From structure to function: insights into the catalytic substrate specificity and thermostability displayed by Bacillus subtilis mannanase BCman.
From structure to function: insights into the catalytic substrate specificity and thermostability displayed by Bacillus subtilis mannanase BCman.
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DOI：
10.1016/j.jmb.2008.03.068
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发表时间：
2008-06
影响因子：
5.6
通讯作者：
Xiao-Xue Yan;X. An;L. Gui;D. Liang
中科院分区：
文献类型：
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作者：
Xiao-Xue Yan;X. An;L. Gui;D. Liang
BCman是一种来源于植物根系有益菌Bacillus subtilis Z-2的β-甘露聚糖酶，具有生产甘露寡糖的潜力，甘露寡糖对甜瓜和烟草都具有防御诱导活性，在植物病害的生物防治中具有重要作用。本文报道了BCman-GH 26酶的生化性质和晶体结构。动力学分析表明，BCman是一种内切β-甘露聚糖酶，对甘露聚糖具有专一性，对甘露寡糖没有活性。催化性酸/碱Glu 167和亲核体Glu 266分别位于β4和β7链上。1.45-ε晶体结构表明BCman为典型的（β/α）8折叠型。与其他内切-β-甘露聚糖酶的鞍形活性中心的一个很大的区别是存在浅碟形活性中心和底物结合位点，这两者都是BCman所特有的。这些差异主要是由于环1（Phe 37-Met 47）、环2（Ser 103-Ala 134）、环3（Phe 162-Asn 185）、环4（Tyr 215-Ile 236）、环5（Pro269-Tyr 278）和环6（Trp 298-Gly 309）的长度和位置的重要变化，所有这些都围绕活性位点。等温滴定量热法和晶体学的数据表明，BCman的活性位点内只有两个底物结合亚位点（+1和-1）。这两个位点参与酶的甘露聚糖降解活性并限制甘露寡糖的结合能力。BCman与甘露聚糖的结合和催化作用主要由含有Trp 302、Trp 298、Trp 172和Trp 72的溶剂暴露芳环条带的表面介导。此外，BCman含有一个二硫键（Cys 66 Cys 86）和一个特殊的His 1-His 23-Glu 336金属结合位点。这种二级结构是酶稳定性的关键因素。
BCman, a β-mannanase from the plant root beneficial bacterium Bacillus subtilis Z-2, has a potential to be used in the production of mannooligosaccharide, which shows defense induction activity on both melon and tobacco, and plays an important role in the biological control of plant disease. Here we report the biochemical properties and crystal structure of BCman-GH26 enzyme. Kinetic analysis reveals that BCman is an endo-β-mannanase, specific for mannan, and has no activity on mannooligosaccharides. The catalytic acid/base Glu167 and nucleophile Glu266 are positioned on the β4 and β7 strands, respectively. The 1.45-Å crystal structure reveals that BCman is a typical (β/α)8folding type. One large difference from the saddle-shaped active center of other endo-β-mannanases is the presence of a shallow-dish-shaped active center and substrate-binding site that are both unique to BCman. These differences are mainly due to important changes in the length and position of loop 1 (Phe37-Met47), loop 2 (Ser103-Ala134), loop3 (Phe162-Asn185), loop 4 (Tyr215-Ile236), loop 5 (Pro269-Tyr278), and loop 6 (Trp298-Gly309), all of which surround the active site. Data from isothermal titration calorimetry and crystallography indicated only two substrate-binding subsites (+1 and −1) within the active site of BCman. These two sites are involved in the enzyme's mannan degradation activity and in restricting the binding capacity for mannooligosaccharides. Binding and catalysis of BCman to mannan is mediated mainly by a surface containing a strip of solvent-exposed aromatic rings of Trp302, Trp298, Trp172, and Trp72. Additionally, BCman contains a disulfide bond (Cys66Cys86) and a special His1-His23-Glu336 metal-binding site. This secondary structure is a key factor in the enzyme's stability.