*College of Marine Life Sciences, Ocean University of China, Qingdao, China.
†Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao, China.
Biochem J. 2014 Jul 15;461(2):335-45. doi: 10.1042/BJ20140159.
Chitosanase is able to specifically cleave β-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 (GlcN)6 substrate, the pyranose rings form extensive interactions with the chitosanase-binding cleft. Several residues (Ser27, Tyr37, Arg45, Thr58, Asp60, His203 and Asp235) 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 ~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.
壳聚糖酶能够特异性地切割壳聚糖中的β-1,4-糖苷键连接，生成壳寡糖产物，在功能食品和癌症治疗等多个领域都有广泛的应用。尽管已经确定了几种壳聚糖酶的结构，但由于缺乏壳聚糖酶-底物复合物的高分辨率结构，该酶的底物结合机制尚未完全阐明。在本研究中，我们展示了来自 Microbacterium sp. 的新型壳聚糖酶 OU01 与底物六葡萄糖胺（GlcN）6 的复合物晶体结构，该酶属于碳水化合物活性酶数据库（http://www.cazy.org/）中的 GH46（糖苷水解酶 46）家族。该结构首次精确确定了底物结合机制。壳聚糖酶-（GlcN）6 复合物结构表明，从（GlcN）6 底物的-2 位到+1 位，吡喃糖环与壳聚糖酶结合裂隙形成广泛的相互作用。结合裂隙中的几个残基（Ser27、Tyr37、Arg45、Thr58、Asp60、His203 和 Asp235）被发现形成了结合底物所需的重要相互作用。对这些残基进行定点突变表明，Y37F 和 H203A 的突变会使催化活性丧失。相比之下，T58A 和 D235A 的突变只会导致催化活性的适度丧失，而 S27A 的突变保留了约 80%的酶活性。与之前的突变研究相结合，这些结果表明-2、-1 和+1 亚位点在底物结合和催化中起主导作用。DSF（差示扫描荧光法）测定证实这些突变对蛋白质稳定性没有显著影响。总之，我们提出了壳聚糖酶与底物（GlcN）6 结合的第一个机制解释，这对于设计用于生物质转化的新型壳聚糖酶至关重要。
