Regulation of One-Carbon Biosynthesis and Utilization in Escherichia coli

Abstract

The regulation of several enzymes involved in one-carbon metabolism was studied in a mutant of Escherichia coli K-12 defective in S-adenosylmethionine synthetase. The mutant that was reported to have a low endogenous concentration of S-adenosylmethionine had elevated levels of N-5, 10-methylene tetrahydrofolate reductase and serine transhydroxymethylase, but the level of N-5, 10methylene tetrahydrofolate dehydrogenase was not altered. These results suggest that S-adenosylmethionine plays a role in the regulation of one-carbon production and utilization. An enzyme system that cleaved glycine to one-carbon units was demonstrated. The enzymes responsible for the cleavage of glycine were induced by exogenous glycine but were independent of S-adenosylmethionine or purine levels in the cell.The regulation by methionine of several enzymes involved in one-carbon metabolism has been demonstrated by several authors (1,5,10,11,16,26). Generally, their approach was to use methionine-cyanocobalamin (B 12) auxotrophs, whose requirement for methionine was furnished by cyanocobalamin. By increasing the concentration of methionine in the medium, they were able to demonstrate a corresponding decrease in levels of certain enzymes involved in one-carbon metabolism, especially methionine biosynthesis. However, these methods do not distinguish between the effect of methionine and S-adenosylmethionine (SAM) levels, which presumably were increased with increasing methionine concentrations. Unfortunately, the effect of SAM on these enzymes is not directly ascertainable, since it is relatively impermeable to the cells. Recently, however, Greene and co-workers (7,9) and Ahmed (1) isolated mutants of Escherichia coli K-12 defective in SAM synthetase with lowered levels of SAM and increased levels of methionine. With these mutants, one may examine the possibility that SAM levels regulate one-carbon formation and distribution. They may also provide the means to resolve the apparent contradictions between the results of Mansouri et al. (16) and Taylor et al. (26) as to whether the supply of methionine controls the levels of serine transhydroxymethylase (EC 2.1.2.1) and obtain information on the interrelationship between the metabolism of one-carbon units and methionine. Our studies with these mutants showed a pronounced effect of SAM levels on N-5, 10-methylene tetrahydrofolate reductase (EC 1.1.1.68), a much smaller effect on serine transhydroxymethylase, and no effect on N-5, 10-methylene tetrahydrofolate dehydrogenase (EC 1.5.1.5). These results indicate that SAM rather than methionine plays a role in controlling serine transhydroxymethylase levels. They also confirm the results of Kung et al. that N-5, 10-methylene tetrahydrofolate reductase levels are regulated by SAM rather than by methionine (12).As suggested by earlier workers, an alternative means of generating one-carbon units is by cleavage of glycine (23). Serine auxotrophs which cannot synthesize serine via the normal phosphorylated pathway may have their requirement for serine su...

Biosynthetically Directed Fractional 13C-labeling of Proteinogenic Amino Acids. An Efficient Analytical Tool to Investigate Intermediary Metabolism

“…A comparable increase is observed for f ( ' ) { a-Ser} under anaerobic growth conditions (Table 3), showing that the reversible cleavage of Ser is fast relative to the glycine cleavage in the anaerobic regime. This observation is in agreement with the finding that the Gly cleavage enzymes possess only about a tenth of the activity of serine hydroxymethyltransferase which catalyses Ser cleavage (Meedel and Pizer, 1974). Furthermore, Plamann et al (1983) have shown that the absence of a functional glycine cleavage pathway results in the excretion of Gly, thereby demonstrating its relevance for C, metabolism in the aerobic regime.…”

Glycine Cleavage System and cAMP Receptor Protein Co-Regulate CRISPR/cas3 Expression to Resist Bacteriophage

“…Glycine is the substrate catalyzed by gcvP, and was reported to activate gcvTHP operon [28,35]. Nevertheless, our study indicated that supplemented glycine promoted gcvTHP expression in minimal media rather than LB.…”

“…It was reported previously that the expression of gcvTHP operon can be induced by glycine in E. coli as well [28]. Therefore, to further investigate the correlation between gcvTHP expression and cas3 expression, the gcvTHP promoter was cloned into a reporter plasmid (pRCL2) and transformed into WT.…”

The gcvB gene encodes a small untranslated RNA involved in expression of the dipeptide and oligopeptide transport systems in Escherichia coli

“…The new small RNA was discovered in the course of examining the promoter control region for the gcvA gene, encoding the transcriptional regulator of the glycine cleavage (Gcv) operon gcvTHP (Wilson et al, 1993). The Gcv enzyme system catalyses the oxidative cleavage of intracellular glycine to NH 3 , CO 2 and a one-carbon methylene group that is transferred to the acceptor tetrahydrofolate (Kikuchi, 1973;Meedel and Pizer, 1974). This reaction is a major source of the activated methyl compound 5,10-methylenetetrahydrofolate used in the biosynthesis of purines, thymine and methionine and its derivative S-adenosylmethionine, the donor substrate for many methyl transfer reactions (Mudd and Cantoni, 1964).…”