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Iron-sulfur (Fe/S) cluster enzymes are crucial to life. Their assembly requires a suite of proteins, some of which are specific for particular subsets of Fe/S enzymes. One such protein is yeast Iba57p, which aconitase and certain radical S-adenosylmethionine enzymes require for activity. Iba57p homologs occur in all domains of life; they belong to the COG0354 protein family and are structurally similar to various folate-dependent enzymes. We therefore investigated the possible relationship between folates and Fe/S cluster enzymes using the Escherichia coil Iba57p homolog, YgfZ. NMR analysis confirmed that purified YgfZ showed stereoselective folate binding. Inactivating ygfZ reduced the activities of the Fe/S tRNA modification enzyme MiaB and certain other Fe/S enzymes, although not aconitase. When successive steps in folate biosynthesis were ablated, [DELTA]folE (lacking pterins and folates) and [DELTA]folP (lacking folates) mutants mimicked the [DELTA]ygfZ mutant in having low MiaB activities, whereas [DELTA]folE [DELTA]thyA mutants supplemented with 5-formyltetrahydrofolate (lacking pterins and depleted in dihydrofolate) and [DELTA]gcvP [DELTA]glyA mutants (lacking one-carbon tetrahydrofolates) had intermediate MiaB activities. These data indicate that YgfZ requires a folate, most probably tetrahydrofolate. Importantly, the [DELTA]ygfZ mutant was hypersensitive to oxidative stress and grew poorly on minimal media. COG0354 genes of bacterial, archaeal, fungal, protistan, animal, or plant origin complemented one or both of these growth phenotypes as well as the MiaB activity phenotype. Comparative genomic analysis indicated widespread functional associations between COG0354 proteins and Fe/S cluster metabolism. Thus COG0354 proteins have an ancient, conserved, folate-dependent function in the activity of certain Fe/S cluster enzymes. comparative genornics | oxidative stress | YgfZ protein | Iba57 | COG0354 doi/ 10.1073/pnas.0911586107

Tetrahydromonapteriu is a major pterin in Escherichia coli and is hypothesized to be the cofactor for phenylalanine hydroxylase (PhhA) in Pseudomonas aeruginosa, but neither its biosynthetic origin nor its cofactor role has been clearly demonstrated. A comparative genomics analysis implicated the enigmaticfo/X and folM genes in tetrahydromonapterin synthesis via their phyletic distribution and chromosomal clustering patterns, folX encodes dihydroneopterin triphosphate epimerase, which interconverts dihydroneopterin triphosphate and dihydromonapterin tripbosphate, folM encodes an unusual short-chain dehydrogenase/ reductase known to have dihydrofolate and dihydrobiopterin reductase activity. The roles of FolX and FolM were tested experimentally first in E. coli, which lacks PhhA and in which the expression of P. aeruginosa PhhA plus the recycling enzyme pterin 4a-carbinolamine dehydratase, PhhB, rescues tyrosine auxotrophy. This rescue was abrogated by deletingfo/X orfoiM and restored by expressing the deleted gene from a plasmid. The folX deletion selectively eliminated tetrahydromonapterin production, which far exceeded folate production. Purified FolM showed high, NADPH-dependent dihydromonapterin reductase activity. These results were substantiated in P. aeruginosa by deleting tyrA (making PhhA the sole source of tyrosine) and folX. The [DELTA]tyrA strain was, as expected, prototrophic for tyrosine, whereas the [DELTA]tyrA [DELTA]folX strain was auxotrophic. As in E. coli, the foiX deletant lacked tetrahydromonapterin. Collectively, these data establish that tetrahydromonapterin formation requires both FolX and FolM, that tetrahydromonapterin is the physiological cofactor for PhhA, and that tetrahydromonapterin can outrank folate as an end product of pterin biosynthesis. doi: 10.1128/JB.01198-09

Glycine undergoes decarboxylation in the glycine cleavage system (GCS) to yield C[O.sub.2], N[H.sub.3], and a 1-carbon unit. C[O.sub.2] also can be generated from the 2-carbon of glycine by 10-formyltetrahydrofolate-dehydrogenase and, after glycine-to-serine conversion by serine hydroxymethyltransferase, from the tricarboxylic acid cycle. To evaluate the relative fates of glycine carbons in C[O.sub.2] generation in healthy volunteers (3 male, 3 female, aged 21-26 y), primed, constant infusions were conducted using 9.26 [micro]mol x [h.sup.-1] x [kg.sup.-1] of [1,2-[sup.13]C]glycine and 1.87 [micro]mol x [h.sup.-1] x [kg.sup.-1] of [5,5,5-[sup.2][H.sub.3]]leucine, followed by an infusion protocol using [1-[sup.13]C]glycine as the glycine tracer. The time period between the infusion protocols was >6 mo. In vivo rates of whole-body glycine and leucine flux were nearly identical in protocols with [1,2-[sup.13]C]glycine and [5,5,5-[sup.2][H.sub.3]]leucine and with [1-[sup.13]C]glycine and [5,5,5-[sup.2][H.sub.3]]leucine tracers, which showed high reproducibility between the tracer protocols. Using the [1-[sup.13]C]glycine tracer, breath C[O.sub.2] data showed a total rate of glycine decarboxylation of 96 [+ or -] 8 [micro]mol x [h.sup.-1] x [kg.sup.-1], which was 22 [+ or -] 3% of whole-body glycine flux. In contrast, infusion of [1,2-[sup.13]C]glycine yielded a glycine-to-C[O.sub.2] flux of 146 [+ or -] 37 [micro]mol x [h.sup.-1] x [kg.sup.-1] (P = 0.026). By difference, this implies a rate of C[O.sub.2] formation from the glycine 2-carbon of 51 [+ or -] 40 [micro]mol x. [h.sup.-1] x [kg.sup.-1], which accounts for ~35% of the total C[O.sub.2] generated in glycine catabolism. These findings also indicate that ~65% of the C[O.sub.2] generation from glycine occurs by decarboxylation, primarily from the GCS. Further, these results suggest that the GCS is responsible for the entry of 5,10-methylenetetrahydrofolate into 1-carbon metabolism at a very high rate (~96 [micro]mol x [h.sup.-1 x [kg.sup.-1]), which is ~20 times the demand for methyl groups for homocysteine remethylation.

