High-resolution native and complex structures of thermostable β-mannanase from Thermomonospora fusca -: substrate specificity in glycosyl hydrolase family 5
High-resolution native and complex structures of thermostable β-mannanase from Thermomonospora fusca -: substrate specificity in glycosyl hydrolase family 5
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DOI：
10.1016/s0969-2126(98)00142-7
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发表时间：
1998-11-15
期刊：
影响因子：
5.7
通讯作者：
Piontek, K
中科院分区：
文献类型：
--
作者：
Hilge, M;Gloor, SM;Piontek, K
背景：β-甘露聚糖酶水解甘露聚糖中的O-糖苷键，甘露聚糖是植物的半纤维素组分。这些酶在纸浆和纸张生产中具有潜在的用途，并且具有重要的生物技术意义。热稳定的β-甘露聚糖酶由于其高温最适性和宽pH耐受性而将是特别有用的。嗜热放线菌Thermomonospora fusca分泌至少一种β-甘露聚糖酶（分子量38 kDa），最适温度为80 ℃。结果：从T. fusca已经通过多个同晶置换方法确定，并精确到1.5埃分辨率。除天然酶外，已测定该酶的甘露三糖和甘露六糖结合形式的结构，其分辨率分别为1.9埃和1.6埃。fusca甘露聚糖酶既不显示对轴向HO-C（2）的有利相互作用，也不显示对葡萄糖构型底物的赤道羟基的歧视。我们提出，选择性产生于两种可能的机制：一个疏水相互作用的底物与Val 263，保守的家族5细菌甘露聚糖酶，区分不同的构象之间的羟甲基基团在天然甘露聚糖和纤维素;和/或Asp 259和轴向羟基基团之间的特定相互作用在C（2）的底物在-2亚位点。与第5家族纤维素酶相比，T.褐色甘露聚糖酶具有数量大大减少的芳族残基，提供了与底物堆叠的平台。这种每隔一个平台的缺失与甘露聚糖中轴向羟基的取向非常一致。
Background: beta-Mannanases hydrolyse the O-glycosidic bonds in mannan, a hemicellulose constituent of plants. These enzymes have potential use in pulp and paper production and are of significant biotechnological interest. Thermostable beta-mannanases would be particularly useful due to their high temperature optimum and broad pH tolerance. The thermophilic actinomycete Thermomonospora fusca secretes at least one beta-mannanase (molecular mass 38 kDa) with a temperature optimum of 80 degrees C. No three-dimensional structure of a mannan-degrading enzyme has been reported until now.Results: The crystal structure of the thermostable beta-mannanase from T. fusca has been determined by the multiple isomorphous replacement method and refined to 1.5 Angstrom resolution. In addition to the native enzyme, the structures of the mannotriose- and mannohexaose-bound forms of the enzyme have been determined to resolutions of 1.9 Angstrom and 1.6 Angstrom, respectively.Conclusions: Analysis of the -1 subsite of T. fusca mannanase reveals neither a favourable interaction towards the axial HO-C(2) nor a discrimination against the equatorial hydroxyl group of gluco-configurated substrates. We propose that selectivity arises from two possible mechanisms: a hydrophobic interaction of the substrate with Val263, conserved in family 5 bacterial mannanases, which discriminates between the different conformations of the hydroxymethyl group in native mannan and cellulose; and/or a specific interaction between Asp259 and the axial hydroxyl group at the C(2) of the substrate in the -2 subsite. Compared with the catalytic clefts of family 5 cellulases, the groove of T. fusca mannanase has a strongly reduced number of aromatic residues providing platforms for stacking with the substrate. This deletion of every second platform is in good agreement with the orientation of the axial hydroxyl groups in mannan.