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首页> 外文期刊>Biochemistry >A Catalytic Intermediate and Several Flavin Redox States Stabilized by Folate-Dependent tRNA Methyltransferase from Bacillus subtilis
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A Catalytic Intermediate and Several Flavin Redox States Stabilized by Folate-Dependent tRNA Methyltransferase from Bacillus subtilis

机译：枯草芽孢杆菌的叶酸依赖性tRNA甲基转移酶稳定的催化中间体和几种黄素氧化还原态。

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摘要

The flavoprotein TrmFO catalyzes the C5 methylation of uridine 54 in the T Psi C loop of tRNAs using 5,10-methylenetetrahydrofolate (CH(2)THF) as a methylene donor and FAD as a reducing agent. Here, we report biochemical and spectroscopic studies that unravel the remarkable capability of Bacillus subtilis TrmFO to stabilize, in the presence of oxygen, several flavin-reduced forms, including an FADH(center dot) radical, and a catalytic intermediate endowed with methylating activity. The FADH(center dot) radical was characterized by high-field electron paramagnetic resonance and electron nuclear double-resonance spectroscopies. Interestingly, the enzyme exhibited tRNA methylation activity in the absence of both an added carbon donor and an external reducing agent, indicating that a reaction intermediate, containing presumably CH(2)THF and FAD hydroquinone, is present in the freshly purified enzyme. Isolation by acid treatment, under anaerobic conditions, of noncovalently bound molecules, followed by mass spectrometry analysis, confirmed the presence in TrmFO of nonmodified FAD. Addition of formaldehyde to the purified enzyme protects the reduced flavins from decay by probably preventing degradation of CH(2)THF. The absence of air-stable reduced FAD species during anaerobic titration of oxidized TrmFO, performed in the absence or presence of added CH(2)THF, argues against their thermodynamic stabilization but rather implicates their kinetic trapping by the enzyme. Altogether, the unexpected isolation of a stable catalytic intermediate suggests that the flavin-binding pocket of TrmFO is a highly insulated environment, diverting the reduced FAD present in this intermediate from uncoupled reactions.
机译：黄素蛋白TrmFO使用5，10-亚甲基四氢叶酸（CH（2）THF）作为亚甲基供体，FAD作为还原剂，催化tRNA的T Psi C环中尿苷54的C5甲基化。在这里，我们报道了生化和光谱学研究，揭示了枯草芽孢杆菌TrmFO在氧气存在下稳定几种黄素还原形式（包括FADH（中心点）自由基）和具有甲基化活性的催化中间体的出色能力。 FADH（中心点）自由基的特征在于高场电子顺磁共振和电子核双共振光谱学。有趣的是，该酶在不存在添加的碳供体和外部还原剂的情况下均表现出tRNA甲基化活性，这表明新鲜纯化的酶中存在可能包含CH（2）THF和FAD对苯二酚的反应中间体。在厌氧条件下，通过酸处理分离非共价键合的分子，然后进行质谱分析，证实了在TrmFO中存在未修饰的FAD。将甲醛添加到纯化的酶可以通过防止CH（2）THF降解来保护还原的黄素免于降解。在不存在或存在添加的CH（2）THF的情况下进行的氧化TrmFO的厌氧滴定过程中，没有空气稳定的还原FAD种类反对它们的热力学稳定性，而是暗示了它们被酶动态捕获。总而言之，稳定的催化中间体的意外分离表明TrmFO的黄素结合口袋是高度绝缘的环境，使存在于该中间体中的还原FAD脱离了偶联反应。

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