Glycine is a precursor of purines, protein, glutathione, and 1-carbon units as 5,10-methylenetetrahydrofolate. Glycine decarboxylation through the glycine cleavage system (GCS) and glycine-serine transformation by serine hydroxymethyl- transferase (SHMT) require pyridoxal 5'-phosphate (PLP; active form of vitamin B-6) as a coenzyme. The intake of vitamin B-6 is frequently low in humans. Therefore, we determined the effects of vitamin B-6 restriction on whole-body glycine flux, the rate of glycine decarboxylation, glycine-to-serine conversion, use of glycine carbons in nucleoside synthesis, and other aspects of 1-carbon metabolism We used a primed, constant infusion of [1,2-[sup.13][C.sub.2]glycine and [5,5,5- [sup.2][H.sub.3]leucine to quantify in vivo kinetics in healthy adults (7 males, 6 females; 20-39 y) of normal vitamin B-6 status of marginal vitamin B-6 deficiency. Vitamin B-6 restriction lowered the plasma PLP concentration from 55 [+ or -] 4 nmol/L (mean [+ or -] SEM) to 23 [+ or -] 1 nmol/L (P < 0.0001), which is consistent with marginal deficiency, whereas the plasma glycine concentration increased (P< 0.01 ). SHMT-mediated conversion of glycine to serine increased from 182 [+ or -] 7 to 205 [+ or -] 9 [micro]mol x [kg.sup.-1] x [h.sup.-1] (P < 0.05), but serine production using a GCS-derived 1-carbon unit (93 [+ or -] 9 vs. 91 [+ or -] 6 [micro]mol x [kg.sup.-1] x [h.sup.-1]) and glycine cleavage (163 [+ or -] 11 vs. 151 [+ or -] 8 [micro]mol x [kg.sup.-1] x [h.sup.-1]) were not changed by vitamin B-6 restriction. The GCS produced 1- carbon units at a rate (~140-170 [micro]mol x [kg.sup.-1] x [h.sup.-1]) that greatly exceeds the demand for remethylation and transmethylation processes (~4-7 [micro]mol x [kg.sup.-1] x [h.sup.-1]). We conclude that the in vivo GCS and SHMT reactions are quite resilient to the effects of marginal vitamin B-6 deficiency, presumably through a compensatory effect of increasing substrate concentration.

Glycine plays several roles in human metabolism, e.g. as a 1-carbon donor, in purine synthesis, and as a component of glutathione. Glycine is decarboxylated via the glycine cleavage system (GCS) that yields concurrent generation of a 1 -carbon unit as 5,10-methylenetetrahydrofolate (methyleneTHF). Serine hydroxymethyltransferase (SHMT) catalyzes the interconversion of glycine and serine, another 1-carbon donor. The quantitative role of glycine in human 1-carbon metabolism has received little attention. The aim of this protocol was to quantify whole body glycine flux, glycine to serine flux, and rate of glycine cleavage in humans. A primed, constant infusion with 9.26 [micro] mol.[kg.sup.-1] [h.sup.-1] [[1,2-.sup.13] [C.sub.2] ]glycine and 1.87 [micro] mol.[kg.sup.- 1] [h.sup.-1] [[2.sup.H.sub.3]]leucine was used to quantify the kinetic behavior of glycine in young, healthy volunteers (n = 5) in a fed state. The isotopic enrichment of infused tracers and metabolic products in plasma, as well as breath [13.sup.C][O.sub.2] enrichment, were determined for use in kinetic analysis. Serine synthesis by direct conversion from glycine via SHMT occurred at 193 [+ or -] 28 [micro] mol.[kg.sup.-1]. [h.sup.-1] (mean [+ or -] SEM), which comprised 41% of the 463 [+ or -] 55 [micro] mol.[kg.-1] [h.sup.-1] total glycine flux. Nearly one-half (46%) of the glycine-to-serine conversion occurred using GCS-derived methyleneTHF 1-carbon units. Based on breath [[13.sup.C][O.sub.2] measurement, glycine decarboxylation (190 [+ or -] 41 [micro] mol.[kg.sub.-1] [h.sup.-1]) accounted for 39 [+ or -] 6% of whole body glycine flux. This study is the first to our knowledge to quantify human glycine cleavage and glycine-to-serine SHMT kinetics. GCS is responsible for a substantial proportion of whole body glycine flux and constitutes a major route for the generation of 1-carbon units. J. Nutr. 137: 2647-2652, 2007.

Folate deficiency leads to neural tube defects and other human diseases, and is a global health problem. Because plants are major folate sources for humans, we have sought to enhance plant folate levels (biofortification). Folates are synthesized from pteridine, p-aminobenzoate (PABA), and glutamate precursors. Previously, we increased pteridine production in tomato fruit up to 140-fold by overexpressing GTP cyclohydrolase I, the first enzyme of pteridine synthesis. This strategy increased folate levels 2-fold, but engineered fruit were PABA-depleted. We report here the engineering of fruit-specific overexpression of aminodeoxychorismate sYnthase, which catalyzes the first step of PABA synthesis. The resulting fruit contained an average of 19-fold more PABA than controls. When transgenic PABA- and pteridine-overproduction traits were combined by crossing, vine-ripened fruit accumulated up to 25-fold more folate than controls. Folate accumulation was almost as high (up to 15-fold) in fruit harvested green and ripened by ethylene-gassing, as occurs in commerce. The accumulated folates showed normal proportions of one-carbon forms, with 5-methyltetrahydrofolate the most abundant, but were less extensively polyglutamylated than controls. Folate concentrations in developing fruit did not change in controls, but increased continuously throughout ripening in transgenic fruit. Pteridine and PABA levels in transgenic fruit were >20-fold higher than in controls, but the pathway intermediates dihydropteroate and dihydrofolate did not accumulate, pointing to a flux constraint at the dihydropteroate synthesis step. The folate levels we achieved provide the complete adult daily requirement in less than one standard serving. metabolic engineering | vitamin

