Monooxygenation by a thermophilic cytochrome P450 via direct electron donation from NADH
Monooxygenation by a thermophilic cytochrome P450 via direct electron donation from NADH
复制标题
嗜热细胞色素 P450 通过 NADH 的直接电子捐赠进行单氧合
DOI：
10.1039/c0mt00079e
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发表时间：
2011
期刊：
影响因子：
3.4
通讯作者：
H. Matsumura
中科院分区：
文献类型：
--
作者：
Ichiyanagi;M.;Koyama;Y.;Sato;Y. & Hishida;K.;Rai Virendra Kumar;H. Matsumura
细胞色素 P450 的催化需要两个电子供体来激活氧分子。在这里，我们报告了来自嗜热嗜酸菌的细胞色素 P450 CYP119A2 (P450st) 的酶催化作用，Sulfolobus tokodaiistrain 7，以 NAD(P)H 作为电子供体，没有氧化还原伴侣，并以高分辨率对 P450st 进行晶体学分析。 P450st 可以以 NADH 或 NADPH 作为电子供体催化苯乙烯环氧化。 P450st 与 NADH 的反应表现出连续机制。分辨率为 1.94 Å 的 X 射线晶体学揭示了用于 NAD(P)H 结合的足够大的血红素袋，以及远端血红素袋中从活性位点到本体溶剂的新型连续通道。即使当大体积化合物（例如 NAD(P)H）结合在血红素袋中时，狭窄的通道也可以将质子或水转移到血红素袋中。此外，位于底物通道周围的 F/G 环区域 (Leu151-Glu156) 在突变体中被删除，并被构建以提高 NAD(P)H 与血红素口袋的可及性。将 Δ151-156 突变体的动力学特性与野生型 P450st 的动力学特性进行比较。突变体的Km值比野生型低约2倍。结果表明NAD(P)H可以为血红素口袋内的P450st提供电子。
The catalysis of cytochrome P450s requires two-electron donation for the activation of an oxygen molecule. Here, we report the enzymatic catalysis of cytochrome P450, CYP119A2 (P450st), from a thermoacidophilic crenarchaeon,Sulfolobus tokodaiistrain 7, with NAD(P)H as an electron donor and no redox partners and the crystallographic analysis of P450st at high resolution. P450st can catalyse styrene epoxidation with either NADH or NADPH as an electron donor. The P450st reaction with NADH exhibited a sequential mechanism. X-ray crystallography at a resolution of 1.94 Å revealed a sufficiently large heme pocket for NAD(P)H binding and a novel contiguous channel from the active site to bulk solvent in the distal heme pocket. The narrow channel may transfer protons or water to the heme pocket even when a bulky compound, such as NAD(P)H, binds in the pocket. In addition, the F/G loop region (Leu151-Glu156), located around the substrate channel, was deleted in the mutant and constructed to improve the accessibility of NAD(P)H to the heme pocket. Kinetic properties of the Δ151-156 mutant were compared with those of the wild-type P450st. TheKmvalue of the mutant was about 2 times lower than that of the wild-type. The results indicated that NAD(P)H could provide the electrons for P450st within the heme pocket.