FAD-independent methylene-tetrahydrofolate (methylene-H₄F) reductase (Mfr), recently identified in mycobacteria, catalyzes the reduction of methylene-H₄F to methyl-H₄F with NADH as hydride donor by a ternary complex mechanism. This biochemical reaction corresponds to that of the ubiquitous FAD-dependent methylene-H₄F reductase (MTHFR), although the latter uses a ping-pong mechanism with FAD as prosthetic group. Comparative genomics and genetic analyses indicated that Mfr is indispensable for the growth of Mycobacterium tuberculosis, which lacks the MTHFR-encoding gene. Thus, Mfr is an excellent target enzyme for the design of antimycobacterial drugs. Here, we report the heterologous production, enzymological characterization and the crystal structure of Mfr from the thermophilic mycobacterium M. hassiacum (hMfr), which shows 78% sequence identity to Mfr from M. tuberculosis. Although hMfr and MTHFR show very low sequence identity and different catalytic mechanisms, their tertiary structures are highly similar, which suggests a divergent evolution of Mfr and MTHFR from a common ancestor. Most of the important active-site residues of MTHFR are conserved and equivalently positioned in the tertiary structure of hMfr. The Glu9Gln variant of hMfr exhibits a drastic reduction of the catalytic activity, which supports the predicted function of the glutamate residue as proton donor in both Mfr and MTHFR. The predicted nicotinamide binding site of hMfr is substantially narrower than the isoalloxazine binding site of MTHFR, which may reflect an evolutional adaptation to the different sizes of the coenzymes.

中文翻译：

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哈西分枝杆菌不依赖 FAD 的亚甲基四氢叶酸还原酶的晶体结构

最近在分枝杆菌中发现的不依赖于 FAD 的亚甲基-四氢叶酸 (methylene-H ₄ F) 还原酶 (Mfr)通过三元复合机制以 NADH 作为氢化物供体催化亚甲基-H ₄ F还原为甲基-H _{4 F。}这种生化反应对应于普遍存在的 FAD 依赖性亚甲基-H ₄ F 还原酶 (MTHFR)，尽管后者使用以 FAD 作为辅基的乒乓机制。比较基因组学和遗传分析表明，Mfr 对于结核分枝杆菌的生长是必不可少的, 缺少 MTHFR 编码基因。因此，Mfr 是设计抗分枝杆菌药物的极佳目标酶。在这里，我们报告了来自嗜热分枝杆菌M. hassiacum (hMfr)的 Mfr 的异源生产、酶学表征和晶体结构，它与结核分枝杆菌的 Mfr 具有 78% 的序列同一性. 尽管 hMfr 和 MTHFR 显示出非常低的序列同一性和不同的催化机制，但它们的三级结构高度相似，这表明 Mfr 和 MTHFR 从一个共同的祖先进化而来。MTHFR 的大部分重要活性位点残基都是保守的，并且等同地位于 hMfr 的三级结构中。hMfr 的 Glu9Gln 变体表现出催化活性的急剧降低，这支持谷氨酸残基在 Mfr 和 MTHFR 中作为质子供体的预测功能。预测的 hMfr 烟酰胺结合位点比 MTHFR 的异恶嗪结合位点窄得多，这可能反映了对不同大小辅酶的进化适应。