Impaired folate-mediated 1-carbon metabolism has been linked to multiple disease outcomes. A better understanding of the nutritional and genetic influences on this complex biochemical pathway is needed to comprehend their impact on human health. To this end, we created a mathematical model of folate-mediated 1-carbon metabolism. The model uses published data on folate enzyme kinetics and regulatory mechanisms to simulate the impact of genetic and nutritional variation on critical aspects of the pathway. We found that the model predictions match experimental data, while providing novel insights into pathway kinetics. Our primary observations were as follows: 1) the inverse association between folate and homocysteine is strongest at very low folate concentrations, but there is no association at high folate concentrations; 2) the DNA methylation reaction rate is relatively insensitive to changes in folate pool size; and 3) as folate concentrations become very high, enzyme velocities decrease. With regard to polymorphisms in 5,10-methylenetetrahydrofolate reductase (MTHFR), the modeling predicts that decrease MTHFR activity reduces concentrations of S-adenosylmethionine and 5-methyltetrahydrofolate, as well as DNA methylation, while modestly increasing S-adenosylhomocysteine and homocysteine concentrations and thymidine or purine synthesis. Decreased folate together with a simulated vitamin B-12 deficiency results in decreases in DNA methylation and purine and thymidine synthesis. Decreased MTHFR activity superimposed on the B-12 deficiency appears to reverse the declines in purine and thymidine synthesis. These mathematical simulations of folate-mediated 1-carbon metabolism provide a cost-efficient approach to in silico experimentation that can complement and help guide laboratory studies.

The transsulfuration pathway, which aids in regulating homocysteine concentration and mediates cysteine synthesis, may be sensitive to vitamin B-6 status because cystathionine [beta]-synthase (CBS) and cystathionine [gamma]-lyase (CGL) require pyridoxal 5'-phosphate (PLP). To assess relations between vitamin B-6 and transsulfuration, we evaluated the effects of dietary pyridoxine (PN) on the hepatic concentration of relevant metabolites and in vitro activity of CBS and CGL. Growing rats were fed AIN-93G- or AIN-76A-based diets that ranged from adequate to deficient in vitamin B-6 (2, 1, 0.5, 0.1, or 0 mg of PN/kg diet, n = 5). This design allowed assessment of the effects of supplemental methionine (AIN-76A) vs. cysteine (AIN-93G) in common research diets over a range of vitamin B-6 levels. CBS activity, assayed in the presence or absence of added S-adenosylmethionine, was independent of diet type and PN level. CGL activity was independent of diet type but proportional to dietary PN. Rats fed deficient (0 and 0.1 mg PN/kg) diets exhibited only ~30% of the CGL activity of those fed the 2 mg PN/kg diets. Hepatic cystathionine increased from 20 to 30 nmol/g for the 1-2 mg PN/kg diets to ~85 nmol/g for the 0 mg PN/kg diet; however, cysteine was reduced only in B-6--deficient rats consuming the AIN-93G diet (means of 30-40 nmol/g for adequate to 11.6 nmol/g for 0 mg PN/kg AIN-76A diet). In spite of these effects, hepatic glutathione concentration increased in vitamin B-6 deficiency. These results suggest that vitamin B-6-dependent changes in transsulfuration do not limit hepatic glutathione production.

Cysteine synthesis from homocysteine is catalyzed by two pyridoxal 5'-phosphate (PLP)-dependent enzymes. This suggests that vitamin B-6 status might affect cysteine and glutathione homeostasis, but it is unclear whether this occurs in humans. We assessed the effects of vitamin B-6 status on static and kinetic parameters of cysteine and glutathione metabolism in healthy female (n = 5) and male (n = 4) volunteers (20-30 y) before and after 4 wk of dietary vitamin B-6 restriction ( KEY WORDS: * cysteine * glutathione * human * transsulfuration * vitamin B-6

The effects of folate status and the methylenetetrahydrofolate reductase (MTHFR) 677C[right arrow]T polymorphism on the kinetics of homocysteine metabolism are unclear. We measured the effects of dietary folate restriction on the kinetics of homocysteine remethylation and synthesis in healthy women (20-30 y old) with the MTHFR 677 C/C or T/T genotypes (n = 9/genotype) using i.v. primed, constant infusions of [[sup.13][C.sub.5]]methionine, [3-[sup.13]C]serine, and [[sup.2][H.sub.3]]leucine before and after 7 wk of dietary folate restriction (115 [micro]g dietary folate equivalents/ d). Dietary folate restriction significantly reduced folate status (~65% reduction in serum folate) in both genotypes. Total remethylation flux was not affected by dietary folate restriction, the MTHFR 677C[right arrow]T polymorphism, or their combination. However, the percentage of remethylation from serine was reduced ~15% (P = 0.031) by folate restriction in C/C subjects. Further, homocysteine synthesis rates of T/T subjects and folate-restricted C/C subjects were twice that of C/C subjects at baseline. In conclusion, elevated homocysteine synthesis is a cause of mild hyperhomocysteinemia in women with marginal folate status, particularly those with the MTHFR 677 T/T genotype. KEY WORDS: * methionine * one-carbon metabolism * S-adenosylmethionine * S-adenosylhomocysteine * women

Whether folate status and the methylenetetrahydrofolate reductase (MTHFR) 677C[right arrow]T polymorphism interact to affect methionine-cycle metabolite concentrations is uncertain. We evaluated the effects of dietary folate restriction on relations among folate status indices and plasma concentrations of methionine cycle metabolites in women with the MTHFR 677 C/C and T/T genotypes. Healthy, normohomocysteinemic women (n = 18; 20-30 y old) of adequate B vitamin status, and equally divided according to MTHFR 677C[right arrow]T genotype (9 C/C and 9 T/T) were recruited. Folate status indices and methionine cycle metabolites were measured in blood samples collected at baseline and after 7 wk of dietary folate restriction (115 [micro]g dietary folate equivalents/d). Significant negative correlations between plasma total homocysteine concentrations and total or 5-methyl folate concentrations (P = 0.041 and 0.023, respectively) in RBCs were found only in T/T subjects. Formylated folates were detected in RBCs of T/T subjects only, and their abundance was predictive of plasma total homocysteine concentration despite no significant alteration by folate restriction. Plasma concentrations of S-adenosylmethionine and S-adenosylhomocysteine were not significantly affected by dietary folate restriction and the MTHFR 677 T/T genotype. In conclusion, plasma total homocysteine concentrations in subjects with the MTHFR 677 T/T genotype were inversely related to 5-methyl folate concentrations and directly related to formylated folate concentrations in RBCs, even though the latter were not significantly affected by moderate folate restriction. KEY WORDS: * homocysteine * methionine * S-adenosylmethionine * S-adenosylhomocysteine * women

