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PubMed2002

Role of the E45K-reduced folate carrier gene mutation in methotrexate resistance in human leukemia cells.

Resistance to the antifolate methotrexate (MTX) can cause treatment failure in childhood acute lymphoblastic leukemia (ALL). This may result from defective MTX accumulation due to alterations in the human reduced folate carrier (hRFC) gene. We have identified an hRFC gene point mutation in a transport-defective CCRF-CEM human T-ALL cell line resulting in a lysine to glutamic acid substitution at codon 45 (E45K), which has been identified in other antifolate-resistant sublines (JBC 273:30 189, 1998; JBC 275:30 855, 2000). To characterize the role of this mutation in MTX resistance, transfection experiments were performed using hRFC-null CCRF-CEM cells. E45K transfectants demonstrated an initial rate of MTX influx that was approximately 0.5-fold that of CCRF-CEM cells, despite marked protein overexpression. Cytotoxicity studies revealed partial reversal of MTX and raltitrexed resistance in E45K transfectants, while trimetrexate resistance was significantly increased. Kinetic analysis indicated only minor differences in MTX kinetics between wild-type and E45K hRFCs, however, K(i)s for folic acid and 5-formyltetrahydrofolate were markedly reduced for E45K hRFC. This was paralleled by increased folic acid transport and reduced synthesis of MTX polyglutamates. Collectively, the results demonstrate that expression of E45K hRFC leads to increased MTX resistance due to decreased membrane transport and, secondarily, from alterations in binding affinities and transport of folate substrates. However, despite these findings, we could find no evidence of this mutation in 121 childhood ALL samples, suggesting that it does not contribute to clinical MTX resistance in this disease.

Amino Acid Substitution↗
PubMed1972

Effects of testosterone on the metabolism of folate coenzymes in the rat.

1. The effects of castration and testosterone treatment on enzymic activities involved in folate coenzyme metabolism in the liver and in accessory sex organs of male adult rats were studied. 2. In the liver of castrated rats the concentration of 10-formyltetrahydrofolate (10-HCO-H(4)folate) synthetase and tetrahydrofolate (H(4)folate) dehydrogenase were significantly decreased whereas that of 5,10-methylenetetrahydrofolate dehydrogenase increased; the treatment with five doses of testosterone caused a return to normal values of these activities. 3. In the prostate of castrated rats a pronounced decrease in H(4)folate dehydrogenase, serine hydroxymethyltransferase and 10-HCO-H(4)folate synthetase activities was observed. The administration of testosterone restored the enzymic activities to normal values. 4. In the seminal vesicles of castrated rats only 10-HCO-H(4)folate synthetase was markedly depressed; testosterone treatment not only restored activity to normal values but raised it to higher than normal values. The slight changes observed in other enzymic activities also returned to normal values with the hormone treatment. 5. These results are discussed in relation to a possible control mechanism of folate metabolism by testosterone.

Animals↗
PubMed1973

Identification and measurement of the folates in sheep liver.

1. Methods are described for the extraction, separation by ion-exchange chromatography and estimation by microbiological assay of the folates in sheep liver. 2. Injection of [2-(14)C]-pteroylglutamate into a sheep fed on a stock diet led to extensive labelling of chromatographically separable liver folates. About 12% of the label in the liver could not be extracted by the method used. 3. Liver folates were examined in five ewes fed on restricted amounts of a diet of wheaten hay-chaff and gluten and injected weekly with vitamin B(12). Chromatographic separation was followed by microbiological assay with Lactobacillus casei, Streptococcus faecalis R. and Pediococcus cerevisiae both before and after treatment of fractions with conjugase (gamma-glutamylcarboxypeptidase). Evidence was obtained that the folates present were predominantly polyglutamate forms of tetrahydropteroylglutamate, 5-methyltetrahydropteroylglutamate and 5- (and 10-) formyltetrahydropteroylglutamates. Differences in the responses of the assay organisms permitted quantitative distinction between these three main classes of folates. 4. Methyltetrahydrofolates were eluted in seven successive peaks that were separated by constant increments in the logarithm of eluant [P(i)]. A similar relationship existed for seven successive peaks of tetrahydrofolate and may also have existed for each of the two series of formyltetrahydrofolates. 5. Based on these and other observations it is proposed that sheep liver folates consist predominantly of the mono- to hepta-glutamates of each of the reduced pteroates identified. The methods employed allowed quantitative determinations to be made of most of the folates present. The predominant forms were hexaglutamates. 6. Four components active for L. casei were detected that could not be identified. Three of them were polyglutamates.

