National Institute of Advanced Industrial Science and Technology (AIST), Health Research Institute, Ikeda, Osaka, Japan.
Biochem J. 2011 Jul 15;437(2):223-30. doi: 10.1042/BJ20110292.
A hyperthermophilic membrane-related β-1,4-endoglucanase (family 5, cellulase) of the archaeon Pyrococcus horikoshii was found to be capable of hydrolysing cellulose at high temperatures. The hyperthermophilic cellulase has promise for applications in biomass utilization. To clarify its detailed function, we determined the crystal structures of mutants of the enzyme in complex with either the substrate or product ligands. We were able to resolve different kinds of complex structures at 1.65-2.01 Å (1 Å=0.1 nm). The structural analysis of various mutant enzymes yielded a sequence of crystallographic snapshots, which could be used to explain the catalytic process of the enzyme. The substrate position is fixed by the alignment of one cellobiose unit between the two aromatic amino acid residues at subsites +1 and +2. During the enzyme reaction, the glucose structure of cellulose substrates is distorted at subsite -1, and the β-1,4-glucoside bond between glucose moieties is twisted between subsites -1 and +1. Subsite -2 specifically recognizes the glucose residue, but recognition by subsites +1 and +2 is loose during the enzyme reaction. This type of recognition is important for creation of the distorted boat form of the substrate at subsite -1. A rare enzyme-substrate complex was observed within the low-activity mutant Y299F, which suggested the existence of a trapped ligand structure before the formation by covalent bonding of the proposed intermediate structure. Analysis of the enzyme-substrate structure suggested that an incoming water molecule, essential for hydrolysis during the retention process, might be introduced to the cleavage position after the cellobiose product at subsites +1 and +2 was released from the active site.
一种来自古菌 Pyrococcus horikoshii 的与膜相关的嗜热内切 β-1,4-葡聚糖酶（纤维素酶家族 5）能够在高温下水解纤维素。这种嗜热纤维素酶有望应用于生物质利用。为了阐明其详细功能，我们测定了该酶与底物或产物配体复合物的突变体的晶体结构。我们能够解析出不同类型的复合物结构，分辨率为 1.65-2.01 Å（1 Å=0.1 nm）。对各种突变酶的结构分析得到了一系列晶体学快照，可以用来解释酶的催化过程。通过两个芳香族氨基酸残基在+1 和+2 亚位点之间的一个纤维二糖单元的排列，固定了底物的位置。在酶反应过程中，纤维素底物的葡萄糖结构在-1 亚位点处发生扭曲，并且糖苷键在-1 和+1 亚位点之间扭曲。-2 亚位点特异性识别葡萄糖残基，但在酶反应过程中，+1 和+2 亚位点的识别较为松散。这种识别方式对于在-1 亚位点处形成底物的扭曲船型结构非常重要。在低活性突变体 Y299F 中观察到了一种罕见的酶-底物复合物，这表明在形成拟议的中间结构之前，存在一种被捕获的配体结构。对酶-底物结构的分析表明，在从活性位点释放出纤维二糖产物后，必需的水分子可能被引入到+1 和+2 亚位点的糖苷键断裂位置，从而参与水解过程。
