文献类型： 外文期刊

作者： Lyu, Qiangian ¹ ; Wang, Song ² ; Xu, Wenhua ³ ; Han, Baoqin ¹ ; Liu, Wanshun ¹ ; Junes, David N. M. ⁴ ; Liu, Weizhi ¹ ;

作者机构： 1.Ocean Univ China, Coll Marine Life Sci, Qingdao, Peoples R China

2.Chinese Acad Fishery Sci, Yellow Sea Fisheries Res Inst, Qingdao, Peoples R China

3.Qingdao Univ, Publ Hlth Lab Ctr, Qingdao 266071, Peoples R China

4.Univ Colorado, Sch Med, Dept Pharmacol, Aurora, CO 80045 USA

关键词： chitosanase;complex structure;differential scanning fluorimetry (DSF);glycoside hydrolyase;X-ray crystallography

期刊名称：BIOCHEMICAL JOURNAL （ 影响因子：3.857； 五年影响因子：4.868 ）

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收录情况： SCI

摘要： Chitosanase is able to specifically cleave beta-1,4-glycosidic bond linkages in chitosan to produce a chito-oligomer product, which has found a variety of applications in many areas, including functional food and cancer therapy. Although several structures for chitosanase have been determined, the substrate-binding mechanism for this enzyme has not been fully elucidated because of the lack of a high-resolution structure of the chitosanase substrate complex. In the present study we show the crystal structure of a novel chitosanase OU01 from Microbacterium sp. in complex with its substrate hexa-glucosamine (GlcN)(6), which belongs to the GH46 (glycoside hydrolyase 46) family in the Carbohydrate Active Enzymes database (http://www.cazy.org/). This structure allows precise determination of the substrate-binding mechanism for the first time. The chitosanase (GlcN)(6) complex structure demonstrates that, from the 2 to + 1 position of the (GleN)(6) substrate, the pyranose rings form extensive interactions with the chitosanase-binding cleft. Several residues (See(27), Tyr(37), Arg(45), Thr(58), Asp(60), His(203) and Asp(235)) in the binding cleft are found to form important interactions required to bind the substrate. Site-directed mutagenesis of these residues showed that mutations of Y37F and H203A abolish catalytic activity. In contrast, the mutations T58A and D235A only lead to a moderate loss of catalytic activity, whereas the S27A mutation retains similar to 80% of the enzymatic activity. In combination with previous mutagenesis studies, these results suggest that the 2, - 1 and + 1 subsites play a dominant role in substrate binding and catalysis. DSF (differential scanning fluorimetry) assays confirmed that these mutations had no significant effect on protein stability. Taken together, we present the first mechanistic interpretation for the substrate (GlcN)6 binding to chitosanase, which is critical for the design of novel chitosanase used for biomass conversion