Crystal structure of FAD-independent methylene-tetrahydrofolate reductase from Mycobacterium hassiacum

Abstract

FAD-independent methylene-tetrahydrofolate (methylene-H4F) reductase (Mfr), recently identified in mycobacteria, catalyzes the reduction of methylene-H4F to methyl-H4F with NADH as hydride donor by a ternary complex mechanism. This biochemical reaction corresponds to that of the ubiquitous FAD-dependent methylene-H4F reductase (MTHFR), although the latter uses a ping-pong mechanism with FAD as prosthetic group. Comparative genomics and genetic analyses indicated that Mfr is indispensable for the growth of Mycobacterium tuberculosis, which lacks the MTHFR-encoding gene. Thus, Mfr is an excellent target enzyme for the design of antimycobacterial drugs. Here, we report the heterologous production, enzymological characterization and the crystal structure of Mfr from the thermophilic mycobacterium M. hassiacum (hMfr), which shows 78% sequence identity to Mfr from M. tuberculosis. Although hMfr and MTHFR show very low sequence identity and different catalytic mechanisms, their tertiary structures are highly similar, which suggests a divergent evolution of Mfr and MTHFR from a common ancestor. Most of the important active-site residues of MTHFR are conserved and equivalently positioned in the tertiary structure of hMfr. The Glu9Gln variant of hMfr exhibits a drastic reduction of the catalytic activity, which supports the predicted function of the glutamate residue as proton donor in both Mfr and MTHFR. The predicted nicotinamide binding site of hMfr is substantially narrower than the isoalloxazine binding site of MTHFR, which may reflect an evolutional adaptation to the different sizes of the coenzymes.