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一种细菌19家族几丁质酶的晶体结构和酶学特性揭示了其与植物酶的差异。

Crystal structure and enzymatic properties of a bacterial family 19 chitinase reveal differences from plant enzymes.

作者信息

Hoell Ingunn A, Dalhus Bjørn, Heggset Ellinor B, Aspmo Stein I, Eijsink Vincent G H

机构信息

Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, 1432 As, Norway.

出版信息

FEBS J. 2006 Nov;273(21):4889-900. doi: 10.1111/j.1742-4658.2006.05487.x. Epub 2006 Sep 28.

Abstract

We describe the cloning, overexpression, purification, characterization and crystal structure of chitinase G, a single-domain family 19 chitinase from the Gram-positive bacterium Streptomyces coelicolor A3(2). Although chitinase G was not capable of releasing 4-methylumbelliferyl from artificial chitooligosaccharide substrates, it was capable of degrading longer chitooligosaccharides at rates similar to those observed for other chitinases. The enzyme was also capable of degrading a colored colloidal chitin substrate (carboxymethyl-chitin-remazol-brilliant violet) and a small, presumably amorphous, subfraction of alpha-chitin and beta-chitin, but was not capable of degrading crystalline chitin completely. The crystal structures of chitinase G and a related Streptomyces chitinase, chitinase C [Kezuka Y, Ohishi M, Itoh Y, Watanabe J, Mitsutomi M, Watanabe T & Nonaka T (2006) J Mol Biol358, 472-484], showed that these bacterial family 19 chitinases lack several loops that extend the substrate-binding grooves in family 19 chitinases from plants. In accordance with these structural features, detailed analysis of the degradation of chitooligosaccharides by chitinase G showed that the enzyme has only four subsites (- 2 to + 2), as opposed to six (- 3 to + 3) for plant enzymes. The most prominent structural difference leading to reduced size of the substrate-binding groove is the deletion of a 13-residue loop between the two putatively catalytic glutamates. The importance of these two residues for catalysis was confirmed by a site-directed mutagenesis study.

摘要

我们描述了来自革兰氏阳性细菌天蓝色链霉菌A3(2)的单结构域19家族几丁质酶G的克隆、过表达、纯化、表征及晶体结构。虽然几丁质酶G无法从人工几丁寡糖底物中释放4-甲基伞形酮,但它能够以与其他几丁质酶相似的速率降解较长的几丁寡糖。该酶还能够降解一种有色胶体几丁质底物(羧甲基几丁质-瑞玛唑亮紫)以及一小部分可能为无定形的α-几丁质和β-几丁质,但无法完全降解结晶几丁质。几丁质酶G和一种相关的链霉菌几丁质酶——几丁质酶C [Kezuka Y, Ohishi M, Itoh Y, Watanabe J, Mitsutomi M, Watanabe T & Nonaka T (2006) J Mol Biol358, 472 - 484] 的晶体结构表明,这些细菌19家族几丁质酶缺少一些能扩展植物19家族几丁质酶底物结合凹槽的环。根据这些结构特征,对几丁质酶G降解几丁寡糖的详细分析表明,该酶只有四个亚位点(-2至+2),而植物酶有六个(-3至+3)。导致底物结合凹槽尺寸减小的最显著结构差异是两个推定的催化谷氨酸之间缺失了一个13个残基的环。定点诱变研究证实了这两个残基对催化作用的重要性。

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