Defects in Human Methionine Synthase in cblG Patients

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 β-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 disease

Biochemical and genetic characterization of mammalian methionine synthase

Mammalian methionine synthase (5-methyl-homocysteine methyltransferase; EC 2.1.1.13) is a cobalamin dependent enzyme that catalyzes the transfer of a methyl group from methyltetrahydrofolate to homocysteine, and generates methionine and tetrahydrofolate. Methionine synthase activity, in two classes of patients with defects in cobalamin metabolism, cblG and cblE, is compromised. These patients exhibit severe megaloblastic anemia, homocystinuria, homocysteinaemia and hypomethionaemia. Hyperhomocysteinaemia is a risk factor for cardiovascular disease. High levels of homocysteine have also been linked to neural tube defects. In the present study, a combination of biochemical analyses and molecular genetic approaches have been used to identify culpable loci in patients belonging to the two complementation groups, cblE and cblG. Also, preliminary studies on the regulation of methionine synthase activity by B\sb{12} have been conducted

Posttranscriptional Regulation of Mammalian Methionine Synthase by B\u3csub\u3e12\u3c/sub\u3e

Methionine synthase is one of two key enzymes involved in the removal of the metabolite, homocysteine. Elevated homocysteine levels constitute a risk factor for cardiovascular diseases and for neural tube defects. In cell culture, the activity of methionine synthase is enhanced several-fold by supplementation with its cofactor, B12. The mechanism of this regulation is unknown, although it has been ascribed to a shift from apoenzyme to holoenzyme. Using sensitive assay techniques as well as a combination of Northern and Western analyses, we demonstrate that the effect of B,, on induction of methionine synthase activity is paralleled by an increase in the level of the enzyme. These studies exclude conversion of apoenzyme to holoenzyme as a basis for activation that had been described previously. Since the mRNA levels do not change during the same period that the methionine synthase levels increase, regulation of this protein by its cofactor must be exerted post-transcriptionally