PDF(Pubmed)
Abstract：
The glycoside hydrolase family 55 (GH55) includes inverting exo-β-1,3-glucosidases and endo-β-1,3-glucanases, acting on laminarin, which is a β1-3/1-6-glucan consisting of a β1-3/1-6-linked main chain and β1-6-linked branches. Despite their different modes of action toward laminarin, endo-β-1,3-glucanases share with exo-β-1,3-glucosidases conserved residues that form the dead-end structure of subsite -1. Here, we investigated the mechanism of endo-type action on laminarin by GH55 endo-β-1,3-glucanase MnLam55A, identified from Microdochium nivale. MnLam55A, like other endo-β-1,3-glucanases, degraded internal β-d-glucosidic linkages of laminarin, producing more reducing sugars than the sum of d-glucose and gentiooligosaccharides detected. β1-3-Glucans lacking β1-6-linkages in the main chain were not hydrolyzed. NMR analysis of the initial degradation of laminarin revealed that MnLam55A preferentially cleaved the nonreducing terminal β1-3-linkage of the laminarioligosaccharide moiety at the reducing end side of the main chain β1-6-linkage. MnLam55A liberates d-glucose from laminaritriose and longer laminarioligosaccharides, but kcat/Km values to laminarioligosaccharides (≤4.21 s-1 mM-1) were much lower than to laminarin (5920 s-1 mM-1). These results indicate that β-glucan binding to the minus subsites of MnLam55A, including exclusive binding of the gentiobiosyl moiety to subsites -1 and -2, is required for high hydrolytic activity. A crystal structure of MnLam55A, determined at 2.4 Å resolution, showed that MnLam55A adopts an overall structure and catalytic site similar to those of exo-β-1,3-glucosidases. However, MnLam55A possesses an extended substrate-binding cleft that is expected to form the minus subsites. Sequence comparison suggested that other endo-type enzymes share the extended cleft. The specific hydrolysis of internal linkages in laminarin is presumably common to GH55 endo-β-1,3-glucanases.
摘要：
糖苷水解酶家族55（GH55）包括反转外切-β-1,3-葡糖苷酶和内切-β-1,3-葡聚糖酶，作用于海带多糖,其是由β1-3/1-6连接的主链和β1-6连接的分支组成的β1-3/1-6-葡聚糖。尽管它们对laminarin的作用方式不同，内切-β-1,3-葡聚糖酶与外切-β-1,3-葡糖苷酶共享形成亚位点-1的死端结构的保守残基。这里,我们研究了GH55内切β-1,3-葡聚糖酶MnLam55A对海带蛋白的内切型作用机制，从Microdochiumnivale鉴定。MnLam55A,像其他内切β-1,3-葡聚糖酶一样，降解的内部β-d-葡糖苷键，产生比检测到的d-葡萄糖和酮寡糖总和更多的还原糖。主链中缺乏β1-6-键的β1-3-葡聚糖未水解。海带多糖的初始降解的NMR分析表明，MnLam55A优先在主链β1-6-键的还原端侧裂解了拉米寡糖部分的非还原端β1-3-键。MnLam55A从层状三糖和更长的层状寡糖中释放d-葡萄糖，但对laminarioligh有糖(≤4.21s-1mM-1)的kcat/Km值远低于对laminarinario(5,920s-1mM-1)的kcat/Km值。这些结果表明，β-葡聚糖与MnLam55A的负亚位点结合，高水解活性需要包括基因二糖基部分与亚位点-1和-2的排他性结合。MnLam55A的晶体结构，在2.4µ分辨率下确定，表明MnLam55A具有与外切β-1,3-葡萄糖苷酶相似的整体结构和催化位点。然而,MnLam55A具有延伸的底物结合裂隙，有望形成负子位点。序列比较表明，其他内型酶共享扩展的裂隙结构。海带多糖中内部键的特定水解大概是GH55内切-β-1,3-葡聚糖酶所共有的。
