Active site mutations of cytochrome p450cam alter the binding, coupling, and oxidation of the foreign substrates (R)- and (s)-2-ethylhexanol.
Active site mutations of cytochrome p450cam alter the binding, coupling, and oxidation of the foreign substrates (R)- and (s)-2-ethylhexanol.
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DOI：
10.1006/abbi.2001.2732
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发表时间：
2002-02
影响因子：
3.9
通讯作者：
K. French;D. Rock;D. Rock;J. Manchester;B. Goldstein;Jeffrey P. Jones
中科院分区：
文献类型：
--
作者：
K. French;D. Rock;D. Rock;J. Manchester;B. Goldstein;Jeffrey P. Jones
三个因素对于设计有效的P450生物催化剂是最重要的。(1)基质必须以显著的速率被氧化。(2)区域选择性必须非常有利于所需产物。(3)该酶必须使用来自NADH或NADPH的大部分还原当量来产生产物。我们选择研究的反应是2-乙基己醇通过P450 cam氧化为2-乙基己酸。我们检查了四个活性位点突变：F87 W，Y 96 W，T185 F和L244 A。选择突变以通过减少活性位点体积、增加活性位点疏水性和改善立体选择性来改善2-乙基己酸生产。F87 W和Y 96 W突变改善了区域选择性，几乎只产生所需产物。T185 F突变改善了NADH与产物形成的偶联。L244 A突变改变了2-乙基己酸生产的立体选择性。这些结果表明，P450 cam的活性位点突变可以改变2-乙基己醇的催化。
Three factors are of primary importance with respect to designing efficient P450 biocatalysts. (1) The substrate must be oxidized at a significant rate. (2) The regioselectivity must heavily favor the desired product. (3) The enzyme must use the majority of the reducing equivalents from NADH or NADPH to produce product. The reaction we chose to study was oxidation of 2-ethylhexanol to 2-ethylhexanoic acid by P450cam. We examined four active site mutations: F87W, Y96W, T185F, and L244A. The mutations were chosen to improve 2-ethyhexanoic acid production by decreasing active site volume, increasing active site hydrophobicity, and improving stereoselectivity. The F87W and Y96W mutations improved regioselectivity, giving almost exclusively the desired product. The T185F mutation improved coupling of NADH to product formation. The L244A mutation altered the stereoselectivity of 2-ethylhexanoic acid production. These results indicate that active site mutations of P450cam can alter catalysis of 2-ethylhexanol.