Spectroscopic, Steady-State Kinetic, and Mechanistic Characterization of the Radical SAM Enzyme QueE, Which Catalyzes a Complex Cyclization Reaction in the Biosynthesis of 7-Deazapurines
Spectroscopic, Steady-State Kinetic, and Mechanistic Characterization of the Radical SAM Enzyme QueE, Which Catalyzes a Complex Cyclization Reaction in the Biosynthesis of 7-Deazapurines
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DOI：
10.1021/bi301156w
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发表时间：
2013-01-08
期刊：
影响因子：
2.9
通讯作者：
Bandarian, Vahe
中科院分区：
文献类型：
--
作者：
McCarty, Reid M.;Krebs, Carsten;Bandarian, Vahe
7-羧基-7-脱氮鸟嘌呤（CDG）合酶（QueE）催化复杂杂环自由基介导的6-羧基-5，6，7，8-四氢蝶呤（CPH4）在生物合成途径的第三步中转化为所有7-脱氮嘌呤。在这里，我们提出了一个详细的表征QueE从枯草芽孢杆菌描绘的CPH4转化为CDG的机制。QueE是自由基S-腺苷-L-甲硫氨酸（SAM）超家族的成员，所有这些超家族都使用结合的[4Fe-4S]（+）簇来催化SAM辅因子的还原裂解以产生甲硫氨酸和5 '-脱氧腺苷自由基（5'-dAdo（中心点）），其启动需要氢原子提取的酶促转化。的紫外-可见光，电子顺磁共振，穆斯堡尔光谱特征的同源二聚体QueE点的存在下，每个单体的一个单一的[4Fe-4S]集群。稳态动力学实验表明，CPH4的Km为20 +/- 7 μ M，总转化的k（cat）为5.4 +/- 1.2 min（-1）。SAM的动力学测定K-app为45 +/- 1 μ M。QueE也是镁依赖性的，并且对二价金属离子表现出0.21 +/-0.03mMXAM辅因子支持多次转换，表明它在每个催化循环结束时再生。QueE的重排机制用在C-6或在C-7的两个前手性位置含有氘的CPH4同位素体来探测。这些研究暗示5 '-dAdo（中心点）是通过从CPH4的C-6提取H原子而由QueE催化的环收缩反应的引发剂。
7-Carboxy-7-deazaguanine (CDG) synthase (QueE) catalyzes the complex heterocyclic radical-mediated conversion of 6-carboxy-5,6,7,8-tetrahydropterin (CPH4) to CDG in the third step of the biosynthetic pathway to all 7-deazapurines. Here we present a detailed characterization of QueE from Bacillus subtilis to delineate the mechanism of conversion of CPH4 to CDG. QueE is a member of the radical S-adenosyl-L-methionine (SAM) superfamily, all of which use a bound [4Fe-4S](+) cluster to catalyze the reductive cleavage of the SAM cofactor to generate methionine and a 5'-deoxyadenosyl radical (5'-dAdo(center dot)), which initiates enzymatic transformations requiring hydrogen atom abstraction. The ultraviolet-visible, electron paramagnetic resonance, and Mossbauer spectroscopic features of the homodimeric QueE point to the presence of a single [4Fe-4S] cluster per monomer. Steady-state kinetic experiments indicate a K-m of 20 +/- 7 mu M for CPH4 and a k(cat) of 5.4 +/- 1.2 min(-1) for the overall transformation. The kinetically determined K-app for SAM is 45 +/- 1 mu M. QueE is also magnesium-dependent and exhibits a K-app for the divalent metal ion of 0.21 +/- 0.03 mM. The SAM cofactor supports multiple turnovers, indicating that it is regenerated at the end of each catalytic cycle. The mechanism of rearrangement of QueE was probed with CPH4 isotopologs containing deuterium at C-6 or the two prochiral positions at C-7. These studies implicate 5'-dAdo(center dot) as the initiator of the ring contraction reaction catalyzed by QueE by abstraction of the H atom from C-6 of CPH4.