Structural, Mechanistic, and Functional Insights into an Arthrobacter nicotinovorans Molybdenum Hydroxylase Involved in Nicotine Degradation.
Structural, Mechanistic, and Functional Insights into an Arthrobacter nicotinovorans Molybdenum Hydroxylase Involved in Nicotine Degradation.
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参与尼古丁降解的食烟节杆菌钼羟化酶的结构、机制和功能见解
DOI：
10.3390/molecules26144387
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发表时间：
2021-07-20
期刊：
影响因子：
--
通讯作者：
Wu G
中科院分区：
文献类型：
--
作者：
Wang L;Mu X;Li W;Xu Q;Xu P;Zhang L;Zhang Y;Wu G
烟碱类关节杆菌通过吡啶途径分解尼古丁。6- hydroxyseudooxynicotine 2-oxidoreductase（又称酮脱氢酶，Kdh）是A. nicotinovorans尼古丁降解途径中的重要酶，负责尼古丁的二次羟基化。Kdh属于钼羟基化酶家族，可催化6-羟基-假羟基尼古丁（6-HPON）氧化为2,6-二羟基-假羟基尼古丁（2,6- dhpon）。研究人员确定了烟酸酵母Kdh全酶的晶体结构，包括其三个亚基KdhL、KdhM和KdhS，以及它们的辅助因子钼胞嘧啶二核苷酸（MCD）、两个铁硫簇（Fe2S2）和黄素腺嘌呤二核苷酸（FAD）。此外，我们通过分子对接获得了底物6- hphon结合的Kdh的结构模型，并进行了分子动力学（MD）和量子力学/分子力学（QM/MM）计算，揭示了Kdh的催化机理。发现KdhL的Glu345、Try551和Glu748残基参与底物结合，发现KdhL的Phe269和Arg383有助于稳定MCD构象。此外，我们还进行了位点诱变和酶活性分析来支持我们的结构和计算结果，这些结果也揭示了随着缓冲液ph的增加，催化效率有增加的趋势。最后，我们的电化学结果证明了Kdh的各种辅助因子之间的电子转移。因此，我们的工作提供了一个全面的结构，机制和功能研究的钼羟化酶Kdh在烟酸烟碱降解途径。
Arthrobacter nicotinovorans decomposes nicotine through the pyridine pathway. 6-hydroxypseudooxynicotine 2-oxidoreductase (also named ketone dehydrogenase, Kdh) is an important enzyme in nicotine degradation pathway of A. nicotinovorans, and is responsible for the second hydroxylation of nicotine. Kdh belongs to the molybdenum hydroxylase family, and catalyzes the oxidation of 6-hydroxy-pseudooxynicotine (6-HPON) to 2,6-dihydroxy-pseudooxynicotine (2,6-DHPON). We determined the crystal structure of the Kdh holoenzyme from A. nicotinovorans, with its three subunits KdhL, KdhM, and KdhS, and their associated cofactors molybdopterin cytosine dinucleotide (MCD), two iron-sulfur clusters (Fe2S2), and flavin adenine dinucleotide (FAD), respectively. In addition, we obtained a structural model of the substrate 6-HPON-bound Kdh through molecular docking, and performed molecular dynamics (MD) and quantum mechanics/molecular mechanics (QM/MM) calculations to unveil the catalytic mechanism of Kdh. The residues Glu345, Try551, and Glu748 of KdhL were found to participate in substrate binding, and Phe269 and Arg383 of KdhL were found to contribute to stabilize the MCD conformation. Furthermore, site-directed mutagenesis and enzymatic activity assays were performed to support our structural and computational results, which also revealed a trend of increasing catalytic efficiency with the increase in the buffer pH. Lastly, our electrochemical results demonstrated electron transfer among the various cofactors of Kdh. Therefore, our work provides a comprehensive structural, mechanistic, and functional study on the molybdenum hydroxylase Kdh in the nicotine degradation pathway of A. nicotinovorans.
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影响因子：
62.1
作者：
Hille, Russ;Hall, James;Basu, Partha
通讯作者：
Basu, Partha
影响因子：
3.2
作者：
Igloi, GL;Brandsch, R
通讯作者：
Brandsch, R
影响因子：
15
作者：
Heering, HA;Weiner, JH;Armstrong, FA
通讯作者：
Armstrong, FA
DOI：
10.1073/pnas.96.16.8884
发表时间：
1999-08-03
影响因子：
11.1
作者：
Dobbek, H;Gremer, L;Huber, R
通讯作者：
Huber, R
影响因子：
5.7
作者：
Bonin, I;Martins, BM;Dobbek, H
通讯作者：
Dobbek, H