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Protein Science (2007), 16:1588-1595. Published by Cold Spring Harbor Laboratory Press. Copyright © 2007 The Protein Society
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Reactivation of methionine synthase from Thermotoga maritima (TM0268) requires the downstream gene product TM0269

Sha Huang1, Gail Romanchuk2, Katherine Pattridge2, Scott A. Lesley4, Ian A. Wilson4, Rowena G. Matthews1,3, and Martha Ludwig2,5

1 Life Sciences Institute, University of Michigan, Ann Arbor, Michigan 48109, USA
2 Biophysics Research Division, University of Michigan, Ann Arbor, Michigan 48109, USA
3 Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA
4 Joint Center for Structural Genomics, The Scripps Research Institute, La Jolla, California 92037, USA

(RECEIVED April 9, 2007; FINAL REVISION May 23, 2007; ACCEPTED May 23, 2007)

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°C, where the specific activity of the purified protein is ~15% of that of E. coli methionine synthase (MetH) at 37°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.

Keywords: structural genomics; adenosylmethionine; cobalamin; structure and function


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