Hermoso Juan A, Sanz-Aparicio Julia, Molina Rafael, Juge Nathalie, González Ramón, Faulds Craig B
Grupo de Cristalografía Macromolecular y Biología Estructural, Instituto Química-Física Rocasolano C.S.I.C., Serrano 119, 28006 Madrid, Spain.
J Mol Biol. 2004 Apr 30;338(3):495-506. doi: 10.1016/j.jmb.2004.03.003.
As a component of the array of enzymes produced by micro-organisms to deconstruct plant cell walls, feruloyl esterases hydrolyze phenolic groups involved in the cross-linking of arabinoxylan to other polymeric structures. This is important for opening the cell wall structure, making material more accessible to glycosyl hydrolases. Here, we describe the first crystal structure of the non-modular type-A feruloyl esterase from Aspergillus niger (AnFaeA) solved at 2.5A resolution. AnFaeA displays an alpha/beta hydrolase fold similar to that found in fungal lipases and different from that reported for other feruloyl esterases. Crystallographic and site-directed mutagenesis studies allow us to identify the catalytic triad (Ser133-His247-Asp194) that forms the catalytic machinery of this enzyme. The active-site cavity is confined by a lid (residues 68-80), on the analogy of lipases, and by a loop (residues 226-244) that confers plasticity to the substrate-binding site. The lid presents a high ratio of polar residues, which in addition to a unique N-glycosylation site stabilises the lid in an open conformation, conferring the esterase character to this enzyme. A putative model for bound 5,5'-diferulic acid-linked arabinoxylan has been built, pointing to the more relevant residues involved in substrate recognition. Comparison with structurally related lipases reveals that subtle amino acid and conformational changes within a highly conserved protein fold may produce protein variants endowed with new enzymatic properties, while comparison with functionally related proteins points to a functional convergence after evolutionary divergence within the feruloyl esterases family.
作为微生物产生的用于解构植物细胞壁的一系列酶的组成部分，阿魏酸酯酶可水解参与阿拉伯木聚糖与其他聚合物结构交联的酚基团。这对于打开细胞壁结构很重要，能使物质更易被糖基水解酶作用。在此，我们描述了黑曲霉非模块化A型阿魏酸酯酶（AnFaeA）的首个晶体结构，其分辨率为2.5埃。AnFaeA呈现出与真菌脂肪酶中发现的α/β水解酶折叠相似，且与其他阿魏酸酯酶报道的折叠不同。晶体学和定点诱变研究使我们能够鉴定出形成该酶催化机制的催化三联体（Ser133-His247-Asp194）。活性位点腔由一个类似于脂肪酶的盖子（68 - 80位残基）以及一个赋予底物结合位点可塑性的环（226 - 244位残基）所限制。盖子呈现出高比例的极性残基，除了一个独特的N - 糖基化位点外，还使盖子稳定在开放构象，赋予该酶酯酶特性。已构建了一个结合5,5'-二阿魏酸连接的阿拉伯木聚糖的推测模型，指出了参与底物识别的更相关残基。与结构相关的脂肪酶比较表明，在高度保守的蛋白质折叠内细微的氨基酸和构象变化可能产生具有新酶特性的蛋白质变体，而与功能相关蛋白质比较则表明在阿魏酸酯酶家族进化分歧后存在功能趋同。
