Mechanistic characterization of enzymes involved in tetrahydrofolate biosynthesis in Mycobacterium tuberculosis

Abstract

Tetrahydrofolate (THF), the primary one carbon unit carrier in biology, is an essential cofactor that needs to be recycled so that reactions involved in dTMP synthesis and purine metabolism are maintained. Furthermore, inhibition of folate biosynthesis is a therapeutic strategy employed in the treatment of cancer, bacterial infections, and protozoal infections. In this work, we characterized two enzymes involved in THF biosynthesis in Mycobacterium tuberculosis, dihydrofolate reductase (MtDHFR) and dihydroneopterin aldolase (MtDHNA).;We hypothesize that these two enzymes differ from their bacterial (in the case of DHNA), and human (in the case of DHFR) counterparts in terms of kinetic properties and chemical mechanism.;To test this hypothesis, we performed a complete characterization of the reactions catalyzed by these enzymes with a combination of steady-state and pre-steady-state assays, kinetic and equilibrium isotope effects, and pH studies.;For the MtDHFR-catalyzed reaction, we were able to propose a chemical mechanism in which a pre-equilibrium protonation step preceded hydride transfer, so that a protonated intermediate occurs. Additionally, we demonstrated that the hydride transfer step is much slower in comparison to DHFRs from other organisms, and that hydride transfer contributes significantly to the turnover number.;Interestingly, MtDHFR possibly undergoes conformational changes prior to release of its two reaction products, which occurs via two parallel pathways. This is the first wild type DHFR to possess parallel pathways for product release. Importantly, depending on the concentration of its substrates, one pathway predominates over the other.;For the MtDHNA, our kinetic analysis revealed that the enzyme acts as an aldolase, epimerase, and oxygenase. This is unique for a wild type enzyme, and the oxygenase product binds tightly to the enzyme inducing protein oligomerization in a negative cooperative manner. We were able to show that MtDHNA partitions in the first turnover between the three distinct reactions, and that when the reaction reaches equilibrium, only the oxygenase product is present.;Furthermore, two partly rate limiting protonation steps possibly leading to the formation and dissolution of a common intermediate are occurring. In the overall turnover, there is possibly an additional solvent contribution, which can be attributed to conformational changes, further corroborated by the presence of a burst of product formation and smaller values D V compared to DV/K..;In summary, the results presented here revealed unique and important characteristics of these two enzymes involved in THF biosynthesis in M. tuberculosis. We hope that this will aid in the development of new antifolates, and in a better understanding of M. tuberculosis metabolism.