets, was composed of a typical beta-sandwich architecture and was similar to that of other carbohydrate-binding module 2 family proteins, despite low sequence similarity. The chitin-binding surface identified by NMR was flat and contained a strip of three solvent-exposed Trp residues (Trp274, Trp308 and Trp326) flanked by acidic residues (Glu279 and Asp281). These acidic residues form a negatively charged patch and are a characteristic feature of ChBD2. Mutagenesis analysis indicated that hydrophobic interaction was dominant for the recognition of crystalline chitin and that the acidic residues were responsible for a higher substrate specificity of ChBD2 for chitin compared with that of cellulose. These results provide the first structure of a hyperthermostable ChBD and yield new insight into the mechanism of protein-carbohydrate recognition. This is important in the development of technology for the exploitation of biomass. (C) 2008 Elsevier Ltd. All rights reserved.