Animal Feed↗
PubMed1973

Folic acid metabolism in vitamin B12-deficient sheep. Depletion of liver folates.

1. Metabolism of folate was studied in six ewes in an advanced state of vitamin B(12) deficiency as judged by voluntary food intake and in their pair-fed controls receiving vitamin B(12). A group of four animals that were maintained throughout the experiment at pasture was also studied. 2. After 34-40 weeks on the cobalt-deficient diet urinary excretion of formiminoglutamate by four deficient animals was about 3.2mmol/day and this was not significantly decreased by injection of three of them with about 4.5mug of [2-(14)C]folate/kg body weight per day for 5 days. Three days after the last injection retention of [2-(14)C]folate by the livers of the deficient animals (5.5% of the dose) was lower than that of their pair-fed controls (26% of the dose) but there was no evidence of net retention of injected folate in the livers of either group. Urinary excretion of (14)C indicated that renal clearance of folate may have been impaired in very severe vitamin B(12) deficiency. 3. As estimated by microbiological assays total folates in the livers of animals at pasture (12.9mug/g) included about 24% of 5-methyltetrahydrofolate as compared with about 72% of a total of 12.5mug/g in three further ewes fed on a stock diet of wheaten hay-chaff and lucerne-chaff. Liver folates of vitamin B(12)-deficient animals (0.5mug/g) included about 88% of 5-methyltetrahydrofolate as compared with about 51% of a total of 5.2mug/g in pair-fed animals treated with vitamin B(12). 4. Chromatography of liver folates of the pair-fed animals permitted quantitative estimates of the pteroylglutamates present. The results showed that the vitamin B(12)-deficient livers were more severely depleted of tetrahydrofolates and formyltetrahydrofolates than of methyltetrahydrofolates and that as the deficiency developed they were more severely depleted of the higher polyglutamates than of the monoglutamate within each of these classes. Results from animals injected with [2-(14)C]folate indicated an impairment of the exchange between pteroylmonoglutamates and pteroylpolyglutamates in the livers of deficient animals. 5. In vitamin B(12)-deficient animals with food intakes below 200g/day some of the liver folates were not completely reduced and some degradation of pteroylpolyglutamates was detected. The latter condition may have been associated with fatty liver. 6. The results are discussed in relation to current theories of vitamin B(12)-folate interactions.

Animals↗
PubMed1974

Folic acid metabolism in vitamin B12-deficient sheep. Effects of injected methionine on liver constituents associated with folate metabolism.

1. The effects of injected l-methionine (2g every second day for 28 days) on liver folates and other constituents of liver associated with folate metabolism were studied in vitamin B(12)-deficient ewes and their pair-fed controls receiving vitamin B(12). The dose rate of methionine used was sufficient to restore almost to normal the elevated excretion in the urine of formiminoglutamate in the deficient animals. 2. Liver folates active for Lactobacillus casei, Streptococcus faecalis R and Pediococcus cerevisiae were severely depressed in deficient livers and were partly restored by methionine. Analysis of the folates after ion-exchange chromatography showed that the major effect of methionine was to increase the concentrations of tetrahydrofolates and formyltetrahydrofolates. Methyltetrahydrofolates were also increased, but there was no effect of methionine on the small amounts of incompletely reduced folates present in deficient livers. The folates present were predominantly penta-, hexa- and hepta-glutamates whether or not animals received vitamin B(12) or methionine. 3. Concentrations of ATP, NAD(+), NADH and NADPH were lower in freeze-clamped liver from vitamin B(12)-deficient sheep than in liver from pair-fed, vitamin B(12)-treated sheep. These changes were not affected by methionine which was also without effect on the elevated K(+)/Na(+) ratios found in deficient livers. 4. The livers of vitamin B(12)-deficient animals contained lower concentrations of choline and higher concentrations of lipid than their pair-fed controls. These effects were reversed by methionine.

Adenosine Triphosphate↗
PubMed1976

One-carbon metabolism in Neurospora crassa wild-type and in mutants partially deficient in serine hydroxymethyltransferase.

