Modulation of methylenetetrahydrofolate reductase activity by S-adenosylmethionine and by dihydrofolate and its polyglutamate analogues

Methylenetetrahydrofolate reductase catalyzes the reduction of methylenetetrahydrofolate ot methyltetrahydrofolate. This reaction commits one carbon units to the pathways of adenosylmethionine-dependent methylation in mammalian cells. We have purified the pig liver enzyme to homogeneity and shown that it contains FAD as a non-covalently bound prosthetic group. Methylenetetrahydrofolate is not only a substrate for the reductase, but also for thymidylate synthase and for methylenetetrahydrofolate dehydrogenase. The latter reaction leads to utilization of one carbon units in de novo purine biosynthesis. A prior, one might expect that methylenetetrahydrofolate reductase activity would be modulated by cellular requirements for de novo biosynthesis of purines and pyrimidines, as well as by cellular levels of adenosylmethionine. Methylenetetrahydrofolate reductase is inhibited by dihydrofolate and its polyglutamate analogues. The Kl is 6.5 [mu] for dihydrofolate and decreases with each additional glutamyl residue to a minimum value of 0.013 [mu] for dihydropteroylhexaglutamate. The I50 for dihydropteroylhexaglutamate inhibition of reductase activity in the presence of 0.5 [mu] methylenetetrahydropteroylhexaglutamate is 0.07 [mu]. We proposed that stimulation of thymidylate synthease activity (as in the replicating cell) may lead to elevations in the steady state levels of cellular dihydrofolate derivatives and to resultant inhibition of methylenetetrahydrofolate reductase activity. Thus methylenetetrahydrofolate derivatives would be spared for purine and pyrimidine biosynthesis.We have also examined the inhibition of methylenetetrahydrofolate reductase by adenosylmethionine, which serves as an allosteric effector of the enzymatic activity. Adenosylmethionine induces a slow transition in the enzyme, and leads to the inhibition of NADPH-menadione. NADPH-methylenetetrahydrofolate and methyltetrahydrofolate-menadione oxido-reductase activities.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/24098/1/0000355.pd

Cloning of nitroalkane oxidase from Fusarium oxysporum identifies a new member of the acyl-CoA dehydrogenase superfamily

The flavoprotein nitroalkane oxidase (NAO) from Fusarium oxysporum catalyzes the oxidation of nitroalkanes to the respective aldehydes with production of nitrite and hydrogen peroxide. The sequences of several peptides from the fungal enzyme were used to design oligonucleotides for the isolation of a portion of the NAO gene from an F. oxysporum genomic DNA preparation. This sequence was used to clone the cDNA for NAO from an F. oxysporum cDNA library. The sequence of the cloned cDNA showed that NOA is a member of the acyl-CoA dehydrogenase (ACAD) superfamily. The members of this family share with NAO a mechanism that is initiated by proton removal from carbon, suggesting a common chemical reaction for this superfamily. NAO was expressed in Escherichia coli and the recombinant enzyme was characterized. Recombinant NAO has identical kinetic parameters to enzyme isolated from F. oxysporum but is isolated with oxidized FAD rather than the nitrobutyl-FAD found in the fungal enzyme. NAO purified from E. coli or from F. oxysporum has no detectable ACAD activity on short- or medium-chain acyl CoAs, and medium-chain acyl-CoA dehydrogenase and short-chain acyl-CoA dehydrogenase are unable to catalyze oxidation of nitroalkanes