Department of Science and Environment, Roskilde University, Universitetsvej 1, 4000 Roskilde, Denmark.
Int J Mol Sci. 2023 Mar 1;24(5):4728. doi: 10.3390/ijms24054728.
Enzymes carrying Iron-Sulfur (Fe-S) clusters perform many important cellular functions and their biogenesis require complex protein machinery. In mitochondria, the IBA57 protein is essential and promotes assembly of [4Fe-4S] clusters and their insertion into acceptor proteins. YgfZ is the bacterial homologue of IBA57 but its precise role in Fe-S cluster metabolism is uncharacterized. YgfZ is needed for activity of the radical S-adenosyl methionine [4Fe-4S] cluster enzyme MiaB which thiomethylates some tRNAs. The growth of cells lacking YgfZ is compromised especially at low temperature. The RimO enzyme is homologous to MiaB and thiomethylates a conserved aspartic acid in ribosomal protein S12. To quantitate thiomethylation by RimO, we developed a bottom-up LC-MS analysis of total cell extracts. We show here that the in vivo activity of RimO is very low in the absence of YgfZ and independent of growth temperature. We discuss these results in relation to the hypotheses relating to the role of the auxiliary 4Fe-4S cluster in the Radical SAM enzymes that make Carbon-Sulfur bonds.
携带有铁硫簇（Fe-S）的酶执行许多重要的细胞功能，其生物发生需要复杂的蛋白质机器。在线粒体中，IBA57 蛋白是必不可少的，它促进 [4Fe-4S]簇的组装及其插入到受体蛋白中。YgfZ 是 IBA57 的细菌同源物，但它在 Fe-S 簇代谢中的精确作用尚未确定。YgfZ 是活性必需的，该酶为自由基 S-腺苷甲硫氨酸 [4Fe-4S]簇酶 MiaB，它将一些 tRNA 硫甲基化。缺乏 YgfZ 的细胞的生长受到影响，尤其是在低温下。RimO 酶与 MiaB 同源，将核糖体蛋白 S12 中的保守天冬氨酸硫甲基化。为了定量测定 RimO 的硫甲基化，我们开发了一种基于底部向上的 LC-MS 分析总细胞提取物的方法。我们在这里表明，在没有 YgfZ 的情况下，RimO 的体内活性非常低，并且与生长温度无关。我们讨论了这些结果与关于辅助 4Fe-4S 簇在形成碳硫键的 Radical SAM 酶中的作用的假设的关系。
