5-Formyltetrahydrofolate Is an Inhibitory but Well Tolerated Metabolite in Arabidopsis Leaves

Abstract

5-Formyltetrahydrofolate (5-CHO-THF) is formed via a second catalytic activity of serine hydroxymethyltransferase (SHMT) and strongly inhibits SHMT and other folate-dependent enzymes in vitro. The only enzyme known to metabolize 5-CHO-THF is 5-CHO-THF cycloligase (5-FCL), which catalyzes its conversion to 5,10-methenyltetrahydrofolate. Because 5-FCL is mitochondrial in plants and mitochondrial SHMT is central to photorespiration, we examined the impact of an insertional mutation in the Arabidopsis 5-FCL gene (At5g13050) under photorespiratory (30 and 370 μmol of CO2 mol–1) and non-photorespiratory (3200 μmol of CO2 mol–1) conditions. The mutation had only mild visible effects at 370 μmol of CO2 mol–1, reducing growth rate by ∼20% and delaying flowering by 1 week. However, the mutation doubled leaf 5-CHO-THF level under all conditions and, under photorespiratory conditions, quadrupled the pool of 10-formyl-/5,10-methenyltetrahydrofolates (which could not be distinguished analytically). At 370 μmol of CO2 mol–1, the mitochondrial 5-CHO-THF pool was 8-fold larger in the mutant and contained most of the 5-CHO-THF in the leaf. In contrast, the buildup of 10-formyl-/5,10-methenyltetrahydrofolates was extramitochondrial. In photorespiratory conditions, leaf glycine levels were up to 46-fold higher in the mutant than in the wild type. Furthermore, when leaves were supplied with 5-CHO-THF, glycine accumulated in both wild type and mutant. These data establish that 5-CHO-THF can inhibit SHMT in vivo and thereby influence glycine pool size. However, the near-normal growth of the mutant shows that even exceptionally high 5-CHO-THF levels do not much affect fluxes through SHMT or any other folate-dependent reaction, i.e. that 5-CHO-THF is well tolerated in plants. 5-Formyltetrahydrofolate (5-CHO-THF) is formed via a second catalytic activity of serine hydroxymethyltransferase (SHMT) and strongly inhibits SHMT and other folate-dependent enzymes in vitro. The only enzyme known to metabolize 5-CHO-THF is 5-CHO-THF cycloligase (5-FCL), which catalyzes its conversion to 5,10-methenyltetrahydrofolate. Because 5-FCL is mitochondrial in plants and mitochondrial SHMT is central to photorespiration, we examined the impact of an insertional mutation in the Arabidopsis 5-FCL gene (At5g13050) under photorespiratory (30 and 370 μmol of CO2 mol–1) and non-photorespiratory (3200 μmol of CO2 mol–1) conditions. The mutation had only mild visible effects at 370 μmol of CO2 mol–1, reducing growth rate by ∼20% and delaying flowering by 1 week. However, the mutation doubled leaf 5-CHO-THF level under all conditions and, under photorespiratory conditions, quadrupled the pool of 10-formyl-/5,10-methenyltetrahydrofolates (which could not be distinguished analytically). At 370 μmol of CO2 mol–1, the mitochondrial 5-CHO-THF pool was 8-fold larger in the mutant and contained most of the 5-CHO-THF in the leaf. In contrast, the buildup of 10-formyl-/5,10-methenyltetrahydrofolates was extramitochondrial. In photorespiratory conditions, leaf glycine levels were up to 46-fold higher in the mutant than in the wild type. Furthermore, when leaves were supplied with 5-CHO-THF, glycine accumulated in both wild type and mutant. These data establish that 5-CHO-THF can inhibit SHMT in vivo and thereby influence glycine pool size. However, the near-normal growth of the mutant shows that even exceptionally high 5-CHO-THF levels do not much affect fluxes through SHMT or any other folate-dependent reaction, i.e. that 5-CHO-THF is well tolerated in plants. 