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Methylene tetrahydrofolate reductase (MTHFR) is a flavoprotein, involved in one-carbon pathway and is responsible for folate and homocysteine metabolism. Regulation of MTHFR is pivotal for maintaining the cellular concentrations of methionine and SAM (S-adenosyl methionine) which are essential for the synthesis of nucleotides and amino acids, respectively. Therefore, mutations in MTHFR leads to its dysfunction resulting in conditions like homocystinuria, cardiovascular diseases, and neural tube defects in infants. Among these conditions, homocystinuria has been highly explored, as it manifests ocular disorders, cognitive disorders and skeletal abnormalities. Hence, in this study, we intend to explore the mutational landscape of human MTHFR isoform-1 (h.MTHFR-1) to decipher the most pathogenic variants pertaining to homocystinuria. Thus, a multilevel stringent prioritization of non-synonymous mutations in h.MTHFR-1 by integrative machine learning approaches was implemented to delineate highly deleterious variants based on its pathogenicity, impact on structural stability and functionality. Subsequently, extended molecular dynamics simulations and molecular docking studies were also integrated in order to prioritize the mutations that perturbs structural stability and functionality of h.MTHFR-1. In addition, displacement of Loop (Arg157-Tyr174) and helix α9 (His263-Ser272) involved in open/closed conformation of substrate binding domain were also probed to confirm the functional loss. On juxtaposed analysis, it was inferred that among 126 missense mutations screened, along with known pathogenic mutations (H127 T, A222 V, T227 M, F257 V and G387D) predicted that W500C, P254S and D585 N variants could be potentially driving homocystinuria. Thus, uncovering the prospects for inclusion of these mutations in diagnostic panels based on further experimental validations., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Thymidylate is synthesized de novo in all living organisms for replication of genomes. The chemical transformation is reductive methylation of deoxyuridylate at C5 to form deoxythymidylate. All eukaryotes including humans complete this well-understood transformation with thymidylate synthase utilizing 6R-N 5 -N 10 -methylene-5,6,7,8-tetrahydrofolate as both a source of methylene and a reducing hydride. In 2002, flavin-dependent thymidylate synthase was discovered as a new pathway for de novo thymidylate synthesis. The flavin-dependent catalytic mechanism is different than thymidylate synthase because it requires flavin as a reducing agent and methylene transporter. This catalytic mechanism is not well-understood, but since it is known to be very different from thymidylate synthase, there is potential for mechanism-based inhibitors that can selectively inhibit the flavin-dependent enzyme to target many human pathogens with low host toxicity., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The cobalamin-independent methionine synthase enzyme catalyzes a challenging reaction: the direct transfer of a methyl from 5-methyl-tetrahydrofolate-glutamate3 to the l-homocysteine thiol. The enzyme has a dual (βα)8 TIM barrel structure that binds, activates and brings the reactants into reaction proximity by conformational movements. In the previously observed open structures, the substrates bind too far apart to react, but we have captured a ternary complex with both substrates bound in a closed form of the enzyme. The closing is described in terms of a hinge between the N- and C-terminal TIM barrels and a rearrangement of key loops within the C domain. The substrate specificity can now be rationalized and the structure reveals His707 as the acid that protonates the THF leaving group through a water molecule trapped in the closed active site. The substrates are correctly oriented for an in-line attack by l-homocysteine on the N(5)-methyl., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Background: Maintaining folate stability during sample handling is important, yet challenging., Objective: We investigated the effects of suboptimal preanalytical conditions on serum folate stability., Methods: By using an HPLC-tandem MS method we measured folates [5-methyltetrahydrofolate (5-methylTHF), folic acid, MeFox (5-methylTHF oxidation product, pyrazino-s-triazine derivative of 4α-hydroxy-5-methylTHF), and other minor folate forms at or below the limit of detection] in human serum exposed to suboptimal conditions., Results: Whole blood samples (n = 21) stored at 32°C for ≤ 3 d (Expt. 1: delayed processing) showed significant decreases in serum total folate (tFOL; sum of folate forms: 11-32%, 5.5-15.9 nmol/L) and 5-methylTHF (36-62%, 14.5-25.1 nmol/L) and a significant increase in MeFox (346-415%, 7.17-8.63 nmol/L). Serum samples (n = 21) stored at 11°C for 7-14 d (Expt. 2: delayed freezing) also showed significant decreases in tFOL (4.6-10.4%, 2.3-5.1 nmol/L) and 5-methylTHF (8.4-29%, 3.4-11.6 nmol/L) and significant increases in MeFox (88-320%, 1.82-6.62 nmol/L). The molar loss in 5-methylTHF exceeded the gain in MeFox in these 2 experiments. When we exposed 3 serum pools (tFOL: 16.7-58.3 nmol/L) for 24 h to an elevated temperature of 37°C (Expt. 3), the significant decrease in 5-methylTHF (33% on average) was compensated for by an equimolar gain in MeFox. Repeated freeze/thaw cycles (≤ 3 cycles) of serum [closed (Expt. 4) and open (Expt. 5) vials] showed generally stable folates with small (<1 nmol/L) changes. Long-term (≤ 12 mo) exposure of 3 serum pools (tFOL: 17.5-63.7 nmol/L) to a suboptimal (-20°C) freezing temperature (Expt. 6) showed significant decreases in tFOL (5% on average) already after 3 mo. The molar loss in 5-methylTHF exceeded the gain in MeFox. Folic acid generally showed good stability., Conclusions: To avoid folate losses, unprocessed whole blood should be protected from elevated temperatures and serum should not be refrigerated for >2 d or for a long term stored at -20°C., (© 2014 American Society for Nutrition.)

