Reactivation of methionine synthase from Thermotoga maritima (TM0268) requires the downstream gene product TM0269.
The crystal structure of the Thermotoga maritima gene product TM0269, determined as part of genome-wide structural coverage of T. maritima by the Joint Center for Structural Genomics, revealed structural homology with the fourth module of the cobalamin-dependent methionine synthase (MetH) from Escherichia coli, despite the lack of significant sequence homology. The gene specifying TM0269 lies in close proximity to another gene, TM0268, which shows sequence homology with the first three modules of E. coli MetH. The fourth module of E. coli MetH is required for reductive remethylation of the cob(II)alamin form of the cofactor and binds the methyl donor for this reactivation, S-adenosylmethionine (AdoMet). Measurements of the rates of methionine formation in the presence and absence of TM0269 and AdoMet demonstrate that both TM0269 and AdoMet are required for reactivation of the inactive cob(II)alamin form of TM0268. These activity measurements confirm the structure-based assignment of the function of the TM0269 gene product. In the presence of TM0269, AdoMet, and reductants, the measured activity of T. maritima MetH is maximal near 80 degrees C, where the specific activity of the purified protein is approximately 15% of that of E. coli methionine synthase (MetH) at 37 degrees C. Comparisons of the structures and sequences of TM0269 and the reactivation domain of E. coli MetH suggest that AdoMet may be bound somewhat differently by the homologous proteins. However, the conformation of a hairpin that is critical for cobalamin binding in E. coli MetH, which constitutes an essential structural element, is retained in the T. maritima reactivation protein despite striking divergence of the sequences.
Life Sciences Institute, University of Michigan, Ann Arbor, Michigan 48109-2216, USA.
Sha
S
Huang
Gail
G
Romanchuk
Katherine
K
Pattridge
Scott A
SA
Lesley
Ian A
IA
Wilson
Rowena G
RG
Matthews
Martha
M
Ludwig
D013766:5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D001059:Apoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D004789:Enzyme Activation; D008745:Methylation; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011487:Protein Conformation; D012436:S-Adenosylmethionine / Q000737:chemistry / Q000378:metabolism; D013696:Temperature; D020124:Thermotoga maritima / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D014805:Vitamin B 12 / Q000737:chemistry / Q000378:metabolism
D001059:Apoproteins; D001426:Bacterial Proteins; D012436:S-Adenosylmethionine; D013766:5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase; D014805:Vitamin B 12