Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India.
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India
J Bacteriol. 2020 May 27;202(12). doi: 10.1128/JB.00709-19.
5,10-Methylenetetrahydrofolate reductase (MetF/MTHFR) is an essential enzyme in one-carbon metabolism for biosynthesis of methionine. Our and analyses of MSMEG_6664/MSMEI_6484, annotated as putative MTHFR in , failed to reveal their function as MTHFRs. However, we identified two hypothetical proteins, MSMEG_6596 and MSMEG_6649, as noncanonical MTHFRs in the bacterium. MTHFRs are known to be oligomeric flavoproteins. Both MSMEG_6596 and MSMEG_6649 are monomeric proteins and lack flavin coenzymes. , the catalytic efficiency (/ ) of MSMEG_6596 (MTHFR1) for 5,10-CH-THF and NADH was ∼13.5- and 15.3-fold higher than that of MSMEG_6649 (MTHFR2). Thus, MSMEG_6596 is the major MTHFR. This interpretation was further supported by better rescue of the Δ strain by MTHFR1 than by MTHFR2. As identified by liquid chromatography-tandem mass spectrometry, the product of MTHFR1- or MTHFR2-catalyzed reactions was 5-CH-THF. The Δ strain was partially auxotrophic for methionine and grew only poorly without methionine or without being complemented with a functional copy of MTHFR1 or MTHFR2. Furthermore, the Δ strain was more sensitive to folate pathway inhibitors (sulfachloropyridazine, -aminosalicylic acid, sulfamethoxazole, and trimethoprim). The studies reveal that MTHFR1 and MTHFR2 are two noncanonical MTHFR proteins that are monomeric and lack flavin coenzyme. Both MTHFR1 and MTHFR2 are involved in methionine biosynthesis and required for antifolate resistance in mycobacteria. MTHFR/MetF is an essential enzyme in a one-carbon metabolic pathway for biosynthesis of methionine. MTHFRs are known to be oligomeric flavoproteins. Our and analyses of MSMEG_6664/MSMEI_6484, annotated as putative MTHFR, failed to reveal their function as MTHFRs. However, we identified two of the hypothetical proteins, MSMEG_6596 and MSMEG_6649, as MTHFR1 and MTHFR2, respectively. Interestingly, both MTHFRs are monomeric and lack flavin coenzymes. deleted for the major () was partially auxotroph for methionine and more sensitive to folate pathway inhibitors (sulfachloropyridazine, -aminosalicylic acid, sulfamethoxazole, and trimethoprim). The studies reveal that MTHFR1 and MTHFR2 are novel MTHFRs involved in methionine biosynthesis and required for antifolate resistance in mycobacteria.
5,10-亚甲基四氢叶酸还原酶（MetF/MTHFR）是一碳代谢中用于合成蛋氨酸的必需酶。我们对 MSMEG_6664/MSMEI_6484 的 分析和 分析，该基因被注释为 的推定 MTHFR，未能揭示其作为 MTHFR 的功能。然而，我们在该细菌中鉴定出两个假定蛋白 MSMEG_6596 和 MSMEG_6649 作为非典型 MTHFR。众所周知，MTHFR 是寡聚黄素蛋白。MSMEG_6596 和 MSMEG_6649 均为单体蛋白，缺乏黄素辅酶。有趣的是，MSMEG_6596 的 5,10-CH-THF 和 NADH 的催化效率（/）比 MSMEG_6649（MTHFR2）高约 13.5 倍和 15.3 倍。因此，MSMEG_6596 是主要的 MTHFR。这一解释进一步得到了通过 MTHFR1 而不是 MTHFR2 更好地拯救 缺失菌株的支持。通过液相色谱-串联质谱鉴定，MTHFR1 或 MTHFR2 催化反应的产物是 5-CH-THF。缺失菌株对蛋氨酸部分呈营养缺陷型，缺乏蛋氨酸或不补充功能正常的 MTHFR1 或 MTHFR2 时生长不良。此外，缺失菌株对叶酸途径抑制剂（磺胺吡啶、-氨基水杨酸、磺胺甲噁唑和甲氧苄啶）更敏感。研究表明，MTHFR1 和 MTHFR2 是两种非典型的 MTHFR 蛋白，它们是单体且缺乏黄素辅酶。MTHFR1 和 MTHFR2 都参与了蛋氨酸生物合成，并且是分枝杆菌中抗叶酸的必需蛋白。MTHFR/MetF 是一碳代谢途径中用于合成蛋氨酸的必需酶。众所周知，MTHFR 是寡聚黄素蛋白。我们对 MSMEG_6664/MSMEI_6484 的 分析和 分析，该基因被注释为假定的 MTHFR，未能揭示其作为 MTHFR 的功能。然而，我们鉴定出两个假定蛋白 MSMEG_6596 和 MSMEG_6649 分别为 MTHFR1 和 MTHFR2。有趣的是，这两种 MTHFR 都是单体且缺乏黄素辅酶。主要的 缺失菌株（）对蛋氨酸部分呈营养缺陷型，对叶酸途径抑制剂（磺胺吡啶、-氨基水杨酸、磺胺甲噁唑和甲氧苄啶）更敏感。研究表明，MTHFR1 和 MTHFR2 是参与蛋氨酸生物合成和分枝杆菌中抗叶酸所必需的新型 MTHFR。