Dimethylglycine dehydrogenase (DMGDH) is a mammalian mitochondrial enzyme which plays an important role in the utilization of methyl groups derived from choline. DMGDH is a flavin containing enzyme which catalyzes the oxidative demethylation of dimethylglycine in vitro with the formation of sarcosine (N-methylglycine), hydrogen peroxide and formaldehyde. DMGDH binds tetrahydrofolate (THF) in vivo, which serves as an acceptor of formaldehyde and in the cell the product of the reaction is 5,10-methylenetetrahydrofolate instead of formaldehyde. To gain insight into the mechanism of the reaction we solved the crystal structures of the recombinant mature and precursor forms of rat DMGDH and DMGDH-THF complexes. Both forms of DMGDH reveal similar kinetic parameters and have the same tertiary structure fold with two domains formed by N- and C-terminal halves of the protein. The active center is located in the N-terminal domain while the THF binding site is located in the C-terminal domain about 40Å from the isoalloxazine ring of FAD. The folate binding site is connected with the enzyme active center via an intramolecular channel. This suggests the possible transfer of the intermediate imine of dimethylglycine from the active center to the bound THF where they could react producing a 5,10-methylenetetrahydrofolate. Based on the homology of the rat and human DMGDH the structural basis for the mechanism of inactivation of the human DMGDH by naturally occurring His109Arg mutation is proposed., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The tetrahydrofolate (THF) riboswitch regulates folate transport and metabolism in a number of Firmicutes by cooperatively binding two molecules of THF. To further understand this riboswitch's specificity for THF, binding and regulatory activity of a series of THF analogs and antifolates were examined. Our data reveal that although binding is dominated by the RNA's interactions with the pterin moiety, the para-aminobenzoic acid (pABA) moiety plays a significant role in transcriptional regulation. Further, we find that adenine and several other analogs bind with high affinity by an alternative binding mechanism. Despite a similar affinity to THF, adenine is a poor regulator of transcriptional attenuation. These results demonstrate that binding alone does not determine a compound's effectiveness in regulating the activity of the riboswitch-a complication in current efforts to develop antimicrobials that target these RNAs., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Mild and prolonged oxidative degradation of 5-methyltetrahydrofolate (5-methylTHF) leads to the biologically inactive pyrazino-s-triazine derivative of 4α-hydroxy-5-methylTHF (MeFox). MeFox and the biologically active 5-formyltetrahydrofolate (5-formylTHF) are isobaric compounds that behave similarly during chromatographic and mass separation, making coelution and misidentification likely. Our published routine liquid chromatography-tandem MS (LC-MS/MS) method did not discern between 5-formylTHF and MeFox, measuring the sum of these compounds at a mass to charge ratio (m/z) of 474→327 as 5-formylTHF. We modified this method to separate MeFox and 5-formylTHF by either chromatography or unique mass transitions and then applied the 2 methods to serum specimens to determine typical concentrations of these compounds. The 2 unique transitions (m/z: 5-formylTHF, 474→299; MeFox, 474→284) showed good sensitivity [limit of detection (nmol/L): 5-formylTHF, 0.21; MeFox, 0.34], selectivity (no interfering peaks), spiking recovery (mean ± SD: 5-formylTHF, 103 ± 3.4%; MeFox, 94 ± 10%), and low imprecision (CV: 5-formylTHF, 3.9% at 2.4 nmol/L; MeFox, 5.1% at 2.9 nmol/L). The mass separation method detected 5-formylTHF in the same specimens as the chromatographic separation method. Analysis of several thousand serum specimens showed that the majority (∼85%) contained MeFox at <3 nmol/L but no detectable 5-formylTHF concentrations, some (∼14%) contained 5-formylTHF at <0.5 nmol/L, and a few specimens contained 5-formylTHF at >1 nmol/L and MeFox at >10 nmol/L. In summary, serum can contain 5-formylTHF high enough to contribute to total folate and contains MeFox that will bias total folate if not appropriately separated. Including measurements of MeFox and 5-formylTHF along with the other folate vitamers will enhance assessments of the association between biologically active folate and health effects.

