The structure of the C-terminal domain of methionine synthase: presenting S-adenosylmethionine for reductive methylation of B12.
BACKGROUND
In both mammalian and microbial species, B12-dependent methionine synthase catalyzes methyl transfer from methyltetrahydrofolate (CH3-H4folate) to homocysteine. The B12 (cobalamin) cofactor plays an essential role in this reaction, accepting the methyl group from CH3-H4folate to form methylcob(III)alamin and in turn donating the methyl group to homocysteine to generate methionine and cob(I)alamin. Occasionally the highly reactive cob(I)alamin intermediate is oxidized to the catalytically inactive cob(II)alamin form. Reactivation to sustain enzyme activity is achieved by a reductive methylation, requiring S-adenosylmethionine (AdoMet) as the methyl donor and, in Esherichia coli, flavodoxin as an electron donor. The intact system is controlled and organized so that AdoMet, rather than methyltetrahydrofolate, is the methyl donor in the reactivation reaction. AdoMet is not wasted as a methyl donor in the catalytic cycle in which methionine is synthesized from homocysteine. The structures of the AdoMet binding site and the cobalamin-binding domains (previously determined) provide a starting point for understanding the methyl transfer reactions of methionine synthase.


RESULTS
We report the crystal structure of the 38 kDa C-terminal fragment of E.coli methionine synthase that comprises the AdoMet-binding site and is essential for reactivation. The structure, which includes residues 901-1227 of methionine synthase, is a C-shaped single domain whose central feature is a bent antiparallel betasheet. Database searches indicate that the observed polypeptide has no close relatives. AdoMet binds near the center of the inner surface of the domain and is held in place by both side chain and backbone interactions.


CONCLUSIONS
The conformation of bound AdoMet, and the interactions that determine its binding, differ from those found in other AdoMet-dependent enzymes. The sequence Arg-x-x-x-Gly-Tyr is critical for the binding of AdoMet to methionine synthase. The position of bound AdoMet suggests that large areas of the C-terminal and cobalamin-binding fragments must come in contact in order to transfer the methyl group of AdoMet to cobalamin. The catalytic and activation cycles may be turned off and on by alternating physical separation and approach of the reactants.
Dixon
M M
MM
Biophysics Research Division, University of Michigan, 930 N. University, Ann Arbor, MI 48109-1055, USA. ludwig@biop.umich.edu
Huang
S
S
Matthews
R G
RG
Ludwig
M
M
D013766:5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase / Q000737:chemistry*; D000595:Amino Acid Sequence; D001665:Binding Sites; D003198:Computer Simulation; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology*; D008745:Methylation; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry; D011487:Protein Conformation; D012436:S-Adenosylmethionine / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid; D014805:Vitamin B 12 / Q000378:metabolism
D010446:Peptide Fragments; D012436:S-Adenosylmethionine; D013766:5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase; D014805:Vitamin B 12