CMP羟甲基化酶MilA中与甲基羟化过程相关的关键氨基酸定点突变分析

Analysis of key amino acid involved in hydroxylation of methyl group of CMP in CMP hydroxymethylase MilA by site-directed mutagenesis

摘要:

【目的】米多霉素生物合成起始反应的MilA蛋白可以使底物胞苷酸单磷酸(CMP)的胞嘧啶碱基C5位上形成羟甲基化，形成羟甲基化胞苷酸(HmCMP)，MilA是迄今发现的首个能够高效利用CMP的羟甲基化酶。MilA属于脱氧胸苷酸合成酶(TS)和脱氧胞苷酸羟甲基化酶(CH)蛋白超家族，通过三维结构预测发现它们的三维结构非常相似，CH94上的丝氨酸曾被证明与胞嘧啶上甲基变成羟甲基过程有关，MilA102上对应的氨基酸为苏氨酸，TS91上对应的氨基酸为脯氨酸。因此，突变MilA上第102位的苏氨酸，考察该氨基酸对CMP和脱氧胞苷酸(dCMP)羟甲基化反应的影响。【方法】通过定点突变技术，将MilA第102位的保守氨基酸苏氨酸(Threonine，T)分别点突变成缬氨酸(Valine，V)和亮氨酸(Leucine，L)。【结果】体外酶活实验结果显示，相对于MilA，MilA T102V对于CMP的催化活性下降87.1%，对于dCMP的催化活性没有影响；而MilA T102L均丧失了对于两种底物的活性。【结论】这表明Thr102对于MilA催化底物CMP形成HmCMP至关重要；同时，失去活性的仅比缬氨酸和野生型苏氨酸的链长多出了一个碳原子，暗示其在与底物结合时形成了一定的空间位阻效应。Thr在102位点的功能经过自然进化的微调有可能已经达到最优化。

Abstract:

[Objective] MilA, a cytidine 5′-monophosphate (CMP) hydroxymethylase which can convert CMP to 5-hydroxymethylcytidine-5′-monophosphate (HmCMP), was discovered and characterized in the mildiomycin biosynthetic pathway. MilA belongs to the superfamily of thymidylate synthase (TS) and cytidylate hydroxymethylase (CH), and was the only enzyme to date that was able to efficiently convert CMP other than dCMP to HmCMP. Three-dimensional structure prediction of MilA revealed high similarity to that of CH and TS. Ser94 of CH was proved to be a key residue mediating hydroxylation of the 5′-methyl cytosine of dCMP, corresponding residue is Thr102 in MilA and Pro92 in TS, respectively. In order to study the role of Thr102 in MilA, we constructed mutants MilA T102V and MilA T102L. [Methods] Based on above analysis, Thr102 of MilA was site-mutated into valine (Val, V) and leucine (Leu, L) to assay their catalytic efficiency. [Results] Compared to the wild type, activity of MilA T102V for CMP was significantly reduced by 87.1%, while that for dCMP was maintained at a similar level. By contrast, MilA T102L could not use CMP and dCMP any more. As for MilA T102L, the side chain of Leu102 was one carbon atom longer than both of Val102 and Thr102, implying that steric hindrance effect was involved in the substrate binding by MilA T102L. [Conclusion] The results demonstrated that Thr102 in MilA was essential for HmCMP formation. The function of MilAT102 may have reached optimization after naturally evolutionary fine-tuning.