Stereochemical Course of the Reaction Catalyzed by RimO, a Radical SAM Methylthiotransferase
Stereochemical Course of the Reaction Catalyzed by RimO, a Radical SAM Methylthiotransferase
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DOI：
10.1021/jacs.5b11035
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发表时间：
2016-03-09
影响因子：
15
通讯作者：
Booker, Squire J.
中科院分区：
文献类型：
--
作者：
Landgraf, Bradley J.;Booker, Squire J.
RimO是生长自由基S-腺苷甲硫氨酸（SAM）酶超家族的成员，其使用还原的[4Fe-4S]簇来实现SAM的5' C-S键的还原裂解以形成5'-脱氧腺苷5 '-自由基（5'-dA（中心点））中间体。RimO使用这种有效的氧化剂来催化甲硫基（-SCH 3）连接到蛋白S12的天冬氨酸89的C3上，S12是组成细菌核糖体30 S亚基的21种蛋白之一。然而，这种转变发生的确切机制仍然难以捉摸。在此，我们描述了RimO反应的立体化学过程。使用在目标乙酰基残基的3个pro-R或3个pro-S位置上带有氘的S12蛋白的肽模拟物，我们表明来自多形拟杆菌（Bt）的RimO催化提取pro-S氢原子，如通过将氘转移到5 ′-脱氧腺苷（5 ′-dAH）中所证明的。所观察到的动力学同位素对H原子与D原子提取的影响类似于1.9，表明该步骤至少部分是速率决定的。我们还证明了Bt RimO可以利用大肠杆菌的黄素氧还蛋白/黄素氧还蛋白氧化还原酶/NADPH还原系统作为必需电子的来源。使用这种体内还原系统减少了，但没有消除，过量的甲基硫醇化产物中5 '-dAH的形成。
RimO is a member of the growing radical S-adenosylmethionine (SAM) superfamily of enzymes, which use a reduced [4Fe-4S] cluster to effect reductive cleavage of the 5' C-S bond of SAM to form a 5'-deoxyadenosyl 5'-radical (5'-dA(center dot)) intermediate. RimO uses this potent oxidant to catalyze the attachment of a methylthio group (-SCH3) to C3 of aspartate 89 of protein S12, one of 21 proteins that compose the 30S subunit of the bacterial ribosome. However, the exact mechanism by which this transformation takes place has remained elusive. Herein, we describe the stereochemical course of the RimO reaction. Using peptide mimics of the S12 protein bearing deuterium at the 3 pro-R or 3 pro-S positions of the target aspartyl residue, we show that RimO from Bacteroides thetaiotaomicron (Bt) catalyzes abstraction of the pro-S hydrogen atom, as evidenced by the transfer of deuterium into 5'-deoxyadenosine (5'-dAH). The observed kinetic isotope effect on H atom versus D atom abstraction is similar to 1.9, suggesting that this step is at least partially rate determining. We also demonstrate that Bt RimO can utilize the flavodoxin/flavodoxin oxidoreductase/NADPH reducing system from Escherichia coli as a source of requisite electrons. Use of this in vivo reducing system decreases, but does not eliminate, formation of 5'-dAH in excess of methylthiolated product.