Human methionine synthase reductase is a molecular chaperone for human methionine synthase

doi:10.1073/pnas.0603694103

Human methionine synthase reductase is a molecular chaperone for human methionine synthase

*Life Sciences Institute and Department of Biological Chemistry, University of Michigan, Ann Arbor, MI 48109-2216;
^‡Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, AB, Canada T2N 4N1; and
^§Department of Bioscience and Biotechnology, Faculty of Engineering, Okayama University, Tsushima-Naka, Okayama 700-8530, Japan

Contributed by Rowena G. Matthews, May 8, 2006

Abstract

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.

Footnotes

^†To whom correspondence may be sent at the present address:
Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Tokyo 184-8588, Japan.
E-mail: yamadak{at}cc.tuat.ac.jp
^¶To whom correspondence may be addressed at:
4002 Life Sciences Institute, University of Michigan, 210 Washtenaw Avenue, Ann Arbor, MI 48109-2216.
E-mail: rmatthew{at}umich.edu
Author contributions: K.Y., R.A.G., T.T., and R.G.M. designed research; K.Y. performed research; K.Y., R.A.G., and R.G.M. analyzed data; and K.Y., R.A.G., T.T., and R.G.M. wrote the paper.
Conflict of interest statement: No conflicts declared.
↵ ^‖Mangum, J. H. & Scrimgeour, K. G. (1962) Fed. Proc. 21, 242 (abstr.).
Abbreviations:

Abbreviations:

MS,

methionine synthase;

hMS,

human MS;

MSR,

MS reductase;

hMSR,

human MSR;

hMSR,

I-H, Ile-22 variant of His-tagged hMSR;

apoMS,

MS apoenzyme;

holoMS,

MS holoenzyme;

FNR,

ferredoxin (flavodoxin)-NADP+ oxidoreductase;

Met,

methionine;

AdoMet,

S-adenosylmethionine;

Cbl,

cobalamin;

MeCbl,

methylcobalamin;

aqCbl,

aquacobalamin.