Loveridge E Joel, Hroch Lukas, Hughes Robert L, Williams Thomas, Davies Rhidian L, Angelastro Antonio, Luk Louis Y P, Maglia Giovanni, Allemann Rudolf K
School of Chemistry, Cardiff University , Main Building, Park Place, Cardiff CF10 3AT, U.K.
Department of Chemistry, Swansea University , Singleton Park, Swansea SA2 8PP, U.K.
Biochemistry. 2017 Apr 4;56(13):1879-1886. doi: 10.1021/acs.biochem.6b01268. Epub 2017 Mar 24.
Mammalian dihydrofolate reductases (DHFRs) catalyze the reduction of folate more efficiently than the equivalent bacterial enzymes do, despite typically having similar efficiencies for the reduction of their natural substrate, dihydrofolate. In contrast, we show here that DHFR from the hyperthermophilic bacterium Thermotoga maritima can catalyze reduction of folate to tetrahydrofolate with an efficiency similar to that of reduction of dihydrofolate under saturating conditions. Nuclear magnetic resonance and mass spectrometry experiments showed no evidence of the production of free dihydrofolate during either the EcDHFR- or TmDHFR-catalyzed reductions of folate, suggesting that both enzymes perform the two reduction steps without release of the partially reduced substrate. Our results imply that the reaction proceeds more efficiently in TmDHFR than in EcDHFR because the more open active site of TmDHFR facilitates protonation of folate. Because T. maritima lives under extreme conditions where tetrahydrofolate is particularly prone to oxidation, this ability to salvage folate may impart an advantage to the bacterium by minimizing the squandering of a valuable cofactor.
哺乳动物二氢叶酸还原酶（DHFRs）催化叶酸还原的效率比相应的细菌酶更高，尽管它们对天然底物二氢叶酸的还原效率通常相似。相比之下，我们在此表明，嗜热栖热菌（Thermotoga maritima）的DHFR在饱和条件下催化叶酸还原为四氢叶酸的效率与催化二氢叶酸还原的效率相似。核磁共振和质谱实验表明，在EcDHFR或TmDHFR催化叶酸还原过程中，没有证据表明会产生游离二氢叶酸，这表明这两种酶在不释放部分还原底物的情况下完成两个还原步骤。我们的结果表明，该反应在TmDHFR中比在EcDHFR中进行得更有效，因为TmDHFR更开放的活性位点促进了叶酸的质子化。由于嗜热栖热菌生活在极端条件下，在这种条件下四氢叶酸特别容易氧化，这种挽救叶酸的能力可能通过尽量减少宝贵辅因子的浪费而赋予该细菌一种优势。
