Kazuhiro Yamada

Methylenetetrahydrofolate reductase (MTHFR) is one of the enzymes involved in homocysteine metabolism. Despite considerable genetic and clinical attention, the reaction mechanism and regulation of this enzyme are not fully understood because of difficult production and poor stability. While recombinant enzymes from thermophilic organisms are often stable and easy to prepare, properties of thermostable MTHFRs have not yet been reported. MTHFR from Thermus thermophilus HB8, a homologue of Escherichia coli MetF, has been expressed in E. coli and purified. The purified MTHFR was chiefly obtained as a heterodimer of apo- and holo-subunits, that is, one flavin adenine dinucleotide (FAD) prosthetic group bound per dimer. The crystal structure of the holo-subunit was quite similar to the β₈α₈ barrel of E. coli MTHFR, while that of the apo-subunit was a previously unobserved closed form. In addition, the intersubunit interface of the dimer in the crystals was different from any of the subunit interfaces of the tetramer of E. coli MTHFR. Free FAD could be incorporated into the apo-subunit of the purified Thermus enzyme after purification, forming a homodimer of holo-subunits. Comparison of the crystal structures of the heterodimer and the homodimer revealed different intersubunit interfaces, indicating a large conformational change upon FAD binding. Most of the biochemical properties of the heterodimer and the homodimer were the same, except that the homodimer showed ≈50% activity per FAD-bound subunit in folate-dependent reactions. The different intersubunit interfaces and rearrangement of subunits of Thermus MTHFR may be related to human enzyme properties, such as the allosteric regulation by S-adenosylmethionine and the enhanced instability of the Ala222Val mutant upon loss of FAD. Whereas E. coli MTHFR was the only structural model for human MTHFR to date, our findings suggest that Thermus MTHFR will be another useful model for this important enzyme.

Sustained activity of mammalian methionine synthase (MS) requires MS reductase (MSR), but there have been few studies of the interactions between these two proteins. In this study, recombinant human MS (hMS) and MSR (hMSR) were expressed in baculovirus-infected insect cells and purified to homogeneity. hMSR maintained hMS activity at a 1:1 stoichiometric ratio with a K _act value of 71 nM. Escherichia coli MS, however, was not activated by hMSR. Moreover, hMS was not significantly active in the presence of E. coli flavodoxin and flavodoxin reductase, which maintain the activity of E. coli MS. These results indicate that recognition of MS by their reductive partners is very strict, despite the high homology between MS from different species. The effects of hMSR on the formation of hMS holoenzyme also were examined by using crude extracts of baculovirus-infected insect cells containing hMS apoenzyme (apoMS). In the presence of MSR and NADPH, holoenzyme formation from apoMS and methylcobalamin was significantly enhanced. The observed stimulation is shown to be due to stabilization of human apoMS in the presence of MSR. Apoenzyme alone is quite unstable at 37°C. MSR also is able to reduce aquacobalamin to cob(II)alamin in the presence of NADPH, and this reduction leads to stimulation of the conversion of apoMS and aquacobalamin to MS holoenzyme. Based on these findings, we propose that MSR serves as a special chaperone for hMS and as an aquacobalamin reductase, rather than acting solely in the reductive activation of MS.