Crystal structure of FAD-independent methylene-tetrahydrofolate reductase fromMycobacterium 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 ofMycobacterium 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 mycobacteriumM. hassiacum(hMfr), which shows 78% sequence identity to Mfr fromM. 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.

Convergent evolution of (βα)8-barrel fold methylene-tetrahydropterin reductases utilizing a common catalytic mechanism

“…The overall structure of jMer is almost identical to those of previously established methanogenic enzymes characterized by a (βα) 8 - or TIM-barrel fold (Figure 2). The same fold was also found in the structures of MTHFR and Mfr [26,27]. The root mean square deviation (RMSD) values of these enzymes are 4.7 Å between eMTHFR and hMfr (over 240 amino acids), 4.6 Å between hMfr and jMer (over 232 amino acids), and 5.3 Å between jMer and eMTHFR (over 200 amino acids).…”

“…As the first step, the hydride-bearing atoms of FAD (N5) in the eMTHFR structure and of F 420 (C5) in the jMer structure were superposed (Figure 3, step 2) and then the rest of the proteins was aligned without moving the hydride-transferring atoms (Figure 3, step 3). The hMfr and eMTHFR structures could be superimposed by an overall alignment due to their relatively high structural similarities [27] (Figure 3, step 4). In these aligned tertiary structures, the identical or similar amino-acid residues that locate at the same position in space were identified (Figure 3, step 5).…”

“…With this aligned models, the position of methyl-H 4 MPT in jMer could be inferred by the coordination of methyl-H 4 F on eMTHFR. The positions of NADH and methyl-H 4 F in hMfr were modeled by us before [27].…”

“…hMfr mutants were generated by GenScript. The expression, purification and activity assay procedure was performed as previously described [27].…”

Mutational and structural studies of (βα)8‐barrel fold methylene‐tetrahydropterin reductases utilizing a common catalytic mechanism

“…The overall structure of jMer is almost identical to those of previously established methanogenic enzymes characterized by a (βα) 8 -or TIM-barrel fold (Figure 2). The same fold was also found in the structures of MTHFR and Mfr (Gehl et al, 2023;Guenther et al, 1999).…”