Effects of the cofactor binding sites on the activities of secondary alcohol dehydrogenase (SADH).
Effects of the cofactor binding sites on the activities of secondary alcohol dehydrogenase (SADH).
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DOI：
10.1016/j.ijbiomac.2016.03.043
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发表时间：
2016-07
影响因子：
8.2
通讯作者：
Tao Wang;Xiangjun Chen;Jun-Pil Han;Sichun Ma;Jianmei Wang;Xu-feng Li;Hui Zhang;Zhibin Liu
中科院分区：
文献类型：
--
作者：
Tao Wang;Xiangjun Chen;Jun-Pil Han;Sichun Ma;Jianmei Wang;Xu-feng Li;Hui Zhang;Zhibin Liu
来自Thermoanaerobic ethanolicus的SADH是与各种辅因子一起催化羰基化合物可逆还原成其相应醇的酶。为了探讨辅助因子与SADH的结合方式，本研究克隆了SADH的辅助因子TeSADH，并将其Ser 199和Arg 200分别替换为Tyr和Asp。根据计算a预测，预期两个位点都在辅因子结合结构域内部或邻近辅因子结合结构域。对TeSADH活性的分析表明，突变体S199 Y在NADH、NADPH作为辅助因子时，其酶效率（kcat/Km）显著提高，而在NADP+、NAD+作为辅助因子时，其酶效率与野生型相近。相反，活性的R200 D突变体显着降低与所有辅因子。此外，在酵母中，与野生型相比，S199 Y突变体显著提高了乙醇浓度。分子动力学模拟结果表明，S199 Y突变体的TeSADH与辅因子之间的氢键网络更强，结合能也随之增加。荧光结果表明，突变体S199 Y对NAD（P）H的选择性增强，与NAD（P）H的结合更加紧密。
SADHs fromThermoanaerobacter ethanolicusare enzymes that, together with various cofactors, catalyze the reversible reduction of carbonyl compounds to their corresponding alcohols. To explore how cofactors bind to SADH,TeSADHwas cloned in this study, and Ser199and Arg200were replaced by Tyr and Asp, respectively. Both sites were expected to be inside or adjacent to the cofactor-binding domain according to computational a prediction. Analysis of TeSADH activities revealed that the enzymatic efficiency (kcat/Km) of the S199Y mutant was noticeably enhanced using by NADH, NADPH as cofactors, and similar with that of wild-type using by NADP+, NAD+. Conversely, the activity of the R200D mutant significantly decreased with all cofactors. Furthermore, in yeast, the S199Y mutant substantially elevated the ethanol concentration compared with the wild type. Molecular dynamics simulation results indicated the H-bonding network between TeSADH and the cofactors was stronger for the S199Y mutant and the binding energy was simultaneously increased. Moreover, the fluorescence results indicated the S199Y mutant exhibited an increased preference for NAD(P)H, binding with NAD(P)H more compactly compared with wild type.