Effects of Sulfanilamide and Methotrexate on 13C Fluxes through the Glycine Decarboxylase/Serine Hydroxymethyltransferase Enzyme System in Arabidopsis1

Abstract

In C 3 plants large amounts of photorespiratory glycine (Gly) are converted to serine by the tetrahydrofolate (THF)-dependent activities of the Gly decarboxylase complex (GDC) and serine hydroxymethyltransferase (SHMT). Using 13 C nuclear magnetic resonance, we monitored the flux of carbon through the GDC/SHMT enzyme system in Arabidopsis thaliana (L.) Heynh. Columbia exposed to inhibitors of THF-synthesizing enzymes. Plants exposed for 96 h to sulfanilamide, a dihydropteroate synthase inhibitor, showed little reduction in flux through GDC/SHMT. Two other sulfonamide analogs were tested with similar results, although all three analogs competitively inhibited the partially purified enzyme. However, methotrexate or aminopterin, which are confirmed inhibitors of Arabidopsis dihydrofolate reductase, decreased the flux through the GDC/SHMT system by 60% after 48 h and by 100% in 96 h. The uptake of [␣-13 C]Gly was not inhibited by either drug class. The specificity of methotrexate action was shown by the ability of 5-formyl-THF to restore flux through the GDC/SHMT pathway in methotrexate-inhibited plants. The experiments with sulfonamides strongly suggest that the mitochondrial THF pool has a long halflife. The studies with methotrexate support the additional, critical role of dihydrofolate reductase in recycling THF oxidized in thymidylate synthesis.

Metabolism of Homocysteine-thiolactone in Plants

“…This suggests that trimethoprim, an inhibitor of bacterial dihydrofolate reductase enzymes (33), does not inhibit plant mitochondrial dihydrofolate reductase. The observation that Hcy or Hcy-thiolactone levels do not increase in the presence of sulfonamide, an inhibitor of de novo folate synthesis (32), suggests that endogenous methyltetrahydrofolate pools in lupine seedlings are not significantly depleted during growth. This suggestion is consistent with a study of one carbon fluxes in Arabidopsis thaliana, which indicated that cellular folate pools have a relatively long half-life in this plant (33).…”

“…Before extraction of Hcy-thiolactone, 6-day-old seedlings were maintained for additional 60 h on water in the absence and presence of the antifolate drug aminopterin (25 M), an inhibitor of eukaryotic dihydrofolate reductase enzymes (32). To facilitate purification of Hcy-thiolactone from plant tissues, yellow lupine seedlings were metabolically labeled with radiotracers (Fig.…”

Two Arabidopsis Threonine Aldolases Are Nonredundant and Compete with Threonine Deaminase for a Common Substrate Pool

“…In [a-13 C]Gly labeling experiments of whole Arabidopsis thaliana, only labeled Gly and Ser accumulated, indicating that Gly is not converted directly to Thr by Thr aldolase in vivo (Prabhu et al, 1996(Prabhu et al, , 1998. Metabolite profiling of Medicago truncatula cell cultures showed that the production of Gly from Thr, likely by Thr aldolase, is induced by treatment with methyl jasmonate (Broeckling et al, 2005).…”

Folate Metabolism in Plants

“…1). In addition to intracellular transports, in vivo studies in Arabidopsis (10,11) have shown that plants are able to take up C1 derivatives of THF from the medium (Fig. 1).…”