The methylenetetrahydrofolate reductase (MTHFR) 677C [right arrow] T polymorphism is thought to influence the partitioning of 1-carbon units between methylation and other components of 1-carbon metabolism and to influence the risk and etiology of several major cancers and cardiovascular disease. Our objective was to determine the effect of the MTHFR 677C [right arrow] T polymorphism and folate status on the relative rate and extent of in vivo synthesis of DNA precursors. Adequately nourished, healthy women (9 CC, 9 TT) were infused with [3-[sup.13C]]serine and [[sup.13][C.sub.5]]methionine for 9 h before and after 7 wk of consumption of a low-folate diet. Blood was drawn over 5 d for monocyte DNA isolation. Isotopic enrichment of the nucleosides in DNA digests was determined by LC-MS/MS. Maximum thymidine enrichment tended to be higher (P = 0.07) in TT than in CC subjects, suggestive of marginally higher mean thymidylate synthesis. However, the subset of TT subjects who exhibited formyltetrahydrofolate in erythrocytes (an indicator of 1-carbon partitioning) had greater (P = 0.036) thymidine enrichment than CC subjects, who had no erythrocyte formyltetrahydrofolate. Purine enrichment was not affected by genotype or folate depletion. However, the deoxyadenosine to deoxyguanosine enrichment ratio was significantly higher in TT subjects, suggesting a greater relative rate of adenine synthesis. The ~40% greater (P = 0.012) labeling of the methyl group of methyldeoxycytidine during folate depletion suggests a change in the origin of this 1-carbon unit. This is the first time that 1-carbon incorporation into human DNA has been measured in vivo after infusion of [sup.13C]-labeled 1-carbon precursors. These findings support the feasibility of further assessment of factors affecting deoxynucleotide synthesis and DNA methylation in human 1-carbon metabolism. KEY WORDS: * thymidine * methyldeoxycytidine * deoxypurine * LC-MS/MS * isotope

Serine hydroxymethyltransferase (SHMT) is a pyridoxal phosphate (PLP)-dependent enzyme that exists as cytosolic and mitochondrial isozymes that catalyze the reversible interconversion of serine and tetrahydrofolate (THF) to glycine and 5,10-methyleneTHF. SHMT is a major source of one-carbon units for cellular metabolism, but its sensitivity to various degrees of altered vitamin B-6 nutritional status has not been determined. In this study, cytosolic and mitochondrial SHMT activities were measured in liver from rats fed dietary pyridoxine (PN) ranging from adequate to deficient levels (2, 1, 0.5, 0.1, and 0 mg PN/kg diet; n = 10 per group). Both mitochondrial and cytosolic SHMT activities increased (P < 0.001) with increasing dietary PN over this range, and activities were a linear function of liver PLP concentration. Mitochondrial SHMT comprised ~70% of total activity. Assays conducted with and without in vitro addition of PLP indicated that total SHMT (apo- and holoenzyme forms) varied with dietary PN for each isoform, but that the proportion of each present as the apoenzyme was not affected by PN intake. This aspect of SHMT nutritional regulation differs from that of many other PLP-dependent enzymes. Hepatic glycine concentration was inversely related to vitamin B-6 intake (P < 0.05), which suggests a functional effect of altered SHMT activity. Overall these results demonstrate the potential for disruption of SHMT-mediated one-carbon metabolism by inadequate vitamin B-6 intake. KEY WORDS: * one-carbon metabolism * rat * serine hydroxymethyltransferase * vitamin B-6

Methylenetetrahydrofolate reductase (MTHFR) and methionine synthase reductase (MTRR) are important for homocysteine remethylation. This study was designed to determine the influence of genetic variants (MTHFR 677C[right arrow],T, MTHFR 1298A[right arrow]C, and MTRR 66A[right arrow]G), folate, and vitamin B-12 status on plasma homocysteine in women (20-30 y; n = 362). Plasma homocysteine was inversely (P < 0.0001) associated with serum folate and plasma vitamin B-12 regardless of genotype. Plasma homocysteine was higher (P < 0.05) for women with the MTHFR 677 TT/1298 AA genotype combination compared with the CC/AA, CC/AC, and CT/AA genotypes. Women with the MTHFR 677 TT/MTRR 66 AG genotype had higher (P < 0.05) plasma homocysteine than all other genotype combinations except the TT/AA and TT/GG genotypes. There were 5.4-, 4.3-, and 3.8-fold increases (P < 0.001) in risk for plasma homocysteine in the top 5, 10, and 20%, respectively, of the homocysteine distribution for subjects with the MTHFR 677 TT compared with the CC and CT genotypes. Predicted plasma homocysteine was inversely associated with serum folate (P = 0.003) and plasma vitamin B-12 (P = 0.002), with the degree of correlation dependent on MTHFR 677C[right arrow]T genotype. These data suggest that coexistence of the MTHFR 677 TT genotype with the MTRR 66A[right arrow]G polymorphism may exacerbate the effect of the MTHFR variant alone. The potential negative effect of combined polymorphisms of the MTHFR and MTRR genes on plasma homocysteine in at-risk population groups with low folate and/or vitamin B-12 status, such as women of reproductive potential, deserves further investigation. KEY WORDS: * folate * genetic polymorphisms * homocysteine * vitamin B-12

Plants are the main source of folate in human diets, but many fruits, tubers, and seeds are poor in this vitamin, and folate deficiency is a worldwide problem. Plants synthesize folate from pteridine, p-aminobenzoate (PABA), and glutamate moieties. Pteridine synthesis capacity is known to drop in ripening tomato fruit; therefore, we countered this decline by fruit-specific overexpression of GTP cyclohydrolase I, the first enzyme of pteridine synthesis. We used a synthetic gene based on mammalian GTP cyclohydrolase I, because this enzyme is predicted to escape feedback control in planta. This engineering maneuver raised fruit pteridine content by 3- to 140-fold and fruit folate content by an average of 2-fold among 12 independent transformants, relative to vector-alone controls. Most of the folate increase was contributed by 5-methyl-tetrahydrofolate polyglutamates and 5,10-methenyltetrahydrofolate polyglutamates, which were also major forms of folate in control fruit. The accumulated pteridines included neopterin, monapterin, and hydroxyrnethylpterin; their reduced forms, which are folate biosynthesis intermediates; and pteridine glycosides not previously found in plants. Engineered fruit with intermediate levels of pteridine overproduction attained the highest folate levels. PABA pools were severely depleted in engineered fruit that were high in folate, and supplying such fruit with PABA by means of the fruit stalk increased their folate content by up to 10-fold. These results demonstrate that engineering a moderate increase in pteridine production can significantly enhance the folate content in food plants and that boosting the PABA supply can produce further gains.