5-Formyltetrahydrofolate (5-CHO-THF) 1The abbreviations used are: 5-CHO-THF, 5-formyltetrahydrofolate; 10-CHO-THF, 10-formyltetrahydrofolate; 5,10-CH=THF, 5,10-methenyltetrahydrofolate; 5,10-CH2-THF, 5,10-methylenetetrahydrofolate; 5-CH3-THF, 5-methyltetrahydrofolate; 5-FCL, 5-formyltetrahydrofolate cycloligase; SHMT, serine hydroxymethyltransferase; GC-MS, gas chromatography-mass spectrometry; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid; CHES, 2-(cyclohexylamino)ethanesulfonic acid; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. is formed from 5,10-methenyltetrahydrofolate (5,10-CH=THF) by a hydrolytic reaction catalyzed by serine hydroxymethyltransferase (SHMT) in the presence of glycine (1Stover P. Schirch V. J. Biol. Chem. 1990; 265: 14227-14233Abstract Full Text PDF PubMed Google Scholar, 2Holmes W.B. Appling D.R. J. Biol. Chem. 2002; 277: 20205-20213Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Spontaneous chemical hydrolysis of 5,10-CH=THF may be a minor additional source (3Baggott J.E. Biochemistry. 2000; 39: 14647-14653Crossref PubMed Scopus (27) Google Scholar). 5-CHO-THF is the most stable natural folate and the most enigmatic, for it is the only one that does not serve as a cofactor in one-carbon metabolism. Instead, 5-CHO-THF is a potent inhibitor of SHMT and most other folate-dependent enzymes in vitro (4Roje S. Janave M.T. Ziemak M.J. Hanson A.D. J. Biol. Chem. 2002; 277: 42748-42754Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 5Stover P. Schirch V. Trends Biochem. Sci. 1993; 18: 102-106Abstract Full Text PDF PubMed Scopus (111) Google Scholar). 5-CHO-THF probably acts as a stable storage form of folate in seeds and fungal spores (5Stover P. Schirch V. Trends Biochem. Sci. 1993; 18: 102-106Abstract Full Text PDF PubMed Scopus (111) Google Scholar, 6Shin Y.S. Kim J.E. Watson J.E. Stokstad E.L.R. Can. J. Biochem. 1975; 53: 338-343Crossref PubMed Scopus (33) Google Scholar, 7Kruschwitz H.L. McDonald D. Cossins E.A. Schirch V. J. Biol. Chem. 1994; 269: 28757-28763Abstract Full Text PDF PubMed Google Scholar), but it is not clear what role, if any, it plays in metabolically active tissues (8Baggott J.E. MacKenzie R.E. Biochem. J. 2003; 374: 773-778Crossref PubMed Google Scholar). This question is particularly pertinent for leaves. Leaf mitochondria have very high levels of SHMT and, during photorespiration, receive a massive influx of glycine (which leads to a matching SHMT-mediated glycine → serine flux) (9Douce R. Bourguignon J. Neuburger M. Rébeillé F. Trends Plant Sci. 2001; 6: 167-176Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar). Conditions in leaf mitochondria therefore favor 5-CHO-THF formation (Fig. 1). Indeed, 5-CHO-THF can comprise 50% of the folate pool in leaf mitochondria (10Chen L. Chan S.Y. Cossins E.A. Plant Physiol. 1997; 115: 299-309Crossref PubMed Scopus (121) Google Scholar, 11Chan S.Y. Cossins E.A. Pteridines. 2003; 14: 67-76Crossref Scopus (5) Google Scholar), which is far more than in mammalian mitochondria (12Horne D.W. Patterson D. Cook R.J. Arch. Biochem. Biophys. 1989; 270: 729-733Crossref PubMed Scopus (91) Google Scholar, 13Horne D.W. Holloway R.S. J. Nutr. 1997; 127: 1772-1775Crossref PubMed Scopus (15) Google Scholar, 14Balaghi M. Horne D.W. Wagner C. Biochem. J. 1993; 291: 145-149Crossref PubMed Scopus (108) Google Scholar). Furthermore, 5-CHO-THF is reported to make up 14–40% of the folate pool in leaves and other metabolically active plant organs (10Chen L. Chan S.Y. Cossins E.A. Plant Physiol. 1997; 115: 299-309Crossref PubMed Scopus (121) Google Scholar, 15Cossins E.A. Can. J. Bot. 2000; 78: 691-708Google Scholar), a much higher proportion than the 3–10% typical of mammals and yeast (2Holmes W.B. Appling D.R. J. Biol. Chem. 2002; 277: 20205-20213Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 16Girgis S. Suh J.R. Jolivet J. Stover P.J. J. Biol. Chem. 1997; 272: 4729-4734Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). 