Introduction: Thymidylate synthase and folate receptors are well-developed targets of cancer therapy. Discovery of a simple and fast method for the conversion of 11CH3Ito[11C]-formaldehyde (11CH2O) encouraged us to label the co-factor of this enzyme. Preliminary studies conducted on cell lines have demonstrated a preferential uptake of [11-14C]-(R)-N5,N10-methylene-5,6,7,8-tetrahydrofolate (14CH2H4folate) by cancerous cell vs. normal cells from the same organ (Saeed M., Sheff D. and Kohen A. Novel positron emission tomography tracer distinguishes normal from cancerous cells. J Biol Chem 2011;286:33872-33878), pointing out 11CH2H4folate as a positron emission tomography (PET) tracer for cancer imaging. Herein we report the synthesis of 11CH2H4folate, which may serve as a potential PET tracer., Methods: In a remotely controlled module, methyl iodide (11CH3I) was bubbled into a reaction vial containing trimethylamine N-oxide in N,N-Dimethylformamide (DMF) and heated to 70°C for 2 min. Formaldehyde (11CH2O) formed after the completion of reaction was then mixed with a solution of freshly prepared tetrahydrofolate (H4folate) by using a fast chemoenzymatic approach to accomplish synthesis of 11CH2H4folate. Purification of the product was carried out by loading the crude reaction mixture on a SAX cartridge, washing with water to remove unbound impurities and finally eluting with a saline solution., Results: The synthesis and purification of 11CH2H4folate were completed within 5 min. High-performance liquid chromatography analysis of the product after SAX purification indicates that more than 90% of the radioactivity that was retained on the SAX cartridge was in 11CH2H4folate, with minor (<10%) radioactivity due to unreacted 11CH2O., Conclusion: We present a fast (∼5 min) synthesis and purification of 11CH2H4folate as a potential PET tracer. The final product is received in physiologically compatible buffer (100 mM sodium phosphate, pH 7.0 containing 500 mM NaCl) and ready for use in vivo., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Folate co-enzymes play a pivotal role in one-carbon transfer cellular processes. Many eukaryotes encode the tri-functional tetrahydrofolate dehydrogenase/cyclohydrolase/synthetase (deh/cyc/syn) enzyme, which consists of a N-terminal bifunctional domain (deh/cyc) and a C-terminal monofunctional domain (syn). Here, we report the first analogous archeal enzyme structures, for the bifunctional methylenetetrahydrofolate dehydrogenase/cyclohydrolase from Thermoplasma acidophilum (TaMTHFDC) as the native protein and also as its NADP complex. The TaMTHFDC structure is a dimer with a polar interface, as well as a NADP binding site that shows minor conformational change. The orientations of the residues in the NADP binding site do not change on ligand binding, incorporating three water molecules which are hydrogen bonded with phosphate groups of NADP in the structure of the complex. Our structural information will contribute to an improved understanding of the basis of THF and one-carbon metabolism., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Folate deficiency is a prevalent phenomenon worldwide especially in underprivileged countries. Polyglutamyl 5-methyltetrahydrofolate (5MTHF) species are the naturally occurring principle folate in store-bought vegetables. Here we report a simple and complete extraction method for the determination of native polyglutamyl 5-methyltetrahydrofolate in vegetables using high performance liquid chromatography with tandem mass spectrometric detection (HPLC-MS/MS). Coarsely chopped samples (18 different vegetables) were steamed to inactivate glutamylase enzymes and liberate folate from binding proteins and extracted in a reducing buffer with (13)C(5) 5MTHF stable isotope added as internal standard. The polyglutamyl 5-methyltetrahydrofolate species were separated in 9 min on a C(18) column using a reversed phase system. HPLC eluate was interfaced with a triple quadrupole mass spectrometer operated in electrospray positive mode. The respective pseudomolecular cation of each polyglutamyl 5-methyltetrahydrofolate species was selected for fragmentation to a common daughter ion for detection. We quantitated polyglutamyl 5-methyltetrahydrofolate in store-bought vegetables from families Brassicaceae, Asteraceae and Amaranthaceae (including mustard greens, romaine lettuce and Swiss chard) of which most have not been quantitated previously. Most vegetables from Asteraceae and those from Amaranthaceae contained similar amounts of monoglutamyl 5MTHF and polyglutamyl 5MTHF while Brassicaceae were dominated by polyglutamyls and endive species (Asteraceae) contained mainly monoglutamyl 5MTHF. The precision of the method for the various polyglutamyl 5-methyltetrahydrofolate forms was 1-9% RSD, recovery 84-91%, limit of detection 64-658 fmol and limit of quantitation 193-1994 fmol. Herein we describe a rapid, sensitive and selective HPLC-MS/MS technique to quantitate polyglutamyl 5-methyltetrahydrofolate species. This method may be suitable for analyzing the polyglutamyl 5-methyltetrahydrofolate profile of inherent folates in a wide range of leafy green vegetables., (Published by Elsevier B.V.)