Townsend, Justin H., Steven R. Davis, Amy D. Mackey, and Jesse F. Gregory III. Folate deprivation reduces homocysteine remethylation in a human intestinal epithelial cell culture model: role of serine in one-carbon donation. Am J Physiol Gastrointest Liver Physiol 286: G588-G595, 2004. First published November 13, 2003; 10.1152/ajpgi.00454.2003.--Little is known about homocysteine metabolism in intestine. To address this question, we investigated homocysteine metabolism under conditions of folate adequacy and folate deprivation in the Caco-2 cell line, a model of human intestinal mucosal cells. Caco-2 cells were cultured in media enriched with [[3-.sup.13]C]serine and [[U-.sup.13][C.sub.5]]methionine tracers, and enrichments of intracellular tree amino acid pools of these amino acids as well as homocysteine, cystathionine, and cysteine were measured by using gas chromatography/mass spectrometry. Homocysteine transsulfuration plus folate-dependent and total remethylation were quantified from these amino acid enrichments. Homocysteine remethylation accounted for 19% of the intracellular free methionine pool in cells cultured with supplemental folate, and nearly all one-carbon units used for remethylation originated from the three carbon of serine via folate-dependent remethylation. Labeling of cystathionine and cysteine indicated the presence of a complete transsulfuration pathway in Caco-2 cells, and this pathway produced 13% of the intracellular free cysteine pool. Appearance of labeled homocysteine and cystathionine in culture medium suggests export of these metabolites from intestinal cells. Remethylation was reduced by one-third in folate-restricted cell cultures (P < 0.001), and only ~50% of the one-carbon units used for remethylation originated from the three carbon of serine under these conditions. In conclusion, the three carbon of serine is the primary source of one-carbon units used for homocysteine remethylation in folate-supplemented Caco-2 cell cultures. Remethylation is reduced as a result of folate restriction in this mucosal cell model, and one-carbon sources other than the three carbon of serine contribute to remethylation under this condition. methionine; methylation; Caco-2; transsulfuration; serine

An important dietary source of vitamin B-6, pyridoxine-5'-[beta]-D-glucoside (PNG), exhibits only partial bioavailability, which is limited by the extent of enzymatic cleavage of the [beta]-glucosidic bond to release metabolically available pyridoxine (PN). This laboratory showed that the intestinal hydrolysis of PNG is catalyzed by cytosolic PNG hydrolase (PNGH) and brush border lactase-phlorizin hydrolase (LPH). LPH-catalyzed PNG hydrolysis in vitro is competitively inhibited by lactose. In the present study, the uptake and hydrolysis of PNG were examined in Caco-2 human colon carcinoma cells, which express a functional LPH but exhibit no PNGH activity. PNG uptake at 37[degrees]C was linear over 5-500 [micro]mol/L PNG. Uptake was not significantly reduced when [Na.sup.+] was substituted with [K.sup.+], [Li.sup.+], or Tris in the medium. Increasing PNG concentration in the medium did not change intracellular concentrations of PN, pyridoxamine (PM), pyridoxamine 5'-phosphate (PMP), or pyridoxal 5'-phosphate (PLP); however, intracellular pyridoxal (PL) concentration increased. Intracellular PNG concentration was not significantly reduced in the presence of lactose, but the concentration of PL declined in proportion to extracellular lactose (P = 0.01). These results indicate that PNG can be absorbed intact in a [Na.sup.+]-independent process and is taken up by passive diffusion. The presence of lactose in this in vitro model of intestinal uptake reduced the enzymatic hydrolysis of PNG by lactase. KEY WORDS: * pyridoxine-5'-[beta]-D-glucoside (PNG) * Caco-2 cells * bioavailability * lactase-phlorizin hydrolase

It is not known how plants synthesize the p-aminobenzoate (PABA) moiety of folates. In Escherichia coli, PABA is made from chorismate in two steps. First, the PabA and PabB proteins interact to catalyze transfer of the amide nitrogen of glutamine to chorismate, forming 4-amino-4-deoxychorismate (ADC). The PabC protein then mediates elimination of pyruvate and aromatization to give PABA. Fungi, actinomycetes, and Plasmodium spp. also synthesize PABA but have proteins comprising fused domains homologous to PabA and PabB. These bipartite proteins are commonly called 'PABA synthases,' although it is unclear whether they produce PABA of ADC. Genomic approaches identified Arabidopsis and tomato cDNAs encoding bipartite proteins containing fused PabA and PabB domains, plus a putative chloroplast targeting peptide. These cDNAs encode functional enzymes, as demonstrated by complementation of an E. coli pabA pabB double mutant and a yeast PABA-synthase deletant. The partially purified recombinant Arabidopsis protein did not produce PABA unless the E. coli PabC enzyme was added, indicating that it forms ADC, not PABA. The enzyme behaved as a monomer in size-exclusion chromatography and was not inhibited by physiological concentrations of PABA, its glucose ester, or folates. When the putative targeting peptide was fused to GFP and expressed in protoplasts, the fusion protein appeared only in chloroplasts, indicating that PABA synthesis is plastidial. In the pericarp of tomato fruit, the PabA-PabB mRNA level fell drastically as ripening advanced, but there was no fall in total PABA content, which stayed between 0.7 and 2.3 nmol-[g.sup.-1] fresh weight.

