Methionine biosynthesis in Ochromonas malhamensis

Abstract

Methyltetrahydrofolate-homocysteine methyltransferase (methionine synthetase) has been partially purified (60- to 80-fold) from the soluble fraction of Ochromonas malhamensis cells. Preliminary studies with the enzyme from cells grown on medium containing cyano-B 12 sufficient for optimum growth were done with and without air. Under anaerobic conditions coenzyme B 12 inhibited methionine formation. In air the coenzyme gave marked stimulation and was much more active than the system under nitrogen. Without coenzyme B 12 the activity under nitrogen and in air was similar. With protein prepared from cells grown with limiting cyano-B 12, more methionine was synthesized anaerobically than in air when coenzyme B 12 was not present. Anaerobically coenzyme B 12 had no effect. In air the coenzyme caused very high activity, equivalent to the B 12-adequate preparation plus coenzyme B 12. Both holoenzyme and apoenzyme are probably present in each preparation, with more of the apoenzyme in the deficient preparation. The adequate enzyme system in air required in addition to coenzyme B 12, ATP, Mg 2+, adenosylmethionine, and NADH. The major work dealt with the B 12-deficient protein. The cofactor requirements were the same as for the adequate system. No other reducing agent was equivalent to NADH. FMNH 2 (H 2) was less effective; dithiothreitol inhibited. The unique preference for NADH may be related to the synthesis of holoenzyme. In this system coenzyme B 12 was the most active B 12 compound. Cyano-B 12 was somewhat less active, but required the same cofactors. Methyl-B 12 was also less active, and at higher levels caused inhibition. This inhibition only occurred in the presence of ATP. Otherwise, the cofactors needed with methyl-B 12 were the same, except that a smaller amount of Mg 2+ and a higher quantity of adenosylmethionine were needed for maximum activity. Ethyl-B 12 was active and propyl-B 12 gave some activity at a low level, but inhibited at a higher level. Some of the activity maybe attributed to its protection of the holoenzyme in air, although the magnitude of the activity suggests that there may have been some enzymatic cleavage of the cobalt-propyl bond. The role of ATP is unclear. It was required for maximum utilization of endogenous B 12 and added coenzyme B 12 and cyano-B 12, and it inhibited the use of methyl-B 12. It is proposed that ATP may alter protein conformation permitting better spacial relationships when the “natural” B 12 coenzyme [formed from coenzyme B 12 and cyano-B 12 ( in vivo or in vitro)]is present, than when methyl-B 12 is the source of the B 12 compound.