5-Formyltetrahydrofolate cycloligase, EC 6.3.3.2 (5-FCL, also known as 5,10-methenyltetrahydrofolate synthetase), is the only enzyme known to recycle 5-CHO-THF to a metabolically active form, which it achieves by catalyzing irreversible, ATP-dependent conversion to 5,10-CH=THF (2Holmes W.B. Appling D.R. J. Biol. Chem. 2002; 277: 20205-20213Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 5Stover P. Schirch V. Trends Biochem. Sci. 1993; 18: 102-106Abstract Full Text PDF PubMed Scopus (111) Google Scholar). This enzyme is also something of an enigma. For one thing, despite the inhibitory effects of its substrate, 5-FCL is not essential in yeast: 5-FCL disruptants had 4-fold more 5-CHO-THF but no other new phenotype (2Holmes W.B. Appling D.R. J. Biol. Chem. 2002; 277: 20205-20213Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). For another, phylogenomic profiling (17von Mering C. Huynen M. Jaeggi D. Schmidt S. Bork P. Snel B. Nucleic Acids Res. 2003; 31: 258-261Crossref PubMed Scopus (1419) Google Scholar, 18Enault F. Suhre K. Poirot O. Abergel C. Claverie J.M. Nucleic Acids Res. 2004; 32: W336-W339Crossref PubMed Scopus (26) Google Scholar) indicates that some bacteria lack 5-FCL even though they have SHMT. Lastly, 5-FCL overexpression in human cells lowered the folate level and raised folate turnover rate, suggesting that 5-FCL may have a second function as a folate-degrading enzyme (19Anguera M.C. Suh J.R. Ghandour H. Nasrallah I.M. Selhub J. Stover P.J. J. Biol. Chem. 2003; 278: 29856-29862Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Another intriguing feature of 5-FCL is that its subcellular location differs among eukaryotes. Whereas the enzyme is largely if not solely cytosolic in yeast and mammals (2Holmes W.B. Appling D.R. J. Biol. Chem. 2002; 277: 20205-20213Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 16Girgis S. Suh J.R. Jolivet J. Stover P.J. J. Biol. Chem. 1997; 272: 4729-4734Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 20Maras B. Stover P. Valiante S. Barra D. Schirch V. J. Biol. Chem. 1994; 269: 18429-18433Abstract Full Text PDF PubMed Google Scholar, 21Bertrand R. Beauchemin M. Dayan A. Ouimet M. Jolivet J. Biochim. Biophys. Acta. 1995; 1266: 245-249Crossref PubMed Scopus (14) Google Scholar), it is exclusively mitochondrial in plants (4Roje S. Janave M.T. Ziemak M.J. Hanson A.D. J. Biol. Chem. 2002; 277: 42748-42754Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Taken with the key role of mitochondrial SHMT in photorespiration, with the inhibition of SHMT by 5-CHO-THF, and with the presence of 5-CHO-THF in leaf mitochondria, the location of plant 5-FCL implies (i) that this enzyme governs mitochondrial 5-CHO-THF levels and (ii) that, via its effect on 5-CHO-THF levels, 5-FCL could regulate the in vivo activities of SHMT and other folate-linked mitochondrial enzymes (Fig. 1). To investigate such regulatory functions for 5-FCL and 5-CHO-THF, we identified and characterized an Arabidopsis 5-FCL insertional mutant. This mutant had greatly elevated mitochondrial 5-CHO-THF levels and hyperaccumulated glycine under photorespiratory conditions. However, the mutant grew almost normally in ambient air, indicating that folate-dependent metabolic reactions in plants are substantially tolerant to 5-CHO-THF. Chemical and Reagents—Folates were from Schircks Laboratories (Jona, Switzerland). 10-Formyldihydrofolate was prepared from (6R,6S)-5-CHO-THF as described (22Baggott J.E. Johanning G.L. Branham K.E. Prince C.W. Morgan S.L. Eto I. Vaughn W.H. Biochem. J. 1995; 308: 1031-1036Crossref PubMed Scopus (33) Google Scholar). [α-32P]dCTP (3000 Ci mmol–1) was from PerkinElmer Life Sciences. Plants and Growth Conditions—Arabidopsis thaliana plants were grown at 22–28 °C in 12-h days (photosynthetic photon flux density 80 μE m–2 s–1) in potting soil irrigated with water. Material was lyophilized to determine dry weight. In experiments at various CO2 levels, plants were grown for 4 weeks in ambient air (∼370 μmol of CO2 mol–1), then either kept in ambient air or transferred to high CO2 (3200 ± 40 μmol mol–1) or low CO2 (30 ± 20 μmol mol–1) conditions for 5 days. CO2 levels were monitored with a Vernier CO2 sensor (Vernier Software and Technology, Beaverton, OR). Arabidopsis Mutant—A 5-FCL mutant (28D07) was identified in the Syngenta T-DNA insertion collection (ecotype Columbia) (23Sessions A. Burke E. Presting G. Aux G. McElver J. Patton D. Dietrich B. Ho P. Bacwaden J. Ko C. Clarke J.D. Cotton D. Bullis D. Snell J. Miguel T. Hutchison D. Kimmerly B. Mitzel