Mammalian folylpoly-gamma-glutamate synthetase. 3. Specificity for folate analogues

A variety of folate analogues were synthesized to explore the specificity of the folate binding site of hog liver folylpolyglutamate synthetase and the requirements for catalysis. Modifications of the internal and terminal glutamate moieties of folate cause large drops in on rates and/or affinity for the protein. The only exceptions are glutamine, homocysteate, and ornithine analogues, indicating a less stringent specificity around the delta-carbon of glutamate. It is proposed that initial folate binding to the enzyme involves low-affinity interactions at a pterin and a glutamate site and that the first glutamate bound is the internal residue adjacent to the benzoyl group. Processive movement of the polyglutamate chain through the glutamate site and a possible conformational change in the protein when the terminal residue is bound would result in tight binding and would position the gamma-carboxyl of the terminal glutamate in the correct position for catalysis. Steric limitations imposed on the internal glutamate residues that loop out and additional steric constraints imposed by binding of different pterin moieties would be expected to effect slight conformational changes in the protein and/or the terminal glutamate and would explain the decrease in on rate and catalytic rate with increased polyglutamate chain length, and the differential effect of one-carbon substitution on the catalytic rate with polyglutamate derivatives. The 4-amino substitution of folate increases the on rate for monoglutamate derivatives but severely impairs catalysis with diglutamate derivatives. Pteroylornithine derivatives are the first potent and specific inhibitors of folylpolyglutamate synthetase to be identified and may act as analogues of reaction intermediates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Polyglutamylation of folate coenzymes is necessary for methionine biosynthesis and maintenance of intact mitochondrial genome in Saccharomyces cerevisiae

One-carbon metabolism is essential to provide activated one-carbon units in the biosynthesis of methionine, purines, and thymidylate. The major forms of folates in vivo are polyglutamylated derivatives. In organisms that synthesize folate coenzymes de novo, the addition of the glutamyl side chains is achieved by the action of two enzymes, dihydrofolate synthetase and folylpolyglutamate synthetase. We report here the characterization and molecular analysis of the two glutamate-adding enzymes of Saccharomyces cerevisiae. We show that dihydrofolate synthetase catalyzing the binding of the first glutamyl side chain to dihydropteroate yielding dihydrofolate is encoded by the YMR113w gene that we propose to rename FOL3. Mutant cells bearing a fol3 mutation require folinic acid for growth and have no dihydrofolate synthetase activity. We show also that folylpolyglutamate synthetase, which catalyzes the extension of the glutamate chains of the folate coenzymes, is encoded by the MET7 gene. Folylpolyglutamate synthetase activity is required for methionine synthesis and for maintenance of mitochondrial DNA. We have tested whether two folylpolyglutamate synthetases could be encoded by the MET7 gene, by the use of alternative initiation codons. Our results show that the loss of mitochondrial functions in met7 mutant cells is not because of the absence of a mitochondrial folylpolyglutamate synthetase.     

Mammalian folylpoly-gamma-glutamate synthetase. 1. Purification and general properties of the hog liver enzyme

Folylpolyglutamate synthetase was purified 30,000-150,000-fold from hog liver. Purification required the use of protease inhibitors, and the protein was purified to homogeneity in two forms. Both forms of the enzyme were monomers of Mr 62,000 and had similar specific activities. The specific activity of the homogeneous protein was over 2000-fold higher than reported for partially purified folylpolyglutamate synthetases from other mammalian sources. Enzyme activity was absolutely dependent on the presence of a reducing agent and a monovalent cation, of which K+ was most effective. The purified enzyme catalyzed a MgATP-dependent addition of glutamate to tetrahydrofolate with the concomitant stoichiometric formation of MgADP and phosphate. Under conditions that resembled the expected substrate and enzyme concentrations in hog liver, tetrahydrofolate was metabolized to long glutamate chain length derivatives with the hexaglutamate, the major in vivo folate derivative, predominating. Enzyme activity was maximal at about pH 9.5. The high-pH optimum was primarily due to an increase in the Km value for the L-glutamate substrate at lower pH values, and the reaction proceeded effectively at physiological pH provided high levels of glutamate were supplied.     

