Mutagenesis and redox partners analysis of the P450 fatty acid decarboxylase OleT(JE).
Mutagenesis and redox partners analysis of the P450 fatty acid decarboxylase OleT(JE).
复制标题
P450 脂肪酸脱羧酶 OleTJE 的诱变和氧化还原伙伴分析
DOI：
10.1038/srep44258
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发表时间：
2017-03-09
影响因子：
4.6
通讯作者：
Li S
中科院分区：
文献类型：
--
作者：
Fang B;Xu H;Liu Y;Qi F;Zhang W;Chen H;Wang C;Wang Y;Yang W;Li S
对来自Jeotgalicoccussp. ATCC 8456能够在H2 O2作为辅因子或使用氧化还原伴侣系统的情况下，通过一步氧化脱羧将游离长链脂肪酸转化为α-烯烃。该酶因其独特的催化机理和在生物燃料生产中的潜在应用而受到广泛关注。在此，我们通过广泛的诱变分析研究了OleTJ E活性位点中一组选定的残基（Arg 245，Cys 365，His 85和Ile 170）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，发现一系列异源氧化还原配偶体能够有效地支持OleTJE的脱羧活性。最佳组合为来自细长聚球藻PCC 7942的SeFdx-6（铁氧还蛋白）和来自谷氨酸棒杆菌ATCC 13032的CgFdR-2（铁氧还蛋白还原酶），肉豆蔻酸转化率最高，达94.4%。测定了OleTJE转化肉豆蔻酸的Michaelis-Menton动力学参数。
The cytochrome P450 enzyme OleTJEfromJeotgalicoccussp. ATCC 8456 is capable of converting free long-chain fatty acids into α-alkenes via one-step oxidative decarboxylation in presence of H2O2as 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 OleTJEthrough 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 OleTJE. The best combination turned out to beSeFdx-6 (ferredoxin) fromSynechococcus elongatusPCC 7942 andCgFdR-2 (ferredoxin reductase) fromCorynebacterium glutamicumATCC 13032, which gave the highest myristic acid conversion rate of 94.4%. Moreover, Michaelis-Menton kinetic parameters of OleTJEtowards myristic acid were determined.