T. Katagiri F. Glazebrook J. Law M. Goff S.A. Plant Cell. 2002; 14: 2985-2994Crossref PubMed Scopus (757) Google Scholar). Segregants, wild type or homozygous for the mutation, were identified by PCR using gene-specific primers located 5′ or 3′ of the T-DNA insertion (5′-CTGAAGTGAGTGGCAACTACA-3′ and 5′-GTCTCACTTCTTCTCTTACCTT-3′, respectively) and the T-DNA-specific primer 5′-GCATCTGAATTTCATAACCAATC-3′. DNA was extracted by the “Shorty” protocol available on the website of the University of Wisconsin Biotechnology Center. The insertion site was confirmed by sequencing. Gel Blot Analyses—Total RNA was extracted from 0.1-g samples of rosette leaves using RNeasy kits (Qiagen, Valencia, CA) and treated with DNase (DNA-free™ kit, Ambion, Austin, TX). RNA samples were separated by and to and for 4 at °C in and and in at The 5-FCL was a of (4Roje S. Janave M.T. Ziemak M.J. Hanson A.D. J. Biol. Chem. 2002; 277: 42748-42754Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar) to the of the 5-FCL (Fig. The was a of a R.J. J. PubMed Scopus Google Scholar). DNA was as described P. Plant Biol. 1989; Scopus Google Scholar) from of leaves from 20 separated by and to were as and in at The was a of the gene using the primers and were with [α-32P]dCTP by the primer was by were extracted from leaf by in of CHES, to with and by for and then The was the and the were were extracted with 5 of were treated with 1 or of at °C for to were then for and to folate prepared as described Biochem. PubMed Scopus Google Scholar). was to 1 for with 5 for of 1 then with 5 for of they were with 5 for of samples were by P.J. Selhub J. Chem. 2000; PubMed Scopus Google Scholar) using a CA) and a with at and The was a of and and a of and with a from to at 1 was with 5,10-CH=THF, 5-CHO-THF, and was in and The was extracted by with of and and was then at °C for and were as For 20 of the was with in a of and a was by to For GC-MS, to dry were under and and in as described J. L. 2003; PubMed Scopus Google Scholar). of the was CA) a was with an gas and a as described J. L. 2003; PubMed Scopus Google Scholar). of and SHMT were prepared from of leaves from Arabidopsis plants as described R. H. Plant Physiol. PubMed Scopus Google Scholar), with the The and mitochondria was in of a 20 1 to a of of of and of and at for in a were from the in 1 and and then at for 20 This was The mitochondrial was in of 1 and under at °C SHMT was in mitochondrial as described (4Roje S. Janave M.T. Ziemak M.J. Hanson A.D. J. Biol. Chem. 2002; 277: 42748-42754Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). 5-CHO-THF wild type and mutant plants weeks were of and were under of the The plants were in or 5-CHO-THF in and in μE m–2 s–1) at °C for The plants were then in and were for as and of an Arabidopsis 5-FCL Mutant—A 5-FCL mutant was identified in the T-DNA mutant collection (23Sessions A. Burke E. Presting G. Aux G. McElver J. Patton D. Dietrich B. Ho P. Bacwaden J. Ko C. Clarke J.D. Cotton D. Bullis D. Snell J. Miguel T. Hutchison D. Kimmerly B. Mitzel T. Katagiri F. Glazebrook J. Law M. Goff S.A. Plant Cell. 2002; 14: 2985-2994Crossref PubMed Scopus (757) Google Scholar) via the the of this confirmed the presence of an to the 3′ of the which is located the of the gene (Fig. Plants homozygous for the mutation and wild type were identified by PCR and to using a T-DNA of the as (Fig. the mutant plants that the T-DNA is only at the 5-FCL The in indicates that T-DNA are at this of leaf RNA no 5-FCL in the mutant (Fig. indicating a The homozygous and wild type were therefore for grown in soil at ambient levels of CO2 (∼370 μmol of CO2 mol–1), visible mutant plants and wild type were The growth rate of mutant plants was ∼20% (Fig. and they a flowering of 1 (Fig. was no in leaf or form (Fig. and were treated with to to form, by Biochem. PubMed Scopus Google Scholar), and then separated by with P.J. Selhub J. Chem. 2000; PubMed Scopus Google Scholar), which was for its and These do not and 5,10-CH=THF, which are during and both as 5,10-CH=THF P.J. Selhub J. Chem. 2000; PubMed