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.

School of Chemical Sciences University of Birmingham Edgbaston Birmingham B15 2TT U K

School of Chemistry Cardiff University Main Building Park Place Cardiff CF10 3AT U 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

School of Chemistry Cardiff University Main Building Park Place Cardiff CF10 3AT U K Department of Pharmaceutical Chemistry and Drug Control Faculty of Pharmacy in Hradec Kralove Charles University Prague Akademika Heyrovskeho 1203 500 05 Hradec Kralove Czech Republic

School of Chemistry Cardiff University Main Building Park Place Cardiff CF10 3AT U K School of Chemical Sciences University of Birmingham Edgbaston Birmingham B15 2TT U K

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