Shandong Provincial Key Laboratory of Synthetic Biology, CAS Key Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, No. 189 Songling Road, Qingdao 266101, China.
University of Chinese Academy of Sciences, Beijing 100049, China.
Sci Rep. 2017 Mar 9;7:44258. doi: 10.1038/srep44258.
The cytochrome P450 enzyme OleT from Jeotgalicoccus sp. ATCC 8456 is capable of converting free long-chain fatty acids into α-alkenes via one-step oxidative decarboxylation in presence of HO as cofactor or using redox partner systems. This enzyme has attracted much attention due to its intriguing but unclear catalytic mechanism and potential application in biofuel production. Here, we investigated the functionality of a select group of residues (Arg245, Cys365, His85, and Ile170) in the active site of OleT through extensive mutagenesis analysis. The key roles of these residues for catalytic activity and reaction type selectivity were identified. In addition, a range of heterologous redox partners were found to be able to efficiently support the decarboxylation activity of OleT. The best combination turned out to be SeFdx-6 (ferredoxin) from Synechococcus elongatus PCC 7942 and CgFdR-2 (ferredoxin reductase) from Corynebacterium glutamicum ATCC 13032, which gave the highest myristic acid conversion rate of 94.4%. Moreover, Michaelis-Menton kinetic parameters of OleT towards myristic acid were determined.
来自 Jeotgalicoccus sp. ATCC 8456 的细胞色素 P450 酶 OleT 能够在 HO 作为辅助因子或使用氧化还原对系统的存在下，通过一步氧化脱羧将游离长链脂肪酸转化为α-烯烃。由于其有趣但不清楚的催化机制以及在生物燃料生产中的潜在应用，该酶引起了广泛关注。在这里，我们通过广泛的突变分析研究了 OleT 活性位点中一组选定残基（Arg245、Cys365、His85 和 Ile170）的功能。确定了这些残基对催化活性和反应类型选择性的关键作用。此外，还发现了一系列异源氧化还原对能够有效地支持 OleT 的脱羧活性。最佳组合是来自 Synechococcus elongatus PCC 7942 的 SeFdx-6（铁氧还蛋白）和来自 Corynebacterium glutamicum ATCC 13032 的 CgFdR-2（铁氧还蛋白还原酶），它们使肉豆蔻酸的转化率最高，达到 94.4%。此外，还确定了 OleT 对肉豆蔻酸的米氏动力学参数。