Scopus Google Scholar). were to 5,10-CH=THF by in H. L. J. Biol. Chem. Full Text PDF PubMed Google Scholar) and to it as an in D.W. Wagner C. PubMed Scopus Google Scholar), but this with plant to from 5,10-CH=THF, both as H. L. J. Biol. Chem. Full Text PDF PubMed Google Scholar). is not to determine the of and 5,10-CH=THF in but only it be that to during that are the of and in to CO2 Plants grown for 4 weeks in ambient air were transferred for 5 days to air or μmol of CO2 or kept in ambient The CO2 which is the CO2 the glycine → serine flux rate in the higher one it Plant Physiol. PubMed Scopus Google Scholar). CO2 had effect on folate that the wild type and mutant leaves were in all (Fig. be 5-CHO-THF levels were higher in the mutant to at levels were also much higher in mutant leaves to 370 and μmol of CO2 the folate of the mutant higher at the CO2 mitochondria in leaves are to be the site of 5-CHO-THF formation and the only site of its by 5-FCL (Fig. we the impact of the 5-FCL mutation on mitochondrial folate levels of plants grown in ambient mitochondrial were from wild type or mutant plants (Fig. some among the mitochondria from the mutant massive 5-CHO-THF to wild type at and a in The mitochondrial 5-CHO-THF from a of of mitochondrial folate in the wild type to a of in the mutant. The mitochondrial of and not in the that of (which from in mitochondria, J.E. Biochemistry. 2002; PubMed Scopus Google Scholar). is that no was in any of the mutant mitochondrial and are the for glycine → serine conversion (Fig. this that the for SHMT is probably very high in leaf mitochondria of the mutant. SHMT in 5-CHO-THF buildup in mutant with to mitochondrial SHMT activities to for a in the mutant. such was SHMT activities in of wild type and mutant mitochondria were ± and ± of ± Acids in to CO2 establish the mitochondrial of 5-CHO-THF inhibits SHMT in we the glycine and serine of the leaves of wild type and mutant plants for 5 days to or μmol of CO2 (Fig. CO2 wild type leaves in glycine mutant leaves much larger that the glycine of mutant leaves was higher than the wild type at 370 μmol of CO2 and 46-fold higher at μmol was also a of serine in the mutant to the wild type at 370 μmol of CO2 mol–1, at μmol mol–1, both at such as and can as in the formation of glycine from in the photorespiratory Plant Physiol. 2003; PubMed Scopus Google Scholar). The levels of and also were substantially in the mutant with wild type at μmol of CO2 but not at the other CO2 of and in leaves to μmol of CO2 dry ± ± ± ± mutant and wild type that are at ± ± mutant and wild type that are at mutant and wild type that are at in a new of 5-CHO-THF on and 5-CHO-THF is up by Arabidopsis and mitochondria V. H. J. Plant Physiol. PubMed Scopus Google Scholar), we examined the effect of 5-CHO-THF via the to wild type and mutant plants. Leaf glycine and serine levels were of (Fig. with in vivo inhibition of SHMT, 5-CHO-THF raised glycine levels in wild type and mutant plants The in glycine was larger in the mutant μmol dry data that 5-CHO-THF can inhibit the activity of mitochondrial SHMT in vivo and for the that 5-CHO-THF one-carbon (5Stover P. Schirch V. Trends Biochem. Sci. 1993; 18: 102-106Abstract Full Text PDF PubMed Scopus (111) Google Scholar). its is for or this and it (8Baggott J.E. MacKenzie R.E. Biochem. J. 2003; 374: 773-778Crossref PubMed Google Scholar). the most of is that 5-FCL and the 5-CHO-THF buildup in mitochondria had impact on plant this impact to be all to leaves (4Roje S. Janave M.T. Ziemak M.J. Hanson A.D. J. Biol. Chem. 2002; 277: 42748-42754Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). therefore that plants are tolerant of 5-CHO-THF, as by of high levels of this folate in leaf mitochondria (10Chen L. Chan S.Y. Cossins E.A. Plant Physiol. 1997; 115: 299-309Crossref PubMed Scopus (121) Google Scholar, 11Chan S.Y. Cossins E.A. Pteridines. 