Varrot A, Schülein M, Davies G J
Structural Biology Laboratory, Department of Chemistry, University of York, U.K.
Biochemistry. 1999 Jul 13;38(28):8884-91. doi: 10.1021/bi9903998.
The mechanisms of crystalline cellulose degradation by cellulases are of paramount importance for the exploitation of these enzymes in applied processes, such as biomass conversion. Cellulases have traditionally been classified into cellobiohydrolases, which are effective in the degradation of crystalline materials, and endoglucanases, which appear to act on "soluble" regions of the substrate. Humicola insolensCel6A (CBH II) is a cellobiohydrolase from glycoside hydrolase family 6 whose native structure has been determined at 1.9 A resolution [Varrot, A., Hastrup, S., Schülein, M., and Davies, G. J. (1999) Biochem. J. 337, 297-304]. Here we present the structure of the catalytic core domain of Humicola insolens cellobiohydrolase II Cel6A in complex with glucose/cellotetraose at 1.7 A resolution. Crystals of Cel6A, grown in the presence of cellobiose, reveal six binding subsites, with a single glucose moiety bound in the -2 subsite and cellotetraose in the +1 to +4 subsites. The complex structure is strongly supportive of the assignment of Asp 226 as the catalytic acid and consistent with proposals that Asp 405 acts as the catalytic base. The structure undergoes several conformational changes upon substrate binding, the most significant of which is a closing of the two active site loops (residues 174-196 and 397-435) with main-chain movements of up to 4.5 A observed. This complex not only defines the polysaccharide-enzyme interactions but also provides the first three-dimensional demonstration of conformational change in this class of enzymes.
纤维素酶降解结晶纤维素的机制对于这些酶在生物质转化等应用过程中的开发利用至关重要。传统上，纤维素酶被分为对结晶材料降解有效的纤维二糖水解酶和似乎作用于底物“可溶性”区域的内切葡聚糖酶。腐质霉Cel6A（CBH II）是一种来自糖苷水解酶家族6的纤维二糖水解酶，其天然结构已在1.9 Å分辨率下确定[Varrot, A., Hastrup, S., Schülein, M., and Davies, G. J. (1999) Biochem. J. 337, 297 - 304]。在此，我们展示了腐质霉纤维二糖水解酶II Cel6A催化核心结构域与葡萄糖/纤维四糖复合物在1.7 Å分辨率下的结构。在纤维二糖存在下生长的Cel6A晶体显示出六个结合亚位点，其中一个葡萄糖部分结合在 -2亚位点，纤维四糖结合在 +1至 +4亚位点。该复合物结构有力地支持了将天冬氨酸226指定为催化酸，并与天冬氨酸405作为催化碱的提议一致。底物结合后，该结构发生了几种构象变化，其中最显著的是两个活性位点环（残基174 - 196和397 - 435）的闭合，观察到主链移动高达4.5 Å。该复合物不仅定义了多糖 - 酶相互作用，还首次提供了这类酶构象变化的三维演示。