Hyperhomocysteinemia in humans is associated with genetic variants of several enzymes of folate and one-carbon metabolism and deficiencies of folate and vitamins [B.sub.12] and [B.sub.6]. In each case, hyperhomocysteinemia might be caused by diminished folate-dependent homocysteine remethylation, but this has not been confirmed in vivo. Because published stable isotopic tracer approaches cannot distinguish folate-dependent from folate-independent remethylation, we developed a dual-tracer procedure in which a [U-[sup.13][C.sub.5]]-methionine tracer is used in conjunction with a [3-[sup.13]C]serine tracer to simultaneously measure rates of total and folate-dependent homocysteine remethylation. In young female subjects, plasma [U-[sup.13][C.sub.4]]homocysteine enrichment, a surrogate measure of intracellular [U-[sup.13][C.sub.5]]methionine enrichment, reached ~90% of the plasma [U-[sup.13][C.sub.5]]methionine enrichment. Methionine-methyl and -carboxyl group fluxes were in the range of previous reports (~25 and ~17 [micro]mol*[kg.sup.-1]*[h.sup.-1], respectively). However, the rate of overall homocysteine remethylation (~8 [micro]mol*[kg.sup.-1]*[h.sup.-1]) was twice that of previous reports, which suggests a larger role for homocysteine remethylation in methionine metabolism than previously thought. By use of estimates of intracellular [3-[sup.13]C]serine enrichment based on a conservative correction of plasma [3-[sup.13]C]serine enrichment, serine was calculated to contribute ~100% of the methyl groups used for total body homocysteine remethylation under the conditions of this protocol. This contribution represented only a small fraction (~2.8%) of total serine flux. Our dual-tracer procedure is well suited to measure the effects of nutrient deficiencies, genetic polymorphisms, and other metabolic perturbations on homocysteine synthesis and total and folate-dependent homocysteine remethylation. methionine; methylation cycle; cystathionine

This study was designed to evaluate the effect of the methylenetetrahydrofolate reductase (MTHFR) 677C [right arrow] T polymorphism on folate and homocysteine response in nonHispanic women consuming a low folate diet followed by a diet providing the Recommended Dietary Allowance (RDA) for folate. Women (aged 20-30 y old) with either the TT (n = 19) or CC (n = 22) MTHFR 677C [right arrow] T genotype participated in a folate depletion-repletion study (7 wk, 115 [micro]g dietary folate equivalents (DFE)/d; 7 wk, 400 [micro]g DFE/d). Overall serum folate decreased (P < 0.0001) during depletion and increased (P < 0.0001) during repletion with lower (P = 0.03) postdepletion serum folate in women with the TT versus CC genotype. Folate status was low (serum folate < 13.6 nmol/L) in more women with the TT (59%) compared with the CC genotype (15%) postdepletion. Red blood cell folate for all subjects decreased during depletion (P < 0.0001) and repletion (P = 0.02) with lower (P = 0.04) red blood cell folate in women with the TT compared with the CC genotype postrepletion. Homocysteine increased (P < 0.0001) for both genotype groups postdepletion and decreased (P = 0.02) postrepletion for the CC genotype group only. Homocysteine concentrations tended to be higher (P = 0.09) in the TT versus CC genotype group postdepletion and postrepletion. These data suggest that the MTHFR 677C [right arrow] T polymorphism negatively affects the folate and homocysteine response in women consuming low folate diets followed by repletion with the RDA. These results may be important when evaluating the impact of the MTHFR 677C [right arrow] T polymorphism in countries in which low folate diets are chronically consumed. KEY WORDS: * folate * 5'10-methylenetetrahydrofolate reductase * genotype * young women

Biotin status and function are decreased during oral carbamazepine (CBZ) administration in both humans and rats, but it is not known whether biotin supplementation can prevent these decreases. To test the effectiveness of pharmacologic biotin supplementation during CBZ administration, 55 rats were randomly divided into 4 groups (0.06 mg biotin/kg diet [+ or -] 3.75 g CB7/kg diet and 6.0 mg biotin/kg diet [+ or -] 3.75 g CB7/kg diet). CBZ and biotin-supplemented diets began on d 5 and 26, respectively, and continued through d 68. Rats (n = 5/group) were killed on d 5, 26, 47 or 68. CBZ reduced serum and liver free biotin (P < 0.05), whereas biotin supplementation during CBZ administration maintained biotin status. CBZ also decreased specific activities and abundance of biotinylated pyruvate and acetyl CoA carboxylases (PC and ACC, P < 0.05) in brain and liver, whereas biotin supplementation prevented these decreases for ACC. Specific activity of PC was maintained upon biotin supplementation, but the abundance of biotinylated PC remained significantly decreased. Brain and serum lactate were elevated after 68 d of CBZ treatment and were reduced to control lactate concentrations upon biotin supplementation (P < 0.05). Conversion of lactate to pyruvate and simultaneous generation of NADH during biotin supplementation could explain how increases in PC activity occur without changes in the abundance of biotinylated PC because we found NADH to be an activator of PC activity in vitro. These results support the use of biotin supplementation as a concurrent strategy during CBZ administration to help maintain biotin status, function of biotin-dependent enzymes and decrease CBZ-induced lactate accumulation. KEY WORDS: * carbamazepine, biotin * rats * lactate * pyruvate carboxylase

Clinical data demonstrate that certain antiepileptic drugs including carbamazepine (CBZ) decrease serum biotin concentration 45-50% and increase urine and serum organic acids, which is suggestive of reduced function of biotin-dependent enzymes. However, little is known about biotin-dependent enzyme function at the tissue level in patients undergoing long-term CBZ treatment. We recently established that dietary CBZ administration to rats increases brain lactate and also decreases specific enzymatic activity and the relative abundance of hepatic biotinylated pyruvate carboxylase (PC). To examine the mechanism of altered activity and abundance of biotinylated PC, the effect of orally administered CBZ on hepatic PC protein and mRNA expression was examined in rats consuming a physiologically relevant level of dietary biotin (0.06 mg/kg). Rats were fed 0 or 3.4 g CBZ/kg diet for 28 d, a dose designed to achieve clinically relevant serum CBZ concentrations. Hepatic biotinylated PC and PC activity were significantly reduced by ~43 and 30%, respectively, in the drug-treated group. Liver PC protein expression and mRNA were ~43 and 35% lower, respectively, in the drug-treated group than in controls. Brain biotinylated PC was significantly lower (29%), whereas specific enzymatic activity was 175% higher in rats consuming the 3.4 g CBZ/kg diet. Brain, but not serum, lactate was significantly higher in rats consuming CBZ. Taken together, the lower PC protein and mRNA expression provide a plausible biochemical mechanism to explain the decreased abundance of biotinylated hepatic PC observed in previous studies. KEY WORDS: * carbamazepine * biotin * rats * antiepileptic drug * pyruvate carboxylase

