Elevated levels of plasma homocysteine is a risk factor in both birth defects and vascular disease. Methionine synthase (MS) is a cobalamin dependent enzyme which catalyzes methylation of homocysteine to methionine. Impaired MS activity is expected to lead to increased levels of plasma homocysteine. In addition, defects in this gene may underlie the methionine-dependence observed in a number of human tumor cell lines. We describe here the isolation and characterization of the human MS cDNA. It contains an open reading frame of 3798 nucleotides encoding a protein of 1265 amino acids with a predicted molecular mass of 140 kDa. The amino acid sequence of the human MS is 55% identical with that of the Escherichia coli enzyme (METH) and 64% identical with the predicted Caenorhabditis elegans enzyme. Seven peptide sequences derived from purified porcine MS have substantial similarity to the human protein. Northern analysis indicates that the MS RNA is present in a wide variety of tissues. We have mapped the human gene to chromosomal location 1q43, a region found monosomic in individuals with deletion 1q syndrome. The isolation of the MS cDNA will now allow the direct determination of whether mutations in this gene contribute to folate-related neural tube defects, cardiovascular diseases, and birth defects.

Inborn errors resulting in isolated functional methionine synthase deficiency fall into two complementation groups, cblG and cblE. Using biochemical approaches we demonstrate that one cblG patient has greatly reduced levels of methionine synthase while in another, the enzyme is specifically impaired in the reductive activation cycle. The biochemical data suggested that low levels of methionine synthase activity in the first patient may result from mutations in the catalytic domains of the enzyme, reduced transcription, or generation of unstable message or protein. Using Northern analysis, we demonstrate that the molecular basis for the biochemical phenotype in this patient is associated with greatly diminished steady-state levels of methionine synthase mRNA. The biochemical data on the second patient cell line implicated mutations specific to reductive activation, a function that is housed in the C-terminal AdoMet-binding domain and the intermediate B12-binding domain, in the highly homologous bacterial enzyme. We have detected two mutations in a compound heterozygous state, one that results in conversion of a conserved proline (1173) to a leucine residue and the other a deletion of an isoleucine residue (881). The crystal structure of the C-terminal domain of the Escherichia coli MS predicts that the Pro to Leu mutation could disrupt activation since it is embedded in a sequence that makes direct contacts with the bound AdoMet. Deletion of isoleucine in the B12-binding domain would result in shortening of a beta-sheet. Our data provide the first evidence for mutations in the methionine synthase gene being culpable for the cblG phenotype. In addition, they suggest directly that mutations in methionine synthase can lead to elevated homocysteine, implicated both in neural tube defects and in cardiovascular diseases.