Reduction of Folate by Dihydrofolate Reductase from Thermotoga maritima

E Joel Loveridge,Rudolf K Allemann,Robert L Hughes,Louis Y P Luk,Giovanni Maglia,Rhidian L Davies,Thomas Williams,Antonio Angelastro,Lukas Hroch

doi:10.1021/acs.biochem.6b01268

Abstract

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.

Highlights

Dihydrofolate reductase (DHFR) is an essential enzyme in many organisms; it catalyzes the NADPH-dependent reduction of 7,8-dihydrofolate (H2F) to 5,6,7,8-tetrahydrofolate (H4F)
Michaelis− Menten kinetics for folate and dihydrofolate were measured with EcDHFR and TmDHFR at 20 °C and pH 7 (Figure S1)
KH (s−1) 159.8 ± 7.918 not determined 0.122 ± 0.00344 0.053 ± 0.001 kinetic isotope effect (KIE) on TmDHFR-catalyzed transfer of hydride to folate, obtained by comparing the kcat observed with NADPH with that observed with 4R-(2H)-NADPH (NADPD), was 3.56 ± 0.52

Summary

Introduction

Dihydrofolate reductase (DHFR) is an essential enzyme in many organisms; it catalyzes the NADPH-dependent reduction of 7,8-dihydrofolate (H2F) to 5,6,7,8-tetrahydrofolate (H4F). In X-ray single-crystal structures of DHFR, folate and dihydrofolate bind almost identically to the enzyme.[4] both molecules present the re face of the pterin at C6 as the most likely candidate for attack by the C4 pro-R hydride of NADPH (Figure 1B). Nuclear magnetic resonance (NMR) studies with DHFR from Lactobacillus casei (LcDHFR) showed that when folate is fully reduced, both transferred hydrides are present on the same face of the product tetrahydrofolate.[32,33] Because initial reduction at C7 would require both an unfavorable hydride transfer geometry and protonation at the solvent-inaccessible N8, it has been suggested that the initial product of folate reduction is 5,6-. We show that in contrast to other bacterial DHFRs, TmDHFR catalyzes reduction of folate with an efficiency similar to that of reduction of dihydrofolate

Methods

Results

Conclusion