Pyridoxine-5'-[beta]-D-glucoside (PNG), a glycosylated form of dietary vitamin B-6, is partially hydrolyzed in the small intestine by the cytosolic enzyme pyridoxine-5'-[beta]-D-glucoside hydrolase (PNG hydrolase) and by the brush border enzyme lactase phlorizin hydrolase (LPH) to release free pyridoxine (PN). This laboratory has previously shown that PNG hydrolase activity is inversely related to dietary vitamin B-6 in rats and guinea pigs. The current investigation was done to examine the effect of dietary PN on PNG hydrolytic activity and its distribution. Nutrient compositional differences between the AIN-76A and AIN-93G purified diets that were unrelated to vitamin B-6 were also examined in relation to PNG hydrolysis in rat small intestinal mucosa. Study one included rats (n = 29) that were fed the AIN-93G diet providing a range of PN concentrations for 5 wk. Rats (n = 49) in study two were fed either AIN-76A or AIN-93G each with graded concentrations of PN. In both studies, rat growth and plasma and liver pyridoxal 5'-phosphate (PLP) concentrations increased (P < 0.05) with increasing concentrations of dietary PN. PNG hydrolytic activity localized to the brush border membrane was five times that measured in the cytosol. Cytosolic PNG hydrolytic activity increased significantly with increasing dietary PN concentration in rats fed the AIN-76A, but not AIN-93G diet. Activity in the mucosal total membrane fraction did not increase in proportion to dietary PN concentration for either diet. Regardless of dietary PN concentration, the basal nutrient composition of the diets affected growth and PNG hydrolytic activity in intestinal mucosa. In contrast to previous results from this laboratory, intestinal hydrolytic activity toward PNG did not increase in vitamin B-6-deficient rats. KEY WORDS: * bioavailability * intestinal hydrolysis * pyridoxine-5'-[beta]-D-glucoside * vitamin B-6

GTP cyclohydrolase I (GCHI) mediates the first and committing step of the pterin branch of the folate-synthesis pathway. In microorganisms and mammals, GCHI is a homodecamer of [approximately equals]26-kDa subunits. Genomic approaches identified tomato and Arabidopsis cDNAs specifying [approximately equals]50-kDa proteins containing two GCHI-like domains in tandem and indicated that such bimodular proteins occur in other plants. Neither domain of these proteins has a full set of the residues involved in substrate binding and catalysis in other GCHIs. The tomato and Arabidopsis cDNAs nevertheless encode functional enzymes, as shown by complementation of a yeast fol2 mutant and by assaying GCHI activity in extracts of complemented yeast cells. Neither domain expressed separately had GCHI activity. Recombinant tomato GCHI formed dihydroneopterin triphosphate as reaction product, as do other GCHIs, but unlike these enzymes it did not show cooperative behavior and was inhibited by its substrate. Denaturing gel electrophoresis verified that the bimodular GCHI polypeptide is not cleaved in vivo into its component domains, and size-exclusion chromatography indicated that the active enzyme is a dimer. The deduced tomato and Arabidopsis GCHI polypeptides lack overt targeting sequences and thus are presumably cytosolic, in contrast to other plant folate-synthesis enzymes, which are mitochondrial proteins with typical signal peptides. GCHI mRNA and protein are strongly in expressed unripe tomato fruits, implying that fruit folate is made in situ rather than imported. As ripening advances, GCHI expression declines sharply, and folate content drops, suggesting that folate synthesis fails to keep pace with turnover.

Pyridoxine-5'-[beta]-D-glucoside (PNG) is a major form of vitamin B-6 in plant foods that exhibits partial bioavailability as vitamin B-6 in humans. We previously identified an intestinal mucosal cytosolic PNG hydrolase that catalyzes the partial hydrolysis of PNG absorbed without prior deglycosylation. Recent observations that the brush border membrane also catalyzes PNG hydrolysis led to the hypothesis that PNG hydrolysis may be another function of the [beta]-glucosidase lactase-phlorizin hydrolase (LPH) and, thus, brush border PNG hydrolysis would undergo a developmental decline similar to that of lactose hydrolysis. In this study, the relationships among hydrolytic activities in small intestinal cytosolic and brush border fractions in rats (n = 9 per group) of various ages (1-2 d and 2, 4, 8, 12 and 24 wk) were examined. In vitro specific activities toward PNG and lactose were greater in brush border than cytosol, and these were greater in newborn rats than in all other age groups (P < 0.01). Brush border activities toward PNG and lactose and were closely correlated (r = 0.84; P < 0.0001). These findings suggest that the hydrolysis of PNG is catalyzed at least partially at the brush border and that the bioavailability Of PNG may be influenced by the residual LPH activity in children and adults. KEY WORDS: * vitamin B-6. pyridoxine-5'-[beta]-D-glucoside * lactase-phlorizin hydrolase * rats * bioavailability

Folate catabolism represents the major route of folate turnover in humans and involves cleavage of the C9-N10 bond producing a pterin and para-aminobenzoylglutamate (pABG). Thus, the quantitation of pABG and its acetylated more predominant counterpart para-acetamidobenzolyglutamate (apABG) may be useful in assessing folate status and requirements. However, until the in vivo fate of dietary pABG is understood, studies using pABG excretion parameters can not be fully interpreted. As part of a larger study, an oral dose (376 nmol or 100 [micro]g) of [[sup.13][C.sub.5]]pABG in 40 mL apple juice was ingested by pregnant women (2nd trimester, n = 2) and nonpregnant controls (n = 2) consuming controlled total folate intakes of 450 or 850 [micro]g/d. Urine collections (24 h) were obtained over the next 4 d and gas chromatography-mass spectrometry was used to measure urinary [[sup.13][C.sub.5]]pABG, [[sup.13][C.sub.5]]apABG and [[sup.13][C.sub.5]]folate. Of the 376 nmol [[sup.13][C.sub.5]]pABG administered, only 17.5 [+ or -] 6.4 nmol; mean [+ or -] SEM) or 4.6 [+ or -] 1.7% of the dose was accounted for in the urine. Most of the excreted [[sup.13][C.sub.5]]pABG, in acetamido form (15.1 [+ or -] 5.3 nmol), was excreted the day after the dose. No urinary [[sup.13][C.sub.5]]folate was detected. Folate intake did not seem to influence the urinary excretion of total pABG derived from oral pABG, whereas pregnancy may lessen total pABG excretion derived from oral pABG. Overall, these results suggest that the contribution of dietary pABG to the urinary excretion of pABG and apABG is small. KEY WORDS: * folate * para-aminobenzoylglutamate * catabolism * stable isotope * humans