Epidemiological studies on folate and chronic diseases often involve the use of frozen serum stored in biorepositories for decades. Folate instability may attenuate associations between folate status and study endpoints. In this cross-sectional study, we retrieved serum samples stored at -25 degrees C for 0, 4, 6, 17, or 29 y in the Janus biobank. Samples were obtained from a total of 650 men aged 40-49 y at the time of blood collection and were evenly distributed according to storage time. Folate was determined by a liquid chromatography tandem MS (LC-MS/MS) assay that measures 5-methyltetrahydrofolate (5mTHF), its oxidation product 4-alpha-hydroxy-5-methyltetrahydrofolate (hmTHF), and other folate species; by a Lactobacillus rhamnosus microbiological assay; and by LC-MS/MS as p-aminobenzoylglutamate (pABG) equivalents after oxidation and mild acid hydrolysis of the folate species. Concentrations of 5mTHF and microbiologically active folate were lower in samples that had been subjected to long-term storage and the data were consistent with a decrease of 3.2 and 2.8%/y, respectively. hmTHF was detected in all specimens but did not accumulate upon long-term storage (>4 y). Folate measured as pABG declined at a slow rate of 0.98%/y and approximately 80% of the folate was recovered after 29 y of storage. B-vitamin status did not differ between individuals delivering samples at different time points, as assessed by measuring total homocysteine, methylmalonic acid, and serum vitamin B-12. In conclusion, folate is substantially degraded in serum frozen for decades but can to a large extent be recovered as pABG equivalents. The pABG assay appears to be the method of choice for the determination of folate status in archival serum samples.

