Structural and Biochemical Characterization of Glycoside Hydrolase Family 79 β-Glucuronidase from Acidobacterium capsulatum
Structural and Biochemical Characterization of Glycoside Hydrolase Family 79 β-Glucuronidase from Acidobacterium capsulatum
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DOI：
10.1074/jbc.m112.346288
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发表时间：
2012-04-20
影响因子：
4.8
通讯作者：
Kaneko, Satoshi
中科院分区：
文献类型：
--
作者：
Michikawa, Mari;Ichinose, Hitomi;Kaneko, Satoshi
我们提出了第一个结构的糖苷水解酶家族79 β-葡萄糖醛酸酶从Acidobacterium capsulatum，无论是作为一个产品复合物与β-D-葡萄糖醛酸（GlcA），并作为其捕获的共价2-氟葡萄糖醛酸中间体。该酶由催化（β/α）（8）桶结构域和具有未知功能的不规则希腊关键基序的β结构域组成。该酶显示出β-葡萄糖醛酸酶活性和痕量水平的β-葡萄糖苷酶和β-木糖苷酶活性。结合诱变研究，这些结构将催化残基鉴定为Glu（173）（酸碱）和Glu（287）（亲核体），与H-1 NMR分析证明的保留机制一致。Glu（45）、Tyr（243）、Tyr（292）-Gly（294）和Tyr（334）形成催化口袋并提供底物区分。与此一致，影响Gln（293）和Gly（294）主链与GlcA羧基之间相互作用的Y292 A突变导致β-葡萄糖醛酸糖苷酶活性显著丧失，同时保留野生型水平的副活性。同样，尽管Y334 F突变体的β-葡糖醛酸糖苷酶活性比野生型酶的活性低200倍（k（cat）/K-m），但β-葡糖苷酶活性实际上比野生型的等效参数高3倍，β-木糖苷酶活性仅低2.5倍，这与Tyr（334）在识别GlcA的C6位置中的作用一致。Glu（45）参与区分GlcA的C4位置处的O-甲基的结合，这一事实揭示了E45 D突变体水解PNP-β-GlcA比野生型酶慢约300倍（k（cat）/K-m），而含4-O-甲基-GlcA的寡糖水解仅慢7倍。
We present the first structure of a glycoside hydrolase family 79 beta-glucuronidase from Acidobacterium capsulatum, both as a product complex with beta-D-glucuronic acid (GlcA) and as its trapped covalent 2-fluoroglucuronyl intermediate. This enzyme consists of a catalytic (beta/alpha)(8)-barrel domain and a beta-domain with irregular Greek key motifs that is of unknown function. The enzyme showed beta-glucuronidase activity and trace levels of beta-glucosidase and beta-xylosidase activities. In conjunction with mutagenesis studies, these structures identify the catalytic residues as Glu(173) (acid base) and Glu(287) (nucleophile), consistent with the retaining mechanism demonstrated by H-1 NMR analysis. Glu(45), Tyr(243), Tyr(292)-Gly(294), and Tyr(334) form the catalytic pocket and provide substrate discrimination. Consistent with this, the Y292A mutation, which affects the interaction between the main chains of Gln(293) and Gly(294) and the GlcA carboxyl group, resulted in significant loss of beta-glucuronidase activity while retaining the side activities at wild-type levels. Likewise, although the beta-glucuronidase activity of the Y334F mutant is similar to 200-fold lower (k(cat)/K-m) than that of the wild-type enzyme, the beta-glucosidase activity is actually 3 times higher and the beta-xylosidase activity is only 2.5-fold lower than the equivalent parameters for wild type, consistent with a role for Tyr(334) in recognition of the C6 position of GlcA. The involvement of Glu(45) in discriminating against binding of the O-methyl group at the C4 position of GlcA is revealed in the fact that the E45D mutant hydrolyzes PNP-beta-GlcA approximately 300-fold slower (k(cat)/K-m) than does the wild-type enzyme, whereas 4-O-methyl-GlcA-containing oligosaccharides are hydrolyzed only 7-fold slower.