Insights into the reactivation of cobalamin-dependent methionine synthase

• Published in: Proceedings of the National Academy of Sciences - PNAS 2009-11, Vol.106 (44), p.18527-18532
• Main Authors: Koutmos, Markos, Datta, Supratim, Pattridge, Katherine A, Smith, Janet L, Matthews, Rowena G
• Format: Article
• Language: English
• Subjects: 5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase - chemistry; 5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase - metabolism; Amino Acid Substitution; Atoms; BASIC BIOLOGICAL SCIENCES; Biochemistry; Biological Sciences; Catalysis; Catalytic Domain; COBALT; Cobalt - metabolism; DISULFIDES; ELECTRON TRANSFER; ELECTRONS; Enzyme Activation; ENZYMES; Escherichia coli - enzymology; GENERAL AND MISCELLANEOUS//MATHEMATICS, COMPUTING, AND INFORMATION SCIENCE; HOMOCYSTEINE; Hydrogen bonds; Ligands; METHIONINE; Models, Biological; Models, Molecular; Molecular structure; Molecules; Mutation - genetics; Oxidation; Oxidation-Reduction; Protein Structure, Secondary; PROTEINS; S-Adenosylhomocysteine - metabolism; Spectrum Analysis; STABILIZATION; TRANSIENTS; Vitamin B 12 - analogs & derivatives; Vitamin B 12 - metabolism; WATER
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.0906132106

Cobalamin-dependent methionine synthase (MetH) is a modular protein that catalyzes the transfer of a methyl group from methyltetrahydrofolate to homocysteine to produce methionine and tetrahydrofolate. The cobalamin cofactor, which serves as both acceptor and donor of the methyl group, is oxidized once every [almost equal to]2,000 catalytic cycles and must be reactivated by the uptake of an electron from reduced flavodoxin and a methyl group from S-adenosyl-L-methionine (AdoMet). Previous structures of a C-terminal fragment of MetH (MetHCT) revealed a reactivation conformation that juxtaposes the cobalamin- and AdoMet-binding domains. Here we describe 2 structures of a disulfide stabilized MetHCT (S₋SMetHCT) that offer further insight into the reactivation of MetH. The structure of S₋SMetHCT with cob(II)alamin and S-adenosyl-L-homocysteine represents the enzyme in the reactivation step preceding electron transfer from flavodoxin. The structure supports earlier suggestions that the enzyme acts to lower the reduction potential of the Co(II)/Co(I) couple by elongating the bond between the cobalt and its upper axial water ligand, effectively making the cobalt 4-coordinate, and illuminates the role of Tyr-1139 in the stabilization of this 4-coordinate state. The structure of S₋SMetHCT with aquocobalamin may represent a transient state at the end of reactivation as the newly remethylated 5-coordinate methylcobalamin returns to the 6-coordinate state, triggering the rearrangement to a catalytic conformation.