Correlation between the Level of Vitamin‐B₁₂‐Dependent Methionine Synthetase and Intracellular Concentration of Vitamin B₁₂ in Some Bacteria

This paper presents several lines of evidence indicating that the synthesis of vitamin‐B 12 ‐dependent 5‐methyltetrahydrofolate‐homocysteine transmethylase (“methionine synthetase”) is closely correlated to the intracellular level of vitamin B 12 in manners different between bacteria which do and do not produce vitamin B 12 . 1. The intracellular level of vitamin B 12 in Rhodospirillum rubrum was reduced to one fifth that of the normal cells when the organism was cultivated in a medium lacking cobalt. In accord with the decrease in vitamin B 12 production, the specific activity of methionine synthetase was lowered to about 50%. It was of much interest that the enzyme was always present as the holoenzyme and the apoenzyme could not be detected even in the vitamin‐B 12 ‐deficient cells containing a markedly decreased level of the holoenzyme. When the vitamin‐B 12 ‐deficient cells were incubated in a cyano‐cobalamin‐supplemented medium, the specific activity of methionine synthetase began to increase after 2 h lag time and reached the maximum in about 6 h. This enhancement of the specific activity was suppressed if an appropriate inhibitor of protein synthesis was added to the medium. These facts strongly suggest that synthesis of protein de novo is essential for the enhancement of the specific activity by incubation with cyanocobalamin. Similar results were obtained in other vitamin B 12 producers such as Streptomyces olivaceus and Corynebacterium simplex. 2. Chromatium D which is known to form cobalt‐free corrinoids but not produce complete vitamin B 12 , was found to possess methionine synthetase as apoenzyme. When the vitamin‐B 12 ‐free cells were incubated in the presence of cyanocobalamin, all the apoenzyme was readily converted to the holoenzyme within 2 h. As the formation of the holoenzyme proceeded, the level of the total enzyme (holo + apo) increased to a certain degree. The enhancement, however, was not affected by the inhibitors of protein synthesis, indicating that the increment could not have resulted from de novo synthesis of protein. A similar phenomenon was observed in Escherichia coli which does not produce vitamin B 12 but possesses apoprotein of methionine synthetase. It is suggested that vitamin B 12 synthesis is closely correlated to that of methionine synthetase in bacteria producing the vitamin, such as Rhodospirillum rubrum, Streptomyces olivaceus and Corynebacterium simplex. On the contrary, the apoprotein of the enzyme is synthesized independently of vitamin B 12 in bacteria not producing the vitamin, such as Chromatium D and Escherichia coli.