1. The concentrations of folate-dependent enzymes in Neurospora crassa Lindegren A wild type (FGSC no. 853), Ser-l mutant, strain H605a (FGSC no. 118), and for mutant, strain C-24 (FGSC no. 9), were compared during exponential growth on defined minimal media. Both mutants were partially lacking in serine hydroxymethyltransferase, but contained higher concentrations of 10-formyltetrahydrofolate synthetase than did the wild type. Mycelia of the mutants contained higher concentrations of these enzymes when growth media were supplemented with 1mM-glycine. In the wild-type, this glycine supplement also increased the specific activities of 5,10-methylenetetrahydrofolate dehydrogenase and 5,10-methylenetetrahydrofolate reductase. 5. During growth, total folate and polyglutamyl folate concentrations were greatest in the wild-type. Methylfolates were not detected in mutant Ser-l, and were only present in the for mutant after growth in glycine-supplemented media. Exogenous glycine increased folate concentration threefold in the wild type, mainly owing to increases in unsubstituted polyglutamyl derivatives. 3. Feeding experiments using 14C-labelled substrates showed that C1 units were generated from formate, glycine and serine in the wild type. Greater incorporation of 14C occurred when mycelia were cultured in glycine-supplemented media. Formate and serine were precursors of C1 units in the mutants, but the ability to cleave glycine was slight or lacking.

Alcohol Oxidoreductases↗
PubMed1978

Rate-limiting factors in urate synthesis and gluconeogenesis in avian liver.

1. Urate synthesis and other metabolic characteristics of isolated chicken hepatocytes were studied. 2. The distinction is made between immediate precursors of the purine ring (glycine, glutamine, aspartate, formyltetrahydrofolate, bicarbonate) and ultimate precursors from which the immediate precursors are formed in the liver. 3. In hepatocytes from well-fed chickens the rate of urate synthesis was not greatly increased by the addition of amino acids or NH(4)Cl, but in hepatocytes from 72h-starved chickens the rate was much increased when alanine or asparagine was added as the only substrate. Other amino acids, when added alone, did not affect the rate. The exceptional effect of alanine and asparagine is due to the ready formation of the immediate precursors. 4. Conditions are described under which glutamine, serine, glycine plus formate, ribose and glucose increased the rate of urate synthesis. 5. At 1mm-NH(4)Cl (a concentration not much higher than that of blood plasma) the rate of urate synthesis in the presence of lactate was increased, but higher concentrations inhibited urate synthesis in the presence of lactate or alanine; with alanine even 1mm-NH(4)Cl was inhibitory. 6. Glucose synthesis from lactate, alanine or dihydroxyacetone was also inhibited by 1mm-NH(4)Cl. 7. NH(4)Cl inhibition of urate and glucose synthesis was paralleled by an increased rate of glutamine synthesis. Thus in the presence of NH(4)Cl the gluconeogenic precursors are diverted from the pathway of gluconeogenesis to that of glutamate and glutamine synthesis. This implies that the synthesis of these amino acids is the primary process in the detoxication of ammonia in the avian liver. 8. Urate synthesis, like urea synthesis, can be looked on as a cyclic process with either phosphoribosyl pyrophosphate or ribose acting as the carrier on which the purine ring is assembled. 9. The energy requirements of urate synthesis depend on whether phosphoribosyl pyrophosphate is regenerated from IMP by pyrophosphorylase or by phosphorylation and pyrophosphorylation of ribose. It is 6 or 9 pyrophosphate bonds of ATP respectively.

Acetates↗
PubMed1980

Purification and physicochemical, kinetic and immunological properties of allosteric serine hydroxymethyltransferase from monkey liver.

The homogeneous serine hydroxymethyltransferase purified from monkey liver, by the use of Blue Sepharose affinity chromatography, exhibited positive homotropic co-operative interactions (h = 2.5) with tetrahydrofolate and heterotropic interactions with L-serine and nicotinamide nucleotides. The enzyme had an unusually high temperature optimum of 60 degrees C and was protected against thermal inactivation by L-serine. The allosteric effects were abolished when the monkey liver enzyme was purified by using a heat-denaturation step in the presence of L-serine, a procedure adopted by earlier workers for the purification of this enzyme from mammalian and bacterial sources. The enzyme activity was inhibited completely by N5-methyltetrahydrofolate, N5-formyltetrahydrofolate, dichloromethotrexate, aminopterin and D-cycloserine, whereas methotrexate and dihydrofolate were partial inhibitors. The insoluble monkey liver enzyme-antibody complex was catalytically active and failed to show positive homotropic co-operative interactions with tetrahydrofolate (h = 1) and heterotropic interactions with NAD+. The enzyme showed a higher heat-stability in a complex with its antibody than as the free enzyme. These results highlight the pitfalls in using a heat-denaturation step in the purification of allosteric enzymes.

Allosteric Regulation↗
PubMed2003

Characterization of a cysteine-less human reduced folate carrier: localization of a substrate-binding domain by cysteine-scanning mutagenesis and cysteine accessibility methods.

