13C Nuclear Magnetic Resonance Detection of Interactions of Serine Hydroxymethyltransferase with C1-Tetrahydrofolate Synthase and Glycine Decarboxylase Complex Activities in Arabidopsis

Abstract

~~ ~~ I n C, plants, serine synthesis is associated with photorespiratory glycine metabolism involving the tetrahydrofolate (THF)-dependent activities of the glycine decarboxylase complex (CDC) and serine hydroxymethyl transferase (SHMT). Alternatively, THF-dependent serine synthesis can occur via the C1 -THF synthase/SHMT pathway. We used 13C nuclear magnetic resonance to examine serine biosynthesis by these two pathways i n Arabidopsis fhaliana (L.) Heynh.Columbia wild type. We confirmed the tight coupling of the CDC/ SHMT system and observed directly i n a higher plant the flux of formate through the C1 -THF synthase/SHMT system. l h e accumulation of 13C-enriched serine over 24 h from the CDC/SHMT activities was 4-fold greater than that from C1 -THF synthase/SHMT activities. Our experiments strongly suggest that the two pathways operate independently in Arabidopsis. Plants exposed to methotrexate and sulfanilamide, powerful inhibitors of THF biosynthesis, reduced serine synthesis by both pathways. l h e results suggest that continuous supply of THF is essential to maintain high rates of serine metabolism. Nuclear magnetic resonance is a powerful tool for the examination of THF-mediated metabolism in its natural cellular environment.Folate coenzymes mediate single-carbon transfers in a variety of cellular processes such as purine biosynthesis, amino acid metabolism, thymidylate synthesis, and chloroplast and mitochondrial protein synthesis. In spite of the pivotal role THF plays in cellular metabolism, studies of THF biosynthesis and THF-dependent metabolism in plants remain sparse. We have recently begun wideranging studies of folate metabolism in Datuva cells and Arabidopsis plants (Wu et al., 1993(Wu et al., , 1994 Prabhu et al., 1994). In this study we addressed the THF-dependent biosynthesis of Ser in intact plants of Arabidopsis tkaliana (L.) Heynh. Columbia wild type using I3C NMR.THF-dependent Gly and Ser metabolism are closely linked in a variety of organisms (Schirch, 1984). The use of a common pool of THF allows transfer of the a-C of Gly

Deletion of Glycine Decarboxylase in Arabidopsis Is Lethal under Nonphotorespiratory Conditions

“…It has been proposed that the mitochondrial Gly-to-Ser conversion, by recycling Gly originating from extramitochondrial SHM reactions, represents an obligatory component of onecarbon metabolism not only in photosynthetic but in all plant tissues (Mouillon et al, 1999). On the other hand, a GDC-independent pathway for the supply of CH 2 -THF, driven by the C1-THF synthase system, has been suggested as an important means of compensating for the lack of GDC activity (Prabhu et al, 1996;Wingler et al, 1999;Li et al, 2003). The existence of such pathways will be difficult to prove because of the very early developmental arrest of the GDC-lacking double mutant.…”

“…It shall be noted that residual GDC activity has been reported for such mutants (for example, Li et al, 2003). Moreover, it was reported that CH 2 -THF and Ser can be synthesized through multiple routes including the so-called C1-THF synthase/SHM pathway (Prabhu et al, 1996;Li et al, 2003). In this GDC-bypassing pathway, formate would be converted into CH 2 -THF via two enzymes, 10-formyl-THF synthetase and the bifunctional enzyme CH 2 -THF dehydrogenase/ CH 2 -THF cyclohydrolase.…”

The Metabolic Status Drives Acclimation of Iron Deficiency Responses in Chlamydomonas reinhardtii as Revealed by Proteomics Based Hierarchical Clustering and Reverse Genetics

“…5,10 methylene tetrahydrofolate is also produced by glycine decarboxylase activity which is then used as the main single carbon donor for synthesis of serine (73). Yet, the synthesis via the 10-formyltetrahydrofolate synthetase provides independent 5,10 methylene tetrahydrofolate for the synthesis of serine (74). Interestingly using 13 C nuclear magnetic resonance, interactions among serine hydroxymethyltransferase, tetrahydrofolate synthase, and glycine decarboxylase complex activities were shown (74).…”

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).…”