Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824, USA.
Department of Chemistry, Michigan State University, East Lansing, MI 48824, USA.
Metallomics. 2022 Mar 25;14(3). doi: 10.1093/mtomcs/mfac014.
LarC catalyzes the CTP-dependent insertion of nickel ion into pyridinium-3,5-bisthiocarboxylic acid mononucleotide (P2TMN), the final biosynthetic step for generating the nickel-pincer nucleotide (NPN) enzyme cofactor. In this study, we characterized a LarC homolog from Moorella thermoacetica (LarCMt) and characterized selected properties of the protein. We ruled out the hypothesis that enzyme inhibition by its product pyrophosphate accounts for its apparent single-turnover activity. Most notably, we identified a cytidinylylated-substrate intermediate that is formed during the reaction of LarCMt. Selected LarCMt variants with substitutions at the predicted CTP-binding site retained substantial amounts of activity, but exhibited greatly reduced levels of the CMP-P2TMN intermediate. In contrast, enhanced amounts of the CMP-P2TMN intermediate were generated when using LarCMt from cells grown on medium without supplemental nickel. On the basis of these results, we propose a functional role for CTP in the unprecedented nickel-insertase reaction during NPN biosynthesis.
LarC 催化 CTP 依赖性的镍离子插入到吡啶-3,5-二硫代羧酸单核苷酸（P2TMN）中，这是生成镍钳核苷酸（NPN）酶辅因子的最后一个生物合成步骤。在这项研究中，我们鉴定了来自 Moorella thermoacetica 的 LarC 同源物（LarCMt），并对该蛋白的部分性质进行了表征。我们排除了其产物焦磷酸酯抑制酶的假说，因为该假说无法解释其明显的单轮活性。最值得注意的是，我们在 LarCMt 的反应中鉴定出了一个胞苷酰化底物中间产物。在预测的 CTP 结合位点有取代的 LarCMt 变体保留了大量的活性，但 CMP-P2TMN 中间产物的水平大大降低。相比之下，当使用在没有补充镍的培养基中生长的细胞中的 LarCMt 时，会生成更多的 CMP-P2TMN 中间产物。基于这些结果，我们提出 CTP 在 NPN 生物合成过程中前所未有的镍插入酶反应中具有功能作用。
