Hilge M, Gloor S M, Rypniewski W, Sauer O, Heightman T D, Zimmermann W, Winterhalter K, Piontek K
Laboratorium für Biochemie ETH Zentrum Universitätstrasse 16 CH-8092 Zürich, Switzerland.
Structure. 1998 Nov 15;6(11):1433-44. doi: 10.1016/s0969-2126(98)00142-7.
. 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 degreesC. No three-dimensional structure of a mannan-degrading enzyme has been reported until now.
. 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 A 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 A and 1.6 A, respectively.
. 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.
β-甘露聚糖酶可水解甘露聚糖中的O-糖苷键，甘露聚糖是植物半纤维素的一种成分。这些酶在纸浆和造纸生产中具有潜在用途，并且具有重大的生物技术意义。由于其最佳温度高且pH耐受性广，耐热β-甘露聚糖酶将特别有用。嗜热放线菌栖热单孢菌分泌至少一种β-甘露聚糖酶（分子量38 kDa），其最佳温度为80℃。到目前为止，尚未报道甘露聚糖降解酶的三维结构。
通过多重同晶置换法确定了栖热单孢菌耐热β-甘露聚糖酶的晶体结构，并将其精修至1.5 Å分辨率。除了天然酶之外，还分别确定了与甘露三糖和甘露六糖结合形式的酶的结构，分辨率分别为1.9 Å和1.6 Å。
对栖热单孢菌甘露聚糖酶的-1亚位点分析表明，其对轴向HO-C(2)既没有良好的相互作用，也没有对葡萄糖构型底物的赤道羟基产生歧视。我们提出选择性来自两种可能的机制：底物与Val263的疏水相互作用，在5家族细菌甘露聚糖酶中保守，可区分天然甘露聚糖和纤维素中羟甲基的不同构象；和/或Asp259与-2亚位点底物C(2)处的轴向羟基之间的特异性相互作用。与5家族纤维素酶的催化裂隙相比，栖热单孢菌甘露聚糖酶的凹槽中芳香族残基的数量大大减少，这些残基为与底物堆积提供平台。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。