Enzymes from the de novo purine biosynthetic pathway have been exploited for the development of anti-cancer drugs, and represent novel targets for anti-bacterial drug development. In Mycobacterium tuberculosis, the cause of tuberculosis, this pathway has been identified as essential for growth and survival. The structure of M. tuberculosis PurN (MtPurN) has been determined in complex with magnesium and iodide at 1.30 A resolution, and with cofactor analogue, 5-methyltetrahydrofolate (5MTHF) at 2.2 A resolution. The structure shows a Rossmann-type fold that is very similar to the known structures of the human and E. coli PurN proteins. In contrast, MtPurN forms a dimer that is quite different from that formed by the Escherichia coli PurN, and which suggests a mechanism whereby communication could take place between the two active sites. Differences are seen in two active site loops and in the binding mode of the 5MTHF cofactor analogue between the two MtPurN molecules of the dimer. A binding site for halide ions is found in the dimer interface, and bound magnesium and iodide ions in the active site suggest sites that might be exploited in potential drug discovery strategies.

A selective and rapid method was developed and validated for determination of 6R-leucovorin (LV), 6S-leucovorin and 5-methyltetrahydrofolate (5-MeTHF) in human plasma using stereoselective liquid chromatography-tandem mass spectrometry. All analytes and the internal standard were extracted from plasma by solid phase extraction using Oasis HLB C(18) cartridges. A macrocyclic glycopeptide teicoplanin column was used for chiral separation of LV and 5-MeTHF isomers with NH(4)TFA or NH(4)OAc in methanol as mobile phase. Detection was performed on an API 4000 tandem mass spectrometer with positive electrospray ionization in multiple reaction monitoring mode. The calibration curves were linear in the range of 0.050-20.0microg/mL for 6R-LV and 6S-LV, and 0.025-10.0microg/mL for 5-MeTHF. The intra- and inter-assay precision was 3.6-13.2%, 3.4-12.9% and 5.3-9.3% for 6R-LV, 6S-LV and 5-MeTHF, respectively. The accuracy was 99.4-102.4%, 95.3-96.8% and 93.0-110% for 6R-LV, 6S-LV and 5-MeTHF, respectively. The lower limit of quantification (LLOQ) was 0.050microg/mL for each LV isomer and 0.025microg/mL for 5-MeTHF. The method was successfully applied to a comparative pharmacokinetic study between leucovorin calcium and levoleucovorin calcium in 10 volunteers. No significant differences between levoleucovorin and leucovorin in pharmacokinetic parameters of 6S-LV and 5-MeTHF were found in volunteers.

Thymidylate synthase (TS) catalyzes the reductive methylation of deoxyuridine monophosphate (dUMP) using methylene tetrahydrofolate (CH(2)THF) as cofactor, the glutamate tail of which forms a water-mediated hydrogen bond with an invariant lysine residue of this enzyme. To understand the role of this interaction, we studied the K48Q mutant of Escherichia coli TS using structural and biophysical methods. The k(cat) of the K48Q mutant was 430-fold lower than wild-type TS in activity, while the K(m) for the (R)-stereoisomer of CH(2)THF was 300 microM, about 30-fold larger than K(m) from the wild-type TS. Affinity constants were determined using isothermal titration calorimetry, which showed that binding was reduced by one order of magnitude for folate-like TS inhibitors, such as propargyl-dideazafolate (PDDF) or compounds that distort the TS active site like BW1843U89 (U89). The crystal structure of the K48Q-dUMP complex revealed that dUMP binding is not impaired in the mutant, and that U89 in a ternary complex of K48Q-nucleotide-U89 was bound in the active site with subtle differences relative to comparable wild-type complexes. PDDF failed to form ternary complexes with K48Q and dUMP. Thermodynamic data correlated with the structural determinations, since PDDF binding was dominated by enthalpic effects while U89 had an important entropic component. In conclusion, K48 is critical for catalysis since it leads to a productive CH(2)THF binding, while mutation at this residue does not affect much the binding of inhibitors that do not make contact with this group.

