A comparative structural analysis of the surface properties of asco-laccases

Laccases of different biological origins have been widely investigated and these studies have elucidated fundamentals of the generic catalytic mechanism. However, other features such as surface properties and residues located away from the catalytic centres may also have impact on enzyme function. Here we present the crystal structure of laccase from Myceliophthora thermophila (MtL) to a resolution of 1.62 Å together with a thorough structural comparison with other members of the CAZy family AA1_3 that comprises fungal laccases from ascomycetes. The recombinant protein produced in A. oryzae has a molecular mass of 75 kDa, a pI of 4.2 and carries 13.5 kDa N-linked glycans. In the crystal, MtL forms a dimer with the phenolic substrate binding pocket blocked, suggesting that the active form of the enzyme is monomeric. Overall, the MtL structure conforms with the canonical fold of fungal laccases as well as the features specific for the asco-laccases. However, the structural comparisons also reveal significant variations within this taxonomic subgroup. Notable differences in the T1-Cu active site topology and polar motifs imply molecular evolution to serve different functional roles. Very few surface residues are conserved and it is noticeable that they encompass residues that interact with the N-glycans and/or are located at domain interfaces. The N-glycosylation sites are surprisingly conserved among asco-laccases and in most cases the glycan displays extensive interactions with the protein. In particular, the glycans at Asn88 and Asn210 appear to have evolved as an integral part of the asco-laccase structure. An uneven distribution of the carbohydrates around the enzyme give unique properties to a distinct part of the surface of the asco-laccases which may have implication for laccase function–in particular towards large substrates.

不同生物来源的漆酶（Laccase）已被广泛研究，相关研究阐明了该类酶通用催化机制的基础原理。然而，诸如表面性质以及远离催化中心的残基等其他特征，同样可能对酶功能产生影响。本研究解析了来自嗜热毁丝霉（Myceliophthora thermophila）的漆酶MtL的晶体结构，分辨率达1.62埃（Å），并对其与碳水化合物活性酶（CAZy）家族AA1_3的其他成员开展了全面的结构比对——该家族包含子囊菌来源的真菌漆酶。在米曲霉（A. oryzae）中异源表达的重组蛋白分子量为75 kDa，等电点（pI）为4.2，且带有13.5 kDa的N-连接聚糖（N-linked glycans）。晶体状态下，MtL以二聚体形式存在，其酚类底物结合口袋处于封闭状态，这提示该酶的活性形式为单体。整体而言，MtL的结构符合真菌漆酶的典型折叠模式，同时具备子囊菌漆酶的特有特征。但结构比对结果同时揭示了该分类亚组内存在显著的结构变异。T1-Cu活性位点的拓扑结构与极性基序存在显著差异，这暗示其分子进化是为了适配不同的功能角色。保守的表面残基数量极少，值得注意的是，这些残基涵盖了与N-连接聚糖相互作用的残基，以及/或者位于结构域界面处的残基。N-连接糖基化位点在子囊菌漆酶中意外地呈现保守性，且多数情况下聚糖与蛋白质存在广泛的相互作用。具体而言，位于Asn88与Asn210位点的聚糖似乎已进化为子囊菌漆酶结构不可或缺的组成部分。酶表面的碳水化合物分布不均，赋予了子囊菌漆酶表面特定区域独特的理化性质，这可能对漆酶的功能——尤其是针对大型底物的催化过程——具有重要意义。