The human reduced folate carrier (hRFC) mediates the transport of reduced folates and classical anti-folates into mammalian cells. Whereas the functionally important domains in hRFC are poorly characterized, previous studies with anti-folate-resistant cells suggest critical roles for transmembrane domain (TMD) 1 and residues (Gly44, Glu45, Ser46 and Ile48) in or flanking this region. An hRFC mutant devoid of cysteine residues was prepared by deleting the C-terminal 56 amino acids, including four cysteine residues, and mutagenizing the remaining cysteine residues to serine residues. A fully functional cysteine-less hRFC protein was expressed in transport-impaired MtxRIIOuaR2-4 Chinese-hamster ovary cells. To explore the role of residues in or flanking TMD1 in transport, all 24 amino acids from Trp25 to Ile48 of hRFC were mutated individually to cysteine residues, and the mutant hRFCs were transfected into MtxRIIOuaR2-4 cells. All of the 24 cysteine mutants were expressed and, with the exception of R42C (Arg42-->Cys), were capable of mediating methotrexate uptake above the low level in MtxRIIOuaR2-4 cells. We found that by treating the transfected cells with the small, water-soluble, thiol-reactive anionic reagent, sodium (2-sulphonatoethyl) methanethiosulphonate, methotrexate transport by several of the cysteine-substituted hRFC mutants was significantly inhibited, including Q40C, G44C, E45C and I48C. Sodium (2-sulphonatoethyl) methanethiosulphonate transport inhibition of the Q40C, G44C and I48C mutants was protected by leucovorin [(6R, S)-5-formyltetrahydrofolate], indicating that these residues lie at or near a substrate-binding site. Using surface-labelling reagents [N-biotinylaminoethyl methanethiosulphonate and 3-(N-maleimidylpropionyl)biocytin, combined with 4-acetamido-4'-maleimidylstilbene-2,2'-disulphonic acid] with cysteine mutants from positions 37-48, the extracellular TMD1 boundary was found to lie between residues 39 and 40, and amino acids 44-46 and 48 were localized to the TMD1 exofacial loop. Collectively, our results imply that amino acids 40, 44, 48 and, possibly, 42 serve important roles in hRFC transport, albeit not as structural components of the putative transmembrane channel for folate substrates.

Amino Acid Sequence↗
PubMed1985

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.

Amide Synthases↗
PubMed1992

Immunoreactive folate-binding proteins from human saliva. Isolation and comparison of two distinct species.

Human saliva contains a single 72,000-M(r) species which specifically reacted with rabbit anti-[human placental folate receptor (PFR)] serum on SDS/PAGE and Western blots. Although a specific radioimmunoassay for human PFR and related folate-binding proteins (FBPs) identified 55 ng of cross-reacting material (CRM) per mg of crude salivary proteins, only a minor fraction (1.6 ng) specifically bound radiolabelled folate. The major fraction of CRM did not contain bound endogenous folate and did not bind radiolabelled folates. On the basis of folate binding, salivary CRM species to PFR were designated as either functional (f-FBP) or non-functional (nf-FBP) species respectively. nf-FBPs and f-FBPs were isolated by different purification schemes. Both purified f-FBPs and nf-FBPs migrated as a single apparent 72,000-M(r) species on SDS/PAGE, but on Sephacryl S-200 gel filtration and sucrose-density-gradient ultracentrifugation they were eluted/sedimented with 40,000-M(r) markers. Each microgram of purified f-FBP and nf-FBP was measured in the radioimmunoassay for PFR as being equivalent to 18 ng and 24 ng of CRM respectively, indicating low epitope-relatedness to PFR. The Kd of f-FBPs was 50 pM and 0.94 mol of folate was bound/mol of protein. f-FBPs exhibited an unusual dependence on Triton X-100 for optimal ligand binding, despite the fact that Triton X-100 micelle binding was not demonstrated. The relative order of affinity of f-FBPs for pteroylglutamate greater than methotrexate greater than 5-formyltetrahydrofolate greater than 5-methyltetrahydrofolate was also distinct from that of purified PFR. Whereas amino acid and carbohydrate analysis revealed that nf-FBP (M(r) 51,400) and f-FBP (M(r) 39,200) were distinct glycoproteins with 8 and 13% carbohydrate respectively, isoelectric focusing and immunological studies suggested some structural identity. The presence of f-FBP and nf-FBP in normal saliva raises new questions about their possible role in vivo.

Adult↗
PubMed1977

Effect of anticonvulsants on the uptake of 5-methyltetrahydrofolic acid by the choroid plexus in rabbits.