Folate-binding triggers the activation of folylpolyglutamate synthetase

Folic acid is an essential vitamin for normal cell growth, primarily through its central role in one-carbon metabolism. Folate analogs (antifolates) are targeted at the same reactions and are widely used as therapeutic drugs for cancer and bacterial infections. Effective retention of folates in cells and the efficacy of antifolate drugs both depend upon the addition of a polyglutamate tail to the folate or antifolate molecule by the enzyme folylpolyglutamate synthetase (FPGS). The reaction mechanism involves the ATP-dependent activation of the free carboxylate group on the folate molecule to give an acyl phosphate intermediate, followed by attack by the incoming L-glutamate substrate. FPGS shares a number of structural and mechanistic details with the bacterial cell wall ligases MurD, MurE and MurF, and these enzymes, along with FPGS, form a subfamily of the ADP-forming amide bond ligase family. High-resolution crystallographic analyses of binary and ternary complexes of Lactobacillus casei FPGS reveal that binding of the first substrate (ATP) is not sufficient to generate an active enzyme. However, binding of folate as the second substrate triggers a large conformational change that activates FPGS and allows the enzyme to adopt a form that is then able to bind the third substrate, L-glutamate, and effect the addition of a polyglutamate tail to the folate.     

Polyglutamate derivatives of folic acid coenzymes and methotrexate

Polyglutamate derivatives of the folate coenzymes are widely distributed in nature. Frequently, they are the predominant form of intracellular folate, and recent investigations have indicated that polyglutamates are the preferred substrates for most of the enzymes of folate metabolism. Dietary folates are mostly polyglutamate conjugates which must be broken down to the mono- or diglutamates before intestinal absorption can occur. Intracellularly, pteroyl monoglutamates are converted to the polyglutamate form, a process which appears to favor their cellular retention. Alterations in polyglutamate chain length may play an important role in the regulation of folate metabolism. Polyglutamate derivatives of the folate antagonist methotrexate have also been isolated. The synthesis and biological properties of these compounds along with their potential chemotherapeutic role are discussed.     

Enzymatic synthesis and function of folylpolyglutamates

Summary Derivatives of folic acid occur in nature predominantly as poly (-glutamyl) derivatives containing 2–8 glutamate residues. The data regarding the function of these derivatives, and their biosynthesis by eucaryotic and procaryotic folylpolyglutamate synthetases, is reviewed.The most universal functions of folylpolyglutamates appear to be (a) as the actual cofactors in vivo for folate dependent enzymes, (b) as inhibitors of folate dependent enzymes for which they are not substrates, and (c) to increase retention of folates after they are transported into cells as monoglutamates. Folylpolyglutamates also have numerous specialized functions in specific organisms, e.g. as structural components of some coliphage, and as allosteric regulators in Neurospora crassa.A single enzyme appears responsible for synthesis of all polyglutamate derivatives, regardless of length. With the recent introduction of sensitive assays this folylpolyglutamate synthetase has begun to be characterized. Although procaryotic and eucaryotic synthetases have many dissimilar properties, both types catalyze the ATP-dependent addition of L-glutamate to the -carboxyl of the glutamate present in the folate. Both types also require a monovalent cation and a relatively high pH. The most significant differences between the two types are in their folate substrate specificity and the product lengths derived from various folates.The mechanism of the bacterial enzyme has been studied and an acyl phosphate intermediate is indicated.     

Folate Metabolism in Datura innoxia (In Vivo and in Vitro Folylpolyglutamate Synthesis in Wild-Type and Methotrexate-Resistant Cells)

In vivo folylpolyglutamate pools of the wild-type (Px4) and methotrexate-resistant (MTX161) Datura innoxia cell lines were detected by incorporation of [14C]p-aminobenzoate into folates. The folylpolyglutamate derivatives were cleaved to p-aminobenzoylpolyglutamates and separated according to glutamyl chain length by high-performance liquid chromatography. Hexaglutamates were the predominant form in both Datura cell lines. The proportions of individual folylpolyglutamates were unaffected by culturing the cells in medium containing products of one-carbon metabolism such as glycine, adenine, thymidine, or methionine. Radiolabeling of the hexaglutamates was greatly reduced in the presence of 10-8 M methotrexate (MTX) in the Px4 cells but not in the MTX161 cells. Tetrahydrofolate, 5, 10-methylenetetrahydrofolate, and folinic acid were effective substrates for the folylpolyglutamate synthetase from Datura cells in vitro, whereas MTX and folate were poor substrates. In vivo, MTX can be slowly converted into its polyglutamate derivatives up to MTXGlu4 or MTXGlu5 in Datura cells in the longer term. Significantly lower levels of MTX polyglutamates in MTX161 cells were found compared with those of Px4 cells during prolonged (10 d) exposure to MTX. Although in vivo and in vitro folylpolyglutamate synthesis was found to be similar in both cell lines, about a 4-fold increase in specific activity of [gamma]-glutamyl hydrolase (GGH) was detected in the MTX161 cell line. The increase in GGH in the resistant cells suggested that breakdown of polyglutamylated forms of MTX may play a role in acquired MTX resistance.     

