The crystal structure of an inverting glycoside hydrolase family 9 exo-β-D-glucosaminidase and the design of glycosynthase
The crystal structure of an inverting glycoside hydrolase family 9 exo-β-D-glucosaminidase and the design of glycosynthase
复制标题
反转糖苷水解酶家族9外切-β-D-氨基葡萄糖苷酶的晶体结构和糖合酶的设计
DOI：
10.1042/bj20150966
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发表时间：
2016
期刊：
影响因子：
--
通讯作者：
and S. Fushinobu
中科院分区：
文献类型：
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作者：
Y. Honda;S. Arai;K. Suzuki;M. Kitaoka;and S. Fushinobu
深发光杆菌（Photobacterium profundum）的外切-β-D-氨基葡萄糖苷酶（EC 3.2.1.165）（PpGlcNase）是一种倒相GH（糖苷水解酶），属于第9家族。我们已经确定了PpGlcNase的三维结构，以描述第一个外切型GH 9糖苷酶的结构-功能关系。与GH 9内切葡聚糖酶的长聚糖结合裂缝相比，PpGlcNase具有窄且直的活性位点口袋。这是因为PpGlcNase有一个长的环，它阻断了内切葡聚糖酶的亚位点-4到-2对应的位置。PpGlcNase的口袋形状解释了其在非还原末端对β 1，4-键的底物偏好。Asp 139、Asp 143和Glu 555位于GlcN（D-氨基葡萄糖）的β-O 1羟基附近，其中Asp 139和Asp 143携带一个亲核水分子，可水解。D139 A、D143 A和E555 A突变体显著降低水解活性，表明它们的重要作用。在这些突变体中，D139 A仅表现出使用α-GlcN-F（α-D-葡糖胺基氟化物）和GlcN作为底物的糖合酶活性，以产生（GlcN）2。利用Asp 139饱和突变，我们获得了D139 E作为最佳的糖合酶。与野生型相比，D139 E的水解活性被显著抑制（<0.1%），F−释放活性也降低（<3%）。因此，D139 E的糖合酶活性低于先前由其它转化GH产生的糖苷酶的活性。在亲核水保持器处的突变是从反转GH产生有效的糖合酶的一般策略。然而，对于GH 9，其中两个酸性残基似乎共享催化碱基的作用，Asp 139的突变可能不可避免地降低F−-释放活性。
Exo-β-D-glucosaminidase (EC 3.2.1.165) fromPhotobacterium profundum(PpGlcNase) is an inverting GH (glycoside hydrolase) belonging to family 9. We have determined the three-dimensional structure of PpGlcNase to describe the first structure–function relationship of an exo-type GH9 glycosidase. PpGlcNase has a narrow and straight active-site pocket, in contrast with the long glycan-binding cleft of a GH9 endoglucanase. This is because PpGlcNase has a long loop, which blocks the position corresponding to subsites −4 to −2 of the endoglucanase. The pocket shape of PpGlcNase explains its substrate preference for a β1,4-linkage at the non-reducing terminus. Asp139, Asp143and Glu555in the active site were located near the β-O1 hydroxy group of GlcN (D-glucosamine), with Asp139and Asp143holding a nucleophilic water molecule for hydrolysis. The D139A, D143A and E555A mutants significantly decreased hydrolytic activity, indicating their essential role. Of these mutants, D139A exclusively exhibited glycosynthase activity using α-GlcN-F (α-D-glucosaminyl fluoride) and GlcN as substrates, to produce (GlcN)2. Using saturation mutagenesis at Asp139, we obtained D139E as the best glycosynthase. Compared with the wild-type, the hydrolytic activity of D139E was significantly suppressed (<0.1%), and the F−-release activity also decreased (<3%). Therefore the glycosynthase activity of D139E was lower than that of glycosynthases created previously from other inverting GHs. Mutation at the nucleophilic water holder is a general strategy for creating an effective glycosynthase from inverting GHs. However, for GH9, where two acidic residues seem to share the catalytic base role, mutation of Asp139might inevitably reduce F−-release activity.