CLONING AND MAPPING OF A CDNA FOR METHIONINE SYNTHASE REDUCTASE, A FLAVOPROTEIN DEFECTIVE IN PATIENTS WITH HOMOCYSTINURIA

Methionine synthase catalyzes the remethylation of homocysteine to met hionine via a reaction in which methylcobalamin serves as an intermedi ate methyl carrier. Over time, the cob(I)alamin cofactor of methionine synthase becomes oxidized to cob(II)alamin rendering the enzyme inact ive. Regeneration of functional enzyme requires reductive methylation via a reaction in which S-adenosylmethionine is utilized as a methyl d onor. Patients of the cblE complementation group of disorders of folat e/cobalamin metabolism who are defective in reductive activation of me thionine synthase exhibit megaloblastic anemia, developmental delay, h yperhomocysteinemia, and hypomethioninemia. Using consensus sequences to predicted binding sites for FMN, FAD, and NADPH, we have cloned a c DNA corresponding to the ''methionine synthase reductase'' reducing sy stem required for maintenance of the methionine synthase in a function al state. The gene MTRR has been localized to chromosome 5p15.2-15.3. A predominant mRNA of 3.6 kb is detected by Northern blot analysis. Th e deduced protein is a novel member of the FNR family of electron tran sferases, containing 698 amino acids with a predicted molecular mass o f 77,700. It shares 38% identity with human cytochrome P450 reductase and 43% with the C. elegans putative methionine synthase reductase. Th e authenticity of the cDNA sequence was confirmed by identification of mutations in cblE patients, including a 4-bp frameshift in two affect ed siblings and a 3-bp deletion in a third patient. The cloning of the cDNA will permit the diagnostic characterization of cblE patients and investigation of the potential role of polymorphisms of this enzyme a s a risk factor in hyperhomocysteinemia-linked vascular disease.