Mammalian folylpoly-gamma-glutamate synthetase. 2. Substrate specificity and kinetic properties

The specificity of hog liver folylpolyglutamate synthetase for folate substrates and for nucleotide and glutamate substrates and analogues has been investigated. The kinetic mechanism, determined by using aminopterin as the folate substrate, is ordered Ter-Ter with MgATP binding first, folate second, and glutamate last. This mechanism precludes the sequential addition of glutamate moieties to enzyme-bound folate. Folate, dihydrofolate, and tetrahydrofolate possess the optimal configurations for catalysis (kcat = 2.5 s-1) while 5- and 10-position substitutions of the folate molecule impair catalysis. kcat values decrease with increasing glutamate chain length, and the rate of decrease varies depending on the state of reduction and substitution of the folate molecule. Folate binding, as assessed by on rates, is slow. Dihydrofolate exhibits the fastest rate, and the rates are slightly reduced for tetrahydrofolate and 10-formyltetrahydrofolate and greatly reduced for 5-methyltetrahydrofolate and folic acid. The on rates for most pteroyldiglutamates are similar to the rates for their respective monoglutamate derivatives, but further extension of the glutamate chain results in a progressive decrease in on rates. Tetrahydrofolate polyglutamates are the only long glutamate chain length folates with detectable substrate activity. The specificity of the L-glutamate binding site is very narrow. L-Homocysteate and 4-threo-fluoroglutamate are alternate substrates and act as chain termination inhibitors in that their addition to the folate molecule prevents or severely retards the further addition of glutamate moieties. The Km for glutamate is dependent on the folate substrate used. MgATP is the preferred nucleotide substrate, and beta,gamma-methylene-ATP, beta,gamma-imido-ATP, adenosine 5'-O-(3-thiotriphosphate), P1,P5-di(adenosine-5') pentaphosphate, and free ATP4- are potent inhibitors of the reaction.     

High-pressure liquid chromatography separation and determination of rat liver folates

The endogenous levels of the various folate compounds in rat liver were determined using high-pressure liquid chromatography for the rapid separation of folate monoglutamate forms with specific quantitation of the folates by microbiological analysis of eluted fractions. The eight folate derivatives that were assayable were tetrahydrofolic acid (H₄PteGlu), 5-methyl-H₄PteGlu, 10-formyl-H₄PteGlu, 5-formyl-H₄PteGlu, 5,10-methenyl-H₄PteGlu, 5,10-methylene-H₄PteGlu, H₂PteGlu, and PteGlu. New techniques for the preparation of tissues were developed in order to reduce the degradation of the folates. Tissue folates were converted to the monoglutamate form by a partially purified hog kidney polyglutamate hydrolase preparation and incubations were carried out at pH 6.0. This minimized folate degradation but still allowed for maximal polyglutamate hydrolase activity. Rapid removal of tissues was compared with freeze-clamping techniques. The major folates in rat liver were H₄PteGlu and 5-methyl-H₄PteGlu, comprising 42 and 39%, respectively, of the total liver folate pool of 27.30 nmol/g liver (about 13 μg/g liver). In addition, 10-formyl-H₄PteGlu and 5-formyl-H₄PteGlu each comprised 10% of the total folate pool. No endogenous PteGlu, H₂PteGlu, or 5,10-methylene-H₄PteGlu was detected in rat liver samples under our conditions. Distribution of ¹⁴C derived from a previous [¹⁴C]folic acid injection paralleled the distribution of folate as determined microbiologically after high-pressure liquid chromatography separation. The importance of these methods for the direct determination and estimation of flux of H₄PteGlu, 5-methyl-H₄PteGlu, and 10-formyl-H₄PteGlu in studies dealing with the folate system was emphasized.     

