Crystal structure and substrate-binding mode of cellulase 12A from Thermotoga maritima
Crystal structure and substrate-binding mode of cellulase 12A from Thermotoga maritima
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DOI：
10.1002/prot.22953
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发表时间：
2011-04-01
影响因子：
2.9
通讯作者：
Guo, Rey-Ting
中科院分区：
文献类型：
--
作者：
Cheng, Ya-Shan;Ko, Tzu-Ping;Guo, Rey-Ting
纤维素酶已被用于处理各种含碳水化合物的材料。黑热菌纤维素酶12A(TmCel12A)属于糖苷水解酶GH12家族。它是一种β-1，4-内切葡聚糖酶，能将纤维素分子降解成更小的片段，促进碳水化合物的进一步利用。由于其超嗜热性，该酶特别适合于工业应用。用活性中心突变体E134C及其含汞衍生物测定了TmCel12A的晶体结构。它采用了典型的GH12家族酶的β-Jellyroll蛋白折叠，具有两个弯曲的β-折叠A和B和一个中心活性部位裂隙。与其他GH12酶的结构比较显示出显著的差异，在两条更长和高度扭曲的β-链B8和B9以及几个环中发现了这一点。一个独特的包含Arg60和Tyr61的A3-B3环通过氢键和堆积来稳定底物，就像在纤维四糖和纤维二糖的复杂晶体中观察到的那样。这些高分辨率结构可以清楚地阐明酶与其底物之间的相互作用网络。与酶结合的糖残基似乎在-2和-1亚位比在+1、+2和-3亚位更有序。在E134C晶体中，裂解部位的结合-1糖始终显示出α-不规则构型，暗示着一种类似中间体的结构。
Cellulases have been used in many applications to treat various carbohydrate-containing materials. Thermotoga maritima cellulase 12A (TmCel12A) belongs to the GH12 family of glycoside hydrolases. It is a beta-1,4-endoglucanase that degrades cellulose molecules into smaller fragments, facilitating further utilization of the carbohydrate. Because of its hyperthermophilic nature, the enzyme is especially suitable for industrial applications. Here the crystal structure of TmCel12A was determined by using an active-site mutant E134C and its mercury-containing derivatives. It adopts a beta-jellyroll protein fold typical of the GH12-family enzymes, with two curved beta-sheets A and B and a central active-site cleft. Structural comparison with other GH12 enzymes shows significant differences, as found in two longer and highly twisted beta-strands B8 and B9 and several loops. A unique Loop A3-B3 that contains Arg60 and Tyr61 stabilizes the substrate by hydrogen bonding and stacking, as observed in the complex crystals with cellotetraose and cellobiose. The high-resolution structures allow clear elucidation of the network of interactions between the enzyme and its substrate. The sugar residues bound to the enzyme appear to be more ordered in the -2 and -1 subsites than in the +1, +2 and -3 subsites. In the E134C crystals the bound -1 sugar at the cleavage site consistently show the alpha-anomeric configuration, implicating an intermediate-like structure.