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 the prosthetic group as intermediate hydride carrier. Comparative genomics and genetic analyses indicated that Mfr is indispensable for the growth of Mycobacterium tuberculosis, which lacks the MTHFR encoding gene. Therefore, Mfr appears to be an excellent target for the design of antimycobacterial drugs. Here, we report the heterologous production, enzymological characterization, and the crystal structure of Mfr from the thermophilic mycobacterium Mycobacterium hassiacum (hMfr), which shows 78% sequence identity to Mfr from M. tuberculosis. Although hMfr and MTHFR have minor sequence identity and different catalytic mechanisms, their structures are highly similar, thus suggesting 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 hMfr and MTHFR. Thus, highly similar binding modes for the C1‐carriers and the reducing agents in hMfr and MTHFR are assumed.

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

“…Accordingly, the hydride‐bearing atoms of FAD (N5) in the eMTHFR structure and of F 420 (C5) in the jMer structure were, at first, superposed (Figure 3 , step 1) and then the rest of the proteins was aligned without moving the hydride‐transferring atoms (Figure 3 , step 2). The hMfr and eMTHFR structures could be superimposed by the normal overall procedure due to their higher structural similarities (Gehl et al, 2023 ) (Figure 3 , step 3). The root mean square deviation (RMSD) values were 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).…”

“…Both enzymes do not contain flavin and catalyze a direct hydride transfer from NADH to methylene‐H 4 F by a ternary complex mechanism (Figure 1b ). The flavin‐independent enzymes, named Mfr (Gehl et al, 2023 ), was currently identified only in mycobacteria as a monomeric enzyme. A knockout strain of M. smegmatis showed impaired growth in the absence of methionine, suggesting that Mfr is involved in the methionine cycle in mycobacteria (Sah et al, 2020 ).…”

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

“…With this aligned models, the position of methyl-H4MPT in jMer could be inferred by the coordination of methyl-H4F on eMTHFR. The positions of NADH and methyl-H4F in hMfr were modeled by us before [27].…”

“…Based on the mutational analysis at position A, it was possible to extend the proposed model of the catalytic mechanism of MTHFR [11] not only to Mfr (Gehl et al 2023) but also to Mer. The mechanism is initiated by the protonation of the C1 carrier to activate this molecule, followed by a hydride transfer from the hydride donor to the activated C1 unit (Figure 7).…”

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