Functional analysis of folate polyglutamylation and its essential role in plant metabolism and development

Cellular folates function as co-enzymes in one-carbon metabolism and are predominantly decorated with a polyglutamate tail that enhances co-enzyme affinity, subcellular compartmentation and stability. Polyglutamylation is catalysed by folylpolyglutamate synthetases (FPGSs) that are specified by three genes in Arabidopsis, FPGS1, 2 and 3, which reportedly encode plastidic, mitochondrial and cytosolic isoforms, respectively. A mutational approach was used to probe the functional importance of folate polyglutamylation in one-carbon metabolism and development. Biochemical analysis of single FPGS loss-of-function mutants established that folate polyglutamylation is essential for organellar and whole-plant folate homeostasis. However, polyglutamylated folates were still detectable, albeit at lower levels, in organelles isolated from the corresponding isozyme knockout lines, e.g. in plastids and mitochondria of the fpgs1 (plastidial) and fpgs2 (mitochondrial) mutants. This result is surprising given the purported single-compartment targeting of each FPGS isozyme. These results indicate redundancy in compartmentalised FPGS activity, which in turn explains the lack of anticipated phenotypic defects for the single FPGS mutants. In agreement with this hypothesis, fpgs1 fpgs2 double mutants were embryo-lethal, fpgs2 fpgs3 mutants exhibited seedling lethality, and fpgs1 fpgs3 mutants were dwarfed with reduced fertility. These phenotypic, metabolic and genetic observations are consistent with targeting of one or more FPGS isozymes to multiple organelles. These data confirm the importance of polyglutamylation in folate compartmentation, folate homeostasis and folate-dependent metabolic processes, including photorespiration, methionine and pantothenate biosynthesis.     

In Vitro Synthesis of Pteroylpoly-{gamma}-glutamates by Cotyledon Extracts of Pisum sativum L.

The formation of folylpolyglutamate derivatives by germinatingpea seeds (Pisum sativum L. cv Homesteader) was examined invivo and in vitro. Differential microbiological assay of cotyledonextracts showed that total folate concentrations increased from163 ng folate equivalents per g fresh weight at day 1 to 680ng per g fresh weight at day 3 of germination. Over a 7 daygermination period, folylpolyglutamate derivatives accountedfor 46–73% of the total cotyledonary folate pool. Theconcentration of these polyglutamate forms of folate increased6.5 fold during the first four days of germination and thenremained relatively constant. Dialyzed extracts of 1–4 day old cotyledons had abilityto incorporate [³H]glutamate and [¹⁴C]tetrahydrofolate intofolylpolyglutamates. This activity was mainly associated withprotein precipitating at 35–45% of saturation with ammoniumsulphate. The folylpolyglutamate synthetase of pea cotyledonshad requirements for ATP and the monoglutamate of tetrahydrofolate.The latter folate was a more effective substrate than 5,10-methylenetetrahydrofolatebut the diglutamate of unsubstituted tetrahydrofolate was notutilized. Ion exchange chromatography of the reaction productssuggested that [³H]glutamate and [¹⁴C]tetrahydrofolate wereincorporated into di-, and tetraglutamates of tetrahydrofolate.Folates of longer glutamyl chain lengths were only detectedwhen the synthetase reaction proceeded for 12 h or longer. (Received August 23, 1985; Accepted January 22, 1986)     

Folate polyglutamate synthetase activity in the cobalamin-inactivated rat.

Exposure to N2O inactivates cob[I]alamin and interferes with the activity of methionine synthetase, of which cob[I]alamin is a coenzyme. Less directly, it stops the formation of folate polyglutamate from tetrahydrofolates. Studies on the activity of folate polyglutamate synthetase in rat liver in vivo were carried out. The synthetase activity increased after exposure to N2O for up to 48 h, but longer exposure was accompanied by a return of activity to baseline values. The rise in synthetase activity was prevented by supplying methionine, 5'-methylthioadenosine or 5-formyltetrahydrofolate. The fall in folate polyglutamate synthetase activity after 48 h was accompanied by a restoration of hepatic synthesis of folate polyglutamate despite continuation of N2O exposure.     