Methylenetetrahydrofolate reductase (MTHFR) polymorphisms may negatively influence one-carbon metabolism and increase health risks in women of reproductive age. The effect of MTHFR single nucleotide polymorphisms at bp 677 and/or 1298 and differences in folate and vitamin B-12 status on plasma homocysteine concentration in women of reproductive age (20-30 y; n = 186) were investigated. From the multivariate regression model, homozygotes (n = 23) for the C677T MTHFR variant had plasma homocysteine concentrations that were higher (P < 0.05) than those observed in the other 5 genotype groups, including those who were heterozygous for both variants (677CT/1298AC; n = 32). Plasma homocysteine was negatively associated with plasma vitamin B-12 concentration (P = 0.015) and serum folate (P = 0.049), with the degree of correlation between plasma vitamin B-12 and homocysteine concentrations dependent on MTHFR genotype. The C677T and A1298C MTHFR polymorphisms were significant predictors (P < 0.05) of plasma homocysteine when regression analysis was used to model plasma homocysteine concentration as a function of genotype, supplement use, serum folate and plasma vitamin B-12 concentration. Plasma homocysteine decreased as vitamin B-12 concentration increased (P = 0.0005) in individuals who were heterozygous for both the C677T and A1298C variants with nonsignificant trends (P = 0.114-0.128) in individuals homozygous for either the C677T or A1298C variants. In contrast, within the group of individuals with the wild-type genotype for both the C677T and A1298C MTHFR variants, homocysteine was not associated with changes in plasma vitamin B-12 concentrations. These data suggest that enhancing vitamin B-12 status may significantly decrease homocysteine in young women with C677T and/or A1298C MTHFR polymorphisms, even when vitamin B-12 concentrations are within the normal range. KEY WORDS: * vitamin B-12 * methylenetetrahydrofolate reductase * polymorphisms * homocysteine * folate * women

Cuskelly, Geraldine J., Peter W. Stacpoole, Jerry Williamson, Thomas G. Baumgartner, and Jesse F. Gregory III. Deficiencies of folate and vitamin [B.sub.6] exert distinct effects on homocysteine, serine, and methionine kinetics. Am J Physiol Endocrinol Metab 281: E1182-E1190, 2001.--Folate and vitamin [B.sub.6] act in generating methyl groups for homocysteine remethylation, but the kinetic effects of folate or vitamin [B.sub.6] deficiency are not known. We used an intravenous primed, constant infusion of stable isotope-labeled serine, methionine, and leucine to investigate one-carbon metabolism in healthy control (n = 5), folate-deficient (n = 4), and vitamin [B.sub.6]-deficient (n = 5) human subjects. The plasma homocysteine concentration in folate-deficient subjects [15.9 [+ or -] 2.1 (SD) [micro] molfl] was approximately two times that of control (7.4 [+ or -] 1.7 [micro] molfl) and vitamin B6-deficient (7.7 [+ or -] 2.1 [micro] mol/1) subjects. The rate of methionine synthesis by homocysteine remethylation was depressed (P = 0.027) in folate deficiency but not in vitamin B6 deficiency. For all subjects, the homocysteine remethylation rate was not significantly associated with plasma homocysteine concentration (r = -0.44, P = 0.12). The fractional synthesis rate of homocysteine from methionine was positively correlated with plasma homocysteine concentration (r = 0.60, P = 0.031), and a model incorporating both homocysteine re-methylation and synthesis rates closely predicted plasma homocysteine levels (r = 0.85, P = 0.0015). Rates of homocysteine remethylation and serine synthesis were inversely correlated (r = -0.89, P < 0.001). These studies demonstrate distinctly different metabolic consequences of vitamin [B.sub.6] and folate deficiencies. one-carbon metabolism; remethylation; stable isotopes; human; vitamin B6; folate Received 5 March 2001; accepted in final form 19 July 2001

A common genetic polymorphism results from a C[arrow right]T substitution in the gene encoding methylenetetrahydrofolate reductase (MTHFR), the enzyme that produces 5-methyltetrahydrofolate (5-methyl-THF) required for the conversion of homocysteine to methionine. In individuals with the T/T genotype (T/T), functional metabolic effects include changes in one-carbon folate derivatives, elevations in plasma homocysteine and differences in response to folic acid supplementation compared with normal (C/C) or heterozygous (C/T) genotypes. The metabolic changes associated with the T/T genotype are postulated to modify risk for chronic disease (e.g., vascular disease and cancer) and neural tube defects (NTD) when accompanied by folate deficiency. The modulation of these metabolic abnormalities by increasing folate intake suggests that folate requirements may be different in affected individuals (T/T) relative to normal (C/C) or heterozygous (C/T) individuals. The complex interaction between this common genetic polymorphism of MTHFR and folate intake is the focus of intense investigation. KEY WORDS: folate; MTHFR polymorphism; homocysteine; vascular disease; cancer; neural tube defects

Folate functions in multiple coenzyme forms in acceptance, redox processing and transfer of one-carbon units, including nucleotides and certain amino acids. Folate-requiring metabolic processes are influenced by folate intake, intake of other essential nutrients, including vitamins B-12 and B-6, and at least one common genetic polymorphism. Estimates of folate requirements have been based on intakes associated with maintenance of normal plasma and erythrocyte folate concentrations and functional tests that reflect abnormalities in folate-dependent reactions. Dietary Reference Intakes for folate that have been developed recently are based primarily on metabolic studies in which erythrocyte folate concentration was considered the major indicator of adequacy. For adults [greater than or equal to]19 y, the Recommended Dietary Allowance (RDA) is 400 [[micro]gram]/d of dietary folate equivalents (DFE); for lactating and pregnant women, the RDAs include an additional 100 and 200 [[micro]gram] of DFE/d, respectively. KEY WORDS: folate; one-carbon metabolism; requirements; dietary reference intakes