2003; 14: 67-76Crossref Scopus (5) Google Scholar). could to this The may be the of an glycine pool to inhibition of SHMT by 5-CHO-THF. a of A. B. E. Neuburger M. R. Plant Physiol. PubMed Google Scholar), the of 5-CHO-THF in leaf mitochondria can be from the data in 4 to be in the wild type and in the mutant. Because 5-CHO-THF levels as low as mitochondrial SHMT activity in vitro (4Roje S. Janave M.T. Ziemak M.J. Hanson A.D. J. Biol. Chem. 2002; 277: 42748-42754Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar), the levels in the wild type be to be inhibitory and in the mutant even more In with this the 5-FCL mutant accumulated more glycine than the wild type. The higher glycine level for the SHMT activity and a flux through the glycine → serine reaction of had it not the 5-FCL mutation have in ambient air, as are glycine or mitochondrial SHMT Biochem. J. PubMed Scopus Google Scholar, Plant Physiol. PubMed Google Scholar). were to the that SHMT activity in the 5-FCL mutant. Another of to 5-CHO-THF may be its in that the of leaves is and that mitochondrial is of the S. S. B. R. Rébeillé F. Plant Physiol. 2003; PubMed Scopus Google Scholar), it can be from the data of and 4 that the mitochondria of the 5-CHO-THF in wild type leaves and in mutant leaves. The for of 5-CHO-THF may be that it is as are the of mitochondrial V. Rébeillé F. Neuburger M. R. Cossins E.A. Biochem. J. 1993; PubMed Scopus Google Scholar), and that mitochondrial folate as do most other folate D.W. Holloway R.S. J. Nutr. PubMed Scopus Google Scholar). much of the 5-CHO-THF mitochondria its to with cytosolic or of folate However, this is only a plant mitochondria various folate-dependent enzymes A.D. Trends Plant Sci. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, A.D. Plant Biol. 2002; PubMed Scopus Google Scholar) that, in other are to 5-CHO-THF P. Schirch V. Biochemistry. 31: PubMed Scopus Google Scholar). These and and of 5,10-CH=THF and the enzyme is therefore to the that of plant to 5-CHO-THF is either of mitochondrial enzymes to this or metabolic in the form of flux from mitochondrial to the for flux from the of in mutant plants. This have mitochondria as the mitochondrial pool not the pool of with mitochondrial one-carbon and then fluxes through and 5,10-CH=THF could not be distinguished it that in vivo the chemical the strongly at H. L. J. Biol. Chem. Full Text PDF PubMed Google Scholar). could be in at to the of mitochondrial folate-dependent SHMT and glycine up to of mitochondrial J.M. S. Bourguignon J. E. R. Rébeillé F. Plant J. PubMed Google Scholar). At such high it can be from the data of 4 that even in mutant mitochondria, of 5-CHO-THF of folate probably 5-CHO-THF Furthermore, of 5-CHO-THF to SHMT and glycine may the inhibition of other folate-dependent enzymes in Because 5-CHO-THF is stable and 5-FCL is the only enzyme known to metabolize the of SHMT in a 5-FCL mutant in the be to much of the folate pool to 5-CHO-THF, under photorespiratory conditions. the leaf 5-CHO-THF pool no more than a of of that may be another, to metabolize 5-CHO-THF, or in effect to and that this to The may of the 5-CHO-THF level only 4-fold in a 5-FCL (2Holmes W.B. Appling D.R. J. Biol. Chem. 2002; 277: 20205-20213Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar), and of bacteria that lack 5-FCL (17von Mering C. Huynen M. Jaeggi D. Schmidt S. Bork P. Snel B. Nucleic Acids Res. 2003; 31: 258-261Crossref PubMed Scopus (1419) Google Scholar, 18Enault F. Suhre K. Poirot O. Abergel C. Claverie J.M. Nucleic Acids Res. 2004; 32: W336-W339Crossref PubMed Scopus (26) Google Scholar). the 5-FCL reaction, which 5-CHO-THF as an any to of the or the (2Holmes W.B. Appling D.R. J. Biol. Chem. 2002; 277: 20205-20213Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, J.M. and Scholar). In of the in a of a in the leaves of 5-FCL mutant plants. we no that 5-FCL as a folate-degrading enzyme in as for the mammalian enzyme (19Anguera M.C. Suh J.R. Ghandour H. Nasrallah I.M. Selhub J. Stover P.J. J. Biol. Chem. 2003; 278: 29856-29862Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). folate in the 5-FCL it only under photorespiratory conditions, suggesting that the was not to the mutation but to its metabolic and for