Regulation of folylpoly-gamma-glutamate synthesis in bacteria: in vivo and in vitro synthesis of pteroylpoly-gamma-glutamates by Lactobacillus casei and Streptococcus faecalis

Lactobacillus casei and Streptococcus faecalis accumulated labeled folic acid and metabolized this compound to poly-gamma-glutamates of chain lengths of up to 11 and 5, respectively. Octa- and nonaglutamates predominated in L. casei, and tetraglutamates predominated in S. faecalis. The most effective monoglutamate substrates for the L. casei and S. faecalis folylpoly-gamma-glutamate (folylpolyglutamate) synthetases were methylene- and formyltetrahydrofolate, respectively. Methylenetetrahydropteroylpoly-gamma-glutamates were the preferred poly-gamma-glutamate substrates for both enzymes and, in each case, the highest activity was observed with the diglutamate substrate. The final distribution of folylpolyglutamates in these bacteria appeared to reflect the ability of folates with various glutamate chain lengths to act as substrates for the bacterial folylpolyglutamate synthetases. The proportions of individual folylpolyglutamates were markedly affected by culturing the bacteria in medium containing adenine, whereas thymine was without effect. Adenine did not affect the level of folylpolyglutamate synthetase in either organism but caused a large increase in the proportion of intracellular folates containing one-carbon units at the oxidation level of formate, folates which are substrates for enzymes involved in purine biosynthesis. The folates with shorter glutamate chain lengths in bacteria cultured in the presence of adenine resulted from primary regulation of the de novo purine biosynthetic pathway, regulation which caused an accumulation of formyltetrahydropteroyl-poly-gamma-glutamates (folate derivatives that are ineffective substrates for folylpolyglutamate synthetases), and did not result from regulation of folylpolyglutamate synthetase per se.     

Folylpoly-γ-glutamate synthesis by bacteria and mammalian cells

Summary The purification and properties of folylpolyglutamate synthetase fromCorynebacterium sp, and some properties of partially purified enzyme fromLactobacillus casei, Streptococcus faecalis, Neurospora crassa, pig liver, and Chinese hamster ovary cells, are described.TheCorynebacterium enzyme catalyzes a MgATP-dependent addition of glutamate to a variety of reduced pteroate and pteroylmono-, di-, and triglutamate substrates, with the concomitant production of MgADP and phosphate. Although glutamate moieties are added in a sequential fashion, the kinetic mechanism, which is Ordered Ter Ter, precludes the sequential addition of glutamate moieties to enzyme-bound folate. It is suggested that catalysis precedes via the formation of a pteroyl--glutamyl phosphate intermediate.Thein vivo distribution of folylpolyglutamates in bacteria and mammalian cells, which differ from source to source, appear to be a reflection of the ability of folylpolyglutamates to act as substrates for folylpolyglutamate synthetases from different sources.Only one enzyme appears to be involved in the conversion of pteroylmonoglutamates to polyglutamate forms in both bacteria and mammalian cells. Bacterial folylpolyglutamate synthetases use a variety of pteroylmonoglutamates as their preferred monoglutamate substrate, but use 5,10-methylenetetrahydropteroylpolyglutamates as their preferred, and sometimes only, polyglutamate substrate. Mono- and polyglutamyl forms of tetrahydrofolate are the preferred substrates of mammalian folylpolyglutamate synthetases.     

An inverse relationship of rat liver folate polyglutamate chain length to nutritional folate sufficiency

The relative concentrations of folylpolyglutamates of differing chain length in rat liver and the uptake of exogenous [3H]folic acid (20 microCi, 20 microgram) into liver folylpolyglutamates were examined in rats maintained on (a) standard and folate-supplemented standard diets and (b) semi-defined folate-sufficient and folate-deficient diets. Folylpolyglutamates extracted from liver were cleaved to p-aminobenzoylpolyglutamates which were separated by ion-exchange chromatography. The relative concentrations and ultimate radiolabeling of longer-chain folylpolyglutamates (six, seven and eight glutamate residues) were greatest in the livers of folate-deficient rats, whereas the intermediate-chain folylpolyglutamates (three, four and five glutamate residues) were the greatest portion of total liver folates of folate-supplemented rats. Thus, the length of the polyglutamate chain added to liver folates is inversely related to the total concentration of liver folates. These data suggest that folylpolyglutamate biosynthesis in the liver may be controlled by the liver folate concentrations. In folate insufficiency such a control mechanism would serve to enhance the affinity of folates for folate-dependent enzymes and to conserve the liver folate concentration.     