The crystal structure of heterotetrameric sarcosine oxidase (TSOX) from Pseudomonas maltophilia has been determined at 1.85 A resolution. TSOX contains three coenzymes (FAD, FMN and NAD+), four different subunits (alpha, 103 kDa; beta, 44 kDa; gamma, 21 kDa; delta, 11 kDa) and catalyzes the oxidation of sarcosine (N-methylglycine) to yield hydrogen peroxide, glycine and formaldehyde. In the presence of tetrahydrofolate, the oxidation of sarcosine is coupled to the formation of 5,10-methylenetetrahydrofolate. The NAD+ and putative folate binding sites are located in the alpha-subunit. The FAD binding site is in the beta-subunit. FMN is bound at the interface of the alpha and beta-subunits. The FAD and FMN rings are separated by a short segment of the beta-subunit with the closest atoms located 7.4 A apart. Sulfite, an inhibitor of oxygen reduction, is bound at the FMN site. 2-Furoate, a competitive inhibitor with respect to sarcosine, is bound at the FAD site. The sarcosine dehydrogenase and 5,10-methylenetetrahydrofolate synthase sites are 35 A apart but connected by a large internal cavity (approximately 10,000 A3). An unexpected zinc ion, coordinated by three cysteine and one histidine side-chains, is bound to the delta-subunit. The N-terminal half of the alpha subunit of TSOX (alphaA) is closely similar to the FAD-binding domain of glutathione reductase but with NAD+ replacing FAD. The C-terminal half of the alpha subunit of TSOX (alphaB) is similar to the C-terminal half of dimethylglycine oxidase and the T-protein of the glycine cleavage system, proteins that bind tetrahydrofolate. The beta-subunit of TSOX is very similar to monomeric sarcosine oxidase. The gamma-subunit is similar to the C-terminal sub-domain of alpha-TSOX. The delta-subunit shows little similarity with any PDB entry. The alphaA domain/beta-subunit sub-structure of TSOX closely resembles the alphabeta dimer of L-proline dehydrogenase, a heteroctameric protein (alphabeta)4 that shows highest overall similarity to TSOX.

In this study, we investigated methionine synthase from Candida albicans (CaMET 6p) and Saccharomyces cerevisiae (ScMET 6p). We describe the cloning of CaMet 6 and ScMet 6, and the expression of both the enzymes in S. cerevisiae. CaMET 6p is able to complement the disruption of met 6 in S. cerevisiae. Following the purification of ScMET 6p and CaMET 6p, kinetic assays were performed to determine substrate specificity. The Michaelis constants for ScMET 6p with CH(3)-H(4)PteGlu(2), CH(3)-H(4)PteGlu(3), CH(3)-H(4)PteGlu(4), and l-homocysteine are 108, 84, 95, and 13 microM, respectively. The Michaelis constants for CaMET 6p with CH(3)-H(4)PteGlu(2), CH(3)-H(4)PteGlu(3), CH(3)-H(4)PteGlu(4), and l-homocysteine are 113, 129, 120, and 14 microM, respectively. Neither enzyme showed activity with CH(3)-H(4)PteGlu(1) as a substrate. We conclude that ScMET 6p and CaMET 6p require a minimum of two glutamates on the methyltetrahydrofolate substrate, similar to the bacterial metE homologs. The cloning, purification, and characterization of these enzymes lay the groundwork for inhibitor-design studies on the cobalamin-independent fungal methionine synthases.

Many microorganisms in the large intestine are capable of synthesizing folate. Preliminary evidence suggests that this folate may be absorbed. The purpose of the 2 experiments reported herein was to estimate the pool of folate in the feces of human infants and piglets and to ascertain, if absorbed, whether the quantity and form of folate are sufficient to potentially affect the folate status of the host organism. The folate content of milk fed to and of fecal solids collected from exclusively human milk-fed (n = 12) and formula-fed (n = 10) term infants (1-6 mo old) was determined microbiologically before (short-chain folates) and after folate conjugase (total folate) treatment. The folate content of formula fed and of feces collected from 10-d-old piglets (n = 10) was also determined microbiologically. The proportion of 5-methyltetrahydrofolate (5-methylTHF) in feces of human infants and piglets that was monoglutamylated was determined by HPLC analysis. The folate content of fecal solids collected from infants was 93.2 +/- 92.8 nmol/d (mean +/- SD), representing on average 50% (8.0-170.1%) of their mean estimated dietary folate intake. Fecal folate was largely present as short-chain folate (66 +/- 21.3%) with the predominant form being 5-methylTHF, 52.5 +/- 30.1% of which was monoglutamylated. In piglets, the folate content of feces was 301.3 +/- 145.7 nmol/d, representing 36% of their dietary folate intake. Piglet fecal folate was largely present as short-chain folate (68.1 +/- 12.6%) with the predominant species being 5-methylTHF, 29.3 +/- 33.2% of which was monoglutamylated. Collectively, these data suggest that the quantity and form of folate (monoglutamylated) in the large intestine of human infants and piglets are sufficiently large to potentially affect folate status.

