Prates J A, Tarbouriech N, Charnock S J, Fontes C M, Ferreira L M, Davies G J
Centro Interdisciplinar de Investigação em Sanidade Animal, Faculdade de Medicina Veteterinária, Pólo Universitário do Alto da Ajuda, 1300-477 Lisboa Codex, Portugal.
Structure. 2001 Dec;9(12):1183-90. doi: 10.1016/s0969-2126(01)00684-0.
Degradation of the plant cell wall requires the synergistic action of a consortium of predominantly modular enzymes. In Clostridiae, these biocatalysts are organized into a supramolecular assembly termed a "cellulosome." This multienzyme complex possesses, in addition to its well-described cellulolytic activity, an apparatus specific for xylan degradation. Cinnamic acid esterases hydrolyze the ferulate groups involved in the crosslinking of arabinoxylans to lignin and thus play a key role in the degradation of the plant cell wall in addition to having promising industrial and medical applications.
We have cloned and overexpressed the feruloyl esterase module from a 5 domain xylanase, Xyn10B from Clostridium thermocellum. The native structure at 1.6 A resolution has been solved with selenomethionine multiple wavelength anomalous dispersion and refined to a final R(free) of 17.8%. The structure of a hydrolytically inactive mutant, S954A, in complex with the reaction product ferulic acid has been refined at a resolution of 1.4 A with an R(free) of 16.0%.
The C. thermocellum Xyn10B ferulic acid esterase displays the alpha/beta-hydrolase fold and possesses a classical Ser-His-Asp catalytic triad. Ferulate esterases are characterized by their specificity, and the active center reveals the binding site for ferulic acid and related compounds. Ferulate binds in a small surface depression that possesses specificity determinants for both the methoxy and hydroxyl ring substituents of the substrate. There appears to be a lack of specificity for the xylan backbone, which may reflect the intrinsic chemical heterogeneity of the natural substrate.
植物细胞壁的降解需要一群主要为模块化酶的协同作用。在梭菌属中，这些生物催化剂被组织成一个称为“纤维小体”的超分子组装体。这种多酶复合物除了具有其广为人知的纤维素分解活性外，还拥有一个专门用于木聚糖降解的装置。肉桂酸酯酶水解参与阿拉伯木聚糖与木质素交联的阿魏酸基团，因此除了具有潜在的工业和医学应用外，在植物细胞壁的降解中也起着关键作用。
我们从嗜热栖热放线菌的一个5结构域木聚糖酶Xyn10B中克隆并过表达了阿魏酸酯酶模块。利用硒代蛋氨酸多波长反常色散解析了分辨率为1.6埃的天然结构，并将其精修至最终的自由R因子为17.8%。一个与反应产物阿魏酸复合的水解无活性突变体S954A的结构已在分辨率为1.4埃、自由R因子为16.0%的条件下进行了精修。
嗜热栖热放线菌Xyn10B阿魏酸酯酶呈现α/β-水解酶折叠结构，并拥有一个经典的丝氨酸-组氨酸-天冬氨酸催化三联体。阿魏酸酯酶以其特异性为特征，活性中心揭示了阿魏酸及相关化合物的结合位点。阿魏酸结合在一个小的表面凹陷处，该凹陷处具有底物甲氧基和羟基环取代基的特异性决定因素。对木聚糖主链似乎缺乏特异性，这可能反映了天然底物固有的化学异质性。
