In vitro oxidative decarboxylation of free fatty acids to terminal alkenes by two new P450 peroxygenases.
In vitro oxidative decarboxylation of free fatty acids to terminal alkenes by two new P450 peroxygenases.
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两种新型 P450 过氧化酶将游离脂肪酸体外氧化脱羧为末端烯烃
DOI：
10.1186/s13068-017-0894-x
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发表时间：
2017
影响因子：
6.3
通讯作者：
Li S
中科院分区：
文献类型：
--
作者：
Xu H;Ning L;Yang W;Fang B;Wang C;Wang Y;Xu J;Collin S;Laeuffer F;Fourage L;Li S
背景以CYP 152环加氧酶家族成员OleTJ为代表的P450脂肪酸脱羧酶由于能够催化简单直接的1-烯烃合成，在生物燃料和生物材料中具有潜在的应用价值，近年来受到了广泛的关注。为了获得更多的机制的见解，更广泛的底物光谱，并提高脱羧活性，它是需要发现和研究更多的P450脂肪酸decarboxylases.ResultsHere，我们首次描述的表达，纯化，并在体外生化特性的两个新的CYP 152过氧酶，CYP-Aa 162和CYP-Sm 46 Δ29，这是能够脱羧直链饱和脂肪酸。发现这两种酶催化广泛的游离脂肪酸（C10-C20）的脱羧和羟基化，具有重叠的底物特异性，但不同的化学选择性。CYP-Sm 46 Δ29主要作为脂肪（月桂）酸脱羧酶（66.1 ± 3.9%的1-十一碳烯产量），而CYP-Aa 162更多地作为脂肪（月桂）酸羟化酶（72.2 ± 0.9%的羟基月桂酸产量）。值得注意的是，功能性CYP-Sm 46 Δ29的光学光谱分析显示没有特征性P450带，表明独特的血红素配位环境。活性位点突变分析表明，用所提出的关键脱羧调节残基His 85和Ile 170取代，增强了CYP-Aa 162和P450 BS β的脱羧活性，强调了这些残基在指导脱羧途径中的重要性。此外，稳态动力学分析表明，CYP-Aa 162和CYP-Sm 46 Δ 29具有底物抑制和协同作用，且与底物碳链长度有关。CYP 152脱羧酶的氧化脱羧化学选择性在很大程度上取决于脂肪酸底物的碳链长度及其在酶活性位点的精确定位。最后，酶的动力学模式分析可以为未来的工艺设计提供重要的指导。
BackgroundP450 fatty acid decarboxylases represented by the unusual CYP152 peroxygenase family member OleTJEhave been receiving great attention recently since these P450 enzymes are able to catalyze the simple and direct production of 1-alkenes for potential applications in biofuels and biomaterials. To gain more mechanistic insights, broader substrate spectra, and improved decarboxylative activities, it is demanded to discover and investigate more P450 fatty acid decarboxylases.ResultsHere, we describe for the first time the expression, purification, and in vitro biochemical characterization of two new CYP152 peroxygenases, CYP-Aa162 and CYP-Sm46Δ29, that are capable of decarboxylating straight-chain saturated fatty acids. Both enzymes were found to catalyze the decarboxylation and hydroxylation of a broad range of free fatty acids (C10–C20) with overlapping substrate specificity, yet distinct chemoselectivity. CYP-Sm46Δ29 works primarily as a fatty (lauric) acid decarboxylase (66.1 ± 3.9% 1-undecene production) while CYP-Aa162 more as a fatty (lauric) acid hydroxylase (72.2 ± 0.9% hydroxy lauric acid production). Notably, the optical spectroscopic analysis of functional CYP-Sm46Δ29 revealed no characteristic P450 band, suggesting a unique heme coordination environment. Active-site mutagenesis analysis showed that substitution with the proposed key decarboxylation-modulating residues, His85 and Ile170, enhanced the decarboxylation activity of CYP-Aa162 and P450BSβ, emphasizing the importance of these residues in directing the decarboxylation pathway. Furthermore, the steady-state kinetic analysis of CYP-Aa162 and CYP-Sm46Δ29 revealed both cooperative and substrate inhibition behaviors which are substrate carbon chain length dependent.ConclusionsOur data identify CYP-Sm46Δ29 as an efficient OleTJE-like fatty acid decarboxylase. Oxidative decarboxylation chemoselectivity of the CYP152 decarboxylases is largely dependent upon the carbon chain length of fatty acid substrates and their precise positioning in the enzyme active site. Finally, the kinetic mode analysis of the enzymes could provide important guidance for future process design.
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影响因子：
14.8
作者：
通讯作者：
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影响因子：
64.8
作者：
Choi, Yong Jun;Lee, Sang Yup
通讯作者：
Lee, Sang Yup
影响因子：
4.8
作者：
Girvan, HM;Marshall, KR;Munro, AW
通讯作者：
Munro, AW
影响因子：
15
作者：
Grant, Job L.;Hsieh, Chun H.;Makris, Thomas M.
通讯作者：
Makris, Thomas M.
聚酮化合物脱羧链终止之前是素硫蛋白A生物合成中的O-磺化。
影响因子：
15
作者：
Gu, Liangcai;Wang, Bo;Kulkarni, Amol;Gehret, Jennifer J.;Lloyd, Kayla R.;Gerwick, Lena;Gerwick, William H.;Wipf, Peter;Hakansson, Kristina;Smith, Janet. L.;Sherman, David H.
通讯作者：
Sherman, David H.