A one-pot synthesis of isotopically labeled R-[6-xH]N5,N10-methylene-5,6,7,8-tetrahydrofolate (CH2H4F) is presented, where x=1, 2, or 3 represents hydrogen, deuterium, or tritium, respectively. The current procedure offers high-yield, high-purity, and microscale-quantity synthesis. In this procedure, two enzymes were used simultaneously in the reaction mixture. The first was Thermoanaerobium brockii alcohol dehydrogenase, which stereospecifically catalyzed a hydride transfer from C-2-labeled isopropanol to the re face of oxidized nicotinamide adenine dinucleotide phosphate to form R-[4-xH]-labeled reduced nicotinamide adenine dinucleotide phosphate. The second enzyme, Escherichia coli dihydrofolate reductase, used the xH to reduce 7,8-dihydrofolate (H2F) to form S-[6-xH]5,6,7,8-tetrahydrofolate (S-[6-xH]H4F). The enzymatic reactions were followed by chemical trapping of S-[6-xH]H4F with formaldehyde to form the final product. Product purification was carried out in a single step by reverse phase high-pressure liquid chromatography separation followed by lyophilization. Two analytical methods were developed to follow the reaction progress. Finally, the utility of the labeled cofactor in mechanistic studies of thymidylate synthase is demonstrated by measuring the tritium kinetic isotope effect on the enzyme's second order rate constant.

A liquid chromatographic-fluorimetric determination of folates in foodstuffs including their extraction, without or with deconjugation, chemical conversion to 5-CH3-H4PteGlu(n) and purification of the extract by affinity chromatography is reported. The conversion enables the analysis of total folates and also of the contents of the different mono- and polyglutamate forms of the folates. The method has a satisfactory day-to-day repeatability (never,more than 10%) and a very low detection limit (0.02 pmol per injection). Depending on the folate studied, the recovery rates varied from 78% (10-CHO-PteGlu) to 98% (5-CHO-H4PteGlu). Furthermore it has been possible to show that the deconjugation of the folates by rat plasma conjugase was incomplete in foodstuffs whereas chicken pancreas conjugase effectively converted the different folate polyglutamates into folate diglutamates. It could not be demonstrated that prior hydrolysis with a protease and amylase was useful for the analysis of the different foodstuffs studied (yeast, spinach, beef liver, beef fillet and peas) when deconjugation was performed with the chicken pancreas conjugase.

10-Formyltetrahydrofolate dehydrogenase has previously been identified as a tight binding protein of the polyglutamate forms of tetrahydrofolate (R. J. Cook and C. Wagner, Biochemistry 21, 4427-4434, 1982). Each subunit contains two independently folded domains connected by a linking peptide. By using the stable substrate and product analogs 10-formyl 5,8-dideazafolate and 5, 8-dideazafolate, respectively, we have determined that the tight binding folate site is separate from the catalytic site and that it is located on the N-terminal domain of the protein. This was achieved by cross-linking 10-formyl 5,8-dideazafolate to the dehydrogenase through the carboxyl group of the substrate analog. The cross-linked substrate analog was converted to the cross-linked product complex by adding either NADP+ or 2-mercaptoethanol, proving that the 10-formyl 5,8-dideazafolate was bound at the active site. With the active site cross-linked to 5,8-dideazafolate and not available for binding, the enzyme still bound 5, 8-dideazafolate-[3H]tetraglutamate tightly but noncovalently. Separation of the large and small domains by limited proteolysis showed that the tightly bound 5,8-dideazafolate-[3H]tetraglutamate was located on the small domain. The location of the cross-linked 10-formyl 5,8-dideazafolate at the active site was determined by amino acid sequencing of an isolated tryptic peptide., (Copyright 1999 Academic Press.)