Folylpolyglutamates in Leishmania major

The intracellular folates of the protozoan parasite Leishmania major have been examined. About 95% of the exogenous [3H]folate accumulated by the protozoan is metabolized to polyglutamate conjugates within 65 hr, and the intracellular folates are about forty-fold concentrated over the folate in the medium. The predominant metabolite of folic acid is the pentaglutamate conjugate (85%), with lessor amounts of the tetraglutamate (approximately 9%) and hexaglutamate (approximately 3%), and trace (less than 2.5%) amounts of di-, tri- and hepta-glutamate conjugates. Chromatographic properties of the products indicate that the conjugates are linked through the gamma-carboxyl groups. The folylpolyglutamate distribution in Leishmania is similar to that found in mammalian tissues.     

Effects of growth rate and methotrexate on folate polyglutamates and folylpolyglutamate hydrolase activity in Krebs ascites cells

The activity of folylpolyglutamate hydrolase was measured throughout intraperitoneal growth of Krebs ascites cells in mice and after exposure to methotrexate. Hydrolase activity was lowest during the log phase of growth. Methotrexate administered intraperitoneally during log growth caused a dose- and time-dependent increase in hydrolase activity. Modest changes were observed in endogenous folate polyglutamate chain length distributions throughout growth and upon exposure to methotrexate, but these changes could not be correlated with hydrolase activity.     

Utilization of yeast polyglutamate folates in man.

Ingestion by healthy humans of small amounts of polyglutamate folates from yeast, equivalent to 300 mug of monoglutamate folate and containing 30 mug of "free folate," resulted in an appreciable elevation of the serum folate corresponding to 300 mug of synthetic pteroylmonoglutamate (PGA). Ingestion of higher amounts of polyglutamate folate did not result in higher serum folate elevations than did 300 mug. It is concluded that small amounts of polyglutamate folate from yeast are fully utilized, presumably by deconjugation in the gut prior to absorption. The relative ineffectiveness of larger doses of polyglutamate folates from yeast may be due to limiting conjugase activity in the gut, unfavorable conditions for its activity (such as unsuitable pH) or to an inhibitor of the enzyme present in impure preparations.     

Regulation of methotrexate polyglutaminate formation in Ehrlich ascites carcinoma cells by endogenous folate pool

The conversion of methotrexate to poly-gamma-glutamyl derivatives in Ehrlich ascites carcinoma cells which are characterized by different pools of endogenous folates is described. The cells in which folate pool was high (the 5-fluorodeoxy-uridine-resistant cell line) the ability to convert methotrexate to its polyglutamate derivatives was much lower than in the cells in which folate pool was smaller (the parental cell line). When the cellular folate pool was reduced by treatment of the cells with lysolecithin, a similar methotrexate polyglutamate concentration in both cell lines was observed. These data suggest that cellular folate pool has a regulatory effect on methotrexate polyglutamate synthesis.     

Effects of deuterium oxide on folate metabolism in Lactobacillus casei

Various folate coenzymes and their polyglutamyl derivatives involved in 1-carbon metabolism are modulated as a result of altered physiological states and also vary with respect to growth conditions. We studied the metabolic changes in folic acid and their conjugated polyglutamyl derivatives in Lactobacillus casei cells grown in the presence of D20. A 40% decrease in methyltetrahydrofolyl polyglutamate derivatives was observed in the cells grown in media prepared with D20-depleted water (D20 content, 8-10 ppm). Chromatographic analysis of folates showed significant alterations in the formyl- and methyltetrahydrofolate derivatives and their polyglutamylation profiles. Higher amounts of oxidized folates were also present in the cells grown in D20-depleted conditions. No significant changes were observed in folates and their polyglutamate derivatives when the cells were grown in the presence of 300, 450 and 600 ppm D20. The altered folate homoestasis is attributed to changes in the metabolic adaptation of cells to D20-depleted environment.