Substrate complex structure, active site labeling and catalytic role of the zinc ion in cysteine glycosidase
Substrate complex structure, active site labeling and catalytic role of the zinc ion in cysteine glycosidase
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DOI：
10.1093/glycob/cwab103
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发表时间：
2022-03-19
期刊：
影响因子：
4.3
通讯作者：
Fushinobu，Shinya
中科院分区：
文献类型：
--
作者：
Maruyama，Shun;Sawano，Kota;Fushinobu，Shinya
长双歧杆菌β-l-阿拉伯呋喃糖苷酶HypBA 1属于糖苷水解酶家族127。在HypBA 1的活性位点，半胱氨酸残基（Cys 417）与Zn 2+原子配位，并作为催化亲核试剂用于保留端基异构体的水解反应。本研究从生物化学和晶体学的角度研究了Zn ~（2+）和半胱氨酸在催化反应中的作用以及底物结合结构。在EDTA和盐酸胍存在下透析后，HypBA 1的酶活性下降，然后通过添加Zn 2+恢复。米氏络合物的结构是使用在酸/碱催化剂残留物（E322 Q）处的突变体的晶体浸泡在含有底物对硝基苯基-β-l-阿拉伯呋喃糖苷的溶液中来确定的。为了研究共价硫代糖基酶中间体结构，使用具有不同端基异构体构型的1-阿拉伯呋喃糖基卤代乙酰胺衍生物的合成抑制剂来靶向亲核半胱氨酸。在HypBA 1的晶体结构中，β-构型的l-阿拉伯呋喃糖酰胺与Cys 417形成共价键，而α-构型的l-阿拉伯呋喃糖酰胺与非催化残基Cys 415连接。质谱分析表明，Cys 415也与探针分子反应。使用β-构型抑制剂，阿拉伯呋喃糖苷部分正确地定位在亚位点处，并且保留活性位点完整性以成功地模拟共价中间体状态。
β-l-Arabinofuranosidase HypBA1 fromBifidobacterium longumbelongs to the glycoside hydrolase family 127. At the active site of HypBA1, a cysteine residue (Cys417) coordinates with a Zn2+atom and functions as the catalytic nucleophile for the anomer-retaining hydrolytic reaction. In this study, the role of Zn2+ion and cysteine in catalysis as well as the substrate-bound structure were studied based on biochemical and crystallographic approaches. The enzymatic activity of HypBA1 decreased after dialysis in the presence of EDTA and guanidine hydrochloride and was then recovered by the addition of Zn2+. The Michaelis complex structure was determined using a crystal of a mutant at the acid/base catalyst residue (E322Q) soaked in a solution containing the substratep-nitrophenyl-β-l-arabinofuranoside. To investigate the covalent thioglycosyl enzyme intermediate structure, synthetic inhibitors ofl-arabinofuranosyl haloacetamide derivatives with different anomer configurations were used to target the nucleophilic cysteine. In the crystal structure of HypBA1, β-configuredl-arabinofuranosylamide formed a covalent link with Cys417, whereas α-configured l-arabinofuranosylamide was linked to a noncatalytic residue Cys415. Mass spectrometric analysis indicated that Cys415 was also reactive with the probe molecule. With the β-configured inhibitor, the arabinofuranoside moiety was correctly positioned at the subsite and the active site integrity was retained to successfully mimic the covalent intermediate state.