Characterization of RimO, a new member of the methylthiotransferase subclass of the radical SAM superfamily.
Characterization of RimO, a new member of the methylthiotransferase subclass of the radical SAM superfamily.
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DOI：
10.1021/bi900939w
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发表时间：
2009-10-27
期刊：
影响因子：
2.9
通讯作者：
Booker, Squire J.
中科院分区：
文献类型：
--
作者：
Lee, Kyung-Hoon;Saleh, Lana;Anton, Brian P.;Madinger, Catherine L.;Benner, Jack S.;Iwig, David F.;Roberts, Richard J.;Krebs, Carsten;Booker, Squire J.
由大肠杆菌中yliG基因编码的RimO最近已在体内被鉴定为负责将甲硫基连接到小核糖体蛋白S12的Asp 88的β-碳上的酶[Anton，B. P.，萨利赫湖，本纳，J.S.，罗利，E.一、Kasif，S.，和Roberts，R. J.（2008）Proc. Natl. Acad. Sci. USA，105，1826-1831].迄今为止，它是唯一已知的催化蛋白质底物甲硫基化的酶;在tRNA上观察到其他四种天然存在的甲硫基修饰。RimO所属的甲硫基转移酶（MTTase）家族的所有成员已被证明在其一级结构中含有典型的CxxxCxxC基序，这是自由基S-腺苷甲硫氨酸（SAM）蛋白家族的典型结构。MiaB是唯一的MTTase，实验证明该酶负责E. coli和海栖热袍菌（Thermotoga maritima）的[4Fe-4S]簇。在此，我们报告在体外的生化和光谱表征的RimO。我们通过分析和光谱方法表明，RimO在E.在棕色固氮菌（Azotobacter vinelandii）铁硫簇生物合成蛋白存在下，大肠杆菌的铁硫簇生物合成蛋白中含有一个[4Fe-4S]2+簇。这种形式的RimO（RimOrcn）与57 Fe和硫化钠的重组产生含有两个[4Fe-4S]2+簇的蛋白质，类似于MiaB。我们还通过质谱分析表明，RimOrcn催化甲硫基与肽底物类似物的连接，该肽底物类似物模拟了E.杆菌该反应的动力学分析表明，在底物类似物存在下，RimOrcn的活性不支持完全转换。我们讨论了在体外完全活跃的RimO组装核糖体的可能要求。我们的研究结果与催化硫插入的其他酶，如生物素合酶，硫辛酰合酶和MiaB的结果一致。
RimO, encoded by the yliG gene in Escherichia coli, has been recently identified in vivo as the enzyme responsible for the attachment of a methylthio group on the β-carbon of Asp88 of the small ribosomal protein S12 [Anton, B. P., Saleh, L., Benner, J. S., Raleigh, E. A., Kasif, S., and Roberts, R. J. (2008) Proc. Natl. Acad. Sci. USA, 105, 1826–1831]. To date, it is the only enzyme known to catalyze methylthiolation of a protein substrate; the four other naturally occurring methylthio modifications have been observed on tRNA. All members of the methylthiotransferase (MTTase) family, to which RimO belongs, have been shown to contain the canonical CxxxCxxC motif in their primary structures that is typical of the radical S-adenosylmethionine (SAM) family of proteins. MiaB, the only characterized MTTase, and the enzyme experimentally shown to be responsible for methylthiolation of N6-isopentenyladenosine of tRNA in E. coli and Thermotoga maritima, has been demonstrated to harbor two distinct [4Fe–4S] clusters. Herein, we report in vitro biochemical, and spectroscopic characterization of RimO. We show by analytical and spectroscopic methods that RimO, heterologously overproduced in E. coli in the presence of iron–sulfur cluster biosynthesis proteins from Azotobacter vinelandii, contains one [4Fe–4S]2+ cluster. Reconstitution of this form of RimO (RimOrcn) with 57Fe and sodium sulfide results in a protein that contains two [4Fe–4S]2+ clusters, similar to MiaB. We also show by mass spectrometry that RimOrcn catalyzes the attachment of a methylthio group to a peptide substrate analog that mimics the loop structure bearing aspartyl 88 of the S12 ribosomal protein from E. coli. Kinetic analysis of this reaction shows that the activity of RimOrcn in the presence of the substrate analog does not support a complete turnover. We discuss the possible requirement for an assembled ribosome for fully active RimO in vitro. Our findings are consistent with those of other enzymes that catalyze sulfur insertion, such as biotin synthase, lipoyl synthase, and MiaB.
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影响因子：
14.8
作者：
Chatterjee, Abhishek;Li, Yue;Zhang, Yang;Grove, Tyler L.;Lee, Michael;Krebs, Carsten;Booker, Squire J.;Begley, Tadhg P.;Ealick, Steven E.
通讯作者：
Ealick, Steven E.
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影响因子：
2.9
作者：
Iwig, DF;Booker, SJ
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Booker, SJ
影响因子：
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Esberg, B;Leung, HCE;Winkler, ME
通讯作者：
Winkler, ME
影响因子：
14.9
作者：
AGRIS, PF;ARMSTRONG, DJ;SOLL, D
通讯作者：
SOLL, D