Two enzymic mechanisms for the methylation of homocysteine by extracts of Escherichia coli

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Abstract
The formation of methionine from homocysteine was studied by using mixtures of cell-free extracts of methionine auxotrophs of E. coli blocked between homocysteine and methionine and giving growth responses (a) only with methionine or (b) with methionine or cobalamin. Methionine formation occurred only when extracts of type (a) and (b) auxotrophs were mixed, indicating that each auxotroph lacks an enzyme present in the other. Enzyme-A, present in the methionine/cobalamin auxotroph, and also in methionine-independent strains of E. coli, was purified 80-fold. It catalyzes the formation of heat-stable intermediates (X1 and X3) from N5N10-methylenetetrahydropteroyl-monoglutamate and -triglutamate respectively. Enzyme-B catalyzes a reaction between X3 (though not X1) and homocysteine yielding methionine. It was purified 15-fold from extracts of an auxotroph responding to methionine only and is also present in strains which synthesize methionine. Methionine formation from homocysteine and X1 requires a cobamide-containing enzyme present in methionine/cobalamin auxotrophs and other strains of E. coli only when they are grown in the presence of cobalamin. This enzyme can also use X3 as substrate. Enzyme-B is free from the cobamide- containing enzyme, contains a negligible amount of cobalamin and can be separated from the cobamide-containing enzyme when both are present in the same extract. A strain of E. coli fully competent to synthesize methionine when harvested from a medium without cobalamin contains a negligible amount of cobalamin. Methionine formation which probably occurs normally by the successive action of enzymes-A and -B is independent of cobalamin but requires tetrahy-dropteroyltriglutamate as cofactor. Growth in the presence of cobalamin induces a second mechanism, obligatory for methionine/cobalamin auxotrophs, that depends on a cobamide-containing enzyme though either tetrahydropteroyl-monoglutamate or -triglutamate serves as cofactor.