Substrate-Assisted Hydroxylation and O-Demethylation in the Peroxidase-like Cytochrome P450 Enzyme CYP121.,ACS Catalysis

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Substrate-Assisted Hydroxylation and O-Demethylation in the Peroxidase-like Cytochrome P450 Enzyme CYP121.
ACS Catalysis ( IF 11.3 ) Pub Date : 2019-12-20 , DOI: 10.1021/acscatal.9b04596
Romie C Nguyen ₁ , Yu Yang ₁ , Yifan Wang ₁ , Ian Davis ₁ , Aimin Liu ₁

Affiliation

CYP121 is a P450 enzyme from Mycobacterium tuberculosis that catalyzes a C-C coupling reaction between the two aromatic rings on its native substrate cyclo(l-Tyr-l-Tyr) (cYY) to form mycocyclosin, a necessary product for cell survival. Unlike the typical P450 enzymes for hydroxylation, CYP121 is believed to behave like a peroxidase and conduct radical-mediated C-C bond formation. Here, we probe whether the phenolic hydrogen of the substrate is the site of the postulated hydrogen atom abstraction for radical formation. We synthesized a singly O-methylated substrate analogue, cYF-4-OMe, and characterized its interaction with CYP121 by ultraviolet-visible and electron paramagnetic resonance spectroscopies and X-ray crystallography. We found that cYF-4-OMe can function as a substrate of CYP121 using the established assay via the peroxide shunt. Analysis of the enzymatic reaction revealed an O-demethylation of cYF-4-OMe instead of cyclization, yielding cYY and formaldehyde. A hydroxylated substrate, cYF-4-OMeOH, is expected to be the intermediate product, which was trapped and structurally characterized by X-ray crystallography. We further determined that the deformylation reaction of cYF-4-OMeOH proceeds via an alkyl-oxygen rather than aryl-oxygen bond cleavage by the 18O-labeling studies. Finally, the pH dependence catalytic study on the native substrate and the methoxy analogue further supports the mechanistic understanding that the hydrogen atom abstraction is the critical first oxidation step exerted by a heme-based oxidant during the cyclization reaction of cYY. The switch in catalytic activity reveals the power of CYP121 as a P450 enzyme and provides insight into the peroxidase-like catalytic mechanism.

中文翻译：

过氧化物酶样细胞色素P450酶CYP121中的底物辅助羟基化和O-去甲基化。

CYP121是一种来自结核分枝杆菌的P450酶，可催化其天然底物环（1-Tyr-1-Tyr）（cYY）上两个芳香环之间的CC偶联反应，从而形成细胞存活所必需的产品Mycocyclosin。与典型的P450羟化酶不同，CYP121的行为像过氧化物酶一样，并能进行自由基介导的CC键形成。在这里，我们探讨了底物的酚氢是否是假定的氢原子抽象为自由基形成的位点。我们合成了一个单独的O-甲基化底物类似物cYF-4-OMe，并通过紫外可见和电子顺磁共振光谱法和X射线晶体学表征了其与CYP121的相互作用。我们发现使用过氧化物分流器建立的测定法可将cYF-4-OMe用作CYP121的底物。酶促反应的分析显示cYF-4-OMe的O-去甲基化而不是环化，产生cYY和甲醛。预期羟基化的底物cYF-4-OMeOH是中间产物，其被捕获并通过X射线晶体学对其结构进行了表征。我们进一步确定，通过18O标记研究，cYF-4-OMeOH的去甲酰基化反应是通过烷基-氧而不是芳基-氧键裂解进行的。最后，对天然底物和甲氧基类似物的pH依赖性催化研究进一步支持了机械学上的理解，即氢原子的提取是cYY环化反应过程中血红素基氧化剂施加的关键的第一步氧化步骤。

更新日期：2020-01-10

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