Previously described methods for the determination of lometrexol in plasma used either fluorescence or ultraviolet detection. An alternative method for the determination of lometrexol utilizing electrochemical detection is described, having comparable sensitivity to fluorometric methods but not requiring pre-analytical oxidation. Following sample clean-up, separation is achieved on a phenyl column with a mobile-phase of 8% acetonitrile in 50 mM sodium acetate buffer, pH 4.0. The calibration curve in plasma is linear from 10 to 200 ng/ml with inter- and intra-day precision of 5.4 and 5.5%, respectively. The recovery of lometrexol from plasma is 81 +/- 1.5%, and the lower limit of detection is 5 ng/ml, using a signal-to-noise ratio of 3.

The protein binding of the tetrahydrofolates folinic acid (FA) and its metabolite 5-methyltetrahydrofolic acid (5-MTHF) to human serum albumin (HSA) is stereoselective. At therapeutically relevant concentrations of tetrahydrofolate (range, 5-100 microM), the protein binding was stereoselective to the (R)-isomers of FA and 5-MTHF. The binding of (R)-FA and (R)-5-MTHF was saturated at a concentration of 7% HSA [(R)-tetrahydrofolate bound, ca. 80%]. In contrast to (S)-FA, which was not bound to HSA, (S)-5-MTHF was bound to 45% under physiological conditions. (R)-FA did not influence the protein binding of (S)-FA. Hypoalbuminemia is common in patients with advanced colorectal cancer and affects differentially the protein binding of the diastereoisomers of FA and 5-MTHF. Thus, an influence on the biochemical modulation of 5-fluorouracil by tetrahydrofolates should be taken into consideration.

Crystal structures of two crystal forms of the complex of Lactobacillus casei (TS) with its substrate dUMP have been solved and refined at 2.55 A resolution. The two crystal forms differ by approximately 5% in the c-axis length. The TS-dUMP complexes are symmetric dimers with dUMP bound equivalently in both active sites. dUMP is non-covalently bound in the same conformation as in ternary complexes of TS with dUMP and cofactor or cofactor analogs. The same hydrogen bonds are made between TS and substrate in the binary and ternary complexes. We have also determined the 2.36 A crystal structure of phosphate-bound L. casei TS. This structure has been refined to an R-factor of 19.3% with highly constrained geometry. Refinement has revealed the locations of all residues in the protein, including the disordered residues 90 to 119, which are part of an insert found only in the L. casei and Staphylococcus aureus transposon Tn4003 TS sequences. The 2.9 A multiple isomorphous replacement (MIR) structure of L. casei TS in a complex with its substrate dUMP has been refined to a crystallographic R-factor of 15.5%. Reducing agents were withheld from crystallization solutions during MIR structure determination to allow heavy-metal labeling of the cysteine residues. Therefore, the active-site cysteine residue in this structure is oxidized and the dUMP is found at half-occupancy in the active site. No significant conformational difference was found between the phosphate-bound and dUMP-bound structures. The TS-dUMP structures were better ordered than the phosphate-bound TS or the oxidized TS-dUMP, particularly Arg23, which is clearly hydrogen-bonded to the phosphate group of dUMP. A large and a small P6(1)22 crystal form are observed for both phosphate-bound and dUMP-bound L. casei TS. The small cell forms of the phosphate-bound and dUMP-bound enzyme are isomorphous, whereas the cell constants of the larger cell form change slightly when dUMP is bound (c = 240 A versus c = 243 A). For both liganded and unliganded enzyme, conversion from the small to the large crystal form sometimes occurs spontaneously, and the crystal packing changes at a single interface. Conversion may be the result of a small change in pH in the mother liquor surrounding the crystal. A single intermolecular contact between symmetry-related Asp287 residues is disrupted on going from the small to the large crystal form.