1. The isolated choroid plexus of the rabbit takes up 5-methyltetrahydrofolate from the incubation medium. 2. Other folate analogues (pteroylglutamic acid, methotrexate, 5-formyltetrahydrofolate = folinic acid) inhibited the uptake of 5-methyltetrahydrofolate. 3. The uptake of 5-methyltetrahydrofolate was inhibited by low temperature, anaerobic conditions and dinitrophenol. 4. The anticonvulsant drugs, diphenylhydantoin and phenobarbital, had no effect on 5-methyltetrahydrofolate uptake. 5. The inhibitory effect of pteroylglutamic acid on the uptake of 5-methyltetrahydrofolate by the choroid plexus may explain the effect of long-term folic acid therapy in aggravating vitamin B12 neuropathy in pernicious anaemia.

Animals↗
PubMed2001

Rapid communication: L-folinic acid versus folic acid for the treatment of hyperhomocysteinemia in hemodialysis patients.

BACKGROUND: The hyperhomocysteinemia found in most hemodialysis patients is refractory to combined oral B-vitamin supplementation featuring supraphysiological doses of folic acid (FA). We evaluated whether a high-dose L-folinic acid-based regimen provided improved total homocysteine (tHcy)-lowering efficacy in chronic hemodialysis patients, as suggested by a recent uncontrolled report. METHODS: We block-randomized 48 chronic, stable hemodialysis patients based on their screening predialysis tHcy levels, sex, and dialysis center into two groups of 24 subjects treated for 12 weeks with oral FA at 15 mg/day or an equimolar amount (20 mg/day) of oral L-folinic acid (FNA) [L-5-formyltetrahydrofolate]. All 48 subjects also received 50 mg/day of oral vitamin B6 and 1.0 mg/day of oral vitamin B12. RESULTS: The mean percentage (%) reductions (with 95% CIs) in predialysis tHcy were not significantly different [FNA = 22.1% (11.8 to 31.4%), FA = 20.7% (11.7 to 30.5%), P = 0.950 by paired t test]. Final on-treatment values (mean with 95% CI) were as follows: FNA, 15.9 micromol/L (14.0 to 18.0); FA, 16.9 micromol/L (14.8 to 18.8). Moreover, in those subjects with baseline tHcy levels >/=14 micromol/L, neither treatment resulted in "normalization" of tHcy levels (that is, final on-treatment values <12 micromol/L) among a significantly different or clinically meaningful number of patients [FNA = 2 out of 22 (9.1%); FA = 2 out of 24 (8.3%); Fisher's exact test of between groups difference, P = 1.000]. CONCLUSIONS: Relative to high-dose FA, high-dose oral L-folinic acid-based supplementation does not afford improved tHcy-lowering efficacy in hemodialysis patients. The preponderance of hemodialysis patients (that is,> 90%) exhibits mild hyperhomocysteinemia refractory to treatment with either regimen.

Aged↗
PubMed1978

Demonstration of high-affinity folate binding activity associated with the brush border membranes of rat kidney.

Folate binding activity of high affinity was identified in the particulate fractions of rat kidney homogenates. This binding activity cofractionated with alkaline phosphatase and maltase, two brush border membranes markers. With an enriched preparation of brush border membranes, freed of endogenous folate by acid treatment, the binding of [3H]olate was found to be saturable (Kb = 4.2 X 10(-11)M) and rapid. Binding was optimal at pH 6.4-7.7. At neutral pH, competition for binding with [3H]folic acid required 1.45 equivalents of pteroylheptaglutamate, 6.25 equivalents of N5-methyltetrathydrofolate, 29 equivalents of methotrexate, and 125 equivalents of N5-formyltetrahydrofolate. At alkaline pH, N5-methyltetrahydrofolate was as effective a competitor as folic acid. In view of reports that renal tubular reabsorption of folate includes an initial tight binding step, the binding activity associated with the brush border membranes may participate in this process.

Animals↗
PubMed1979

Role of thymidylate synthetase activity in development of methotrexate cytotoxicity.

Methotrexate (MTX) inhibition of the growth of mouse or human leukemia cells in culture was partially prevented by either thymidine (dThd) or hypoxanthine. 5-Fluoro-2'-deoxyuridine (FdUrd) also decreased the growth-inhibitory potency of MTX in the presence of small concentrations of 5-formyltetrahydrofolate (citrovorum factor) and sufficient exogenous dThd to support the synthesis of thymidylate nucleotides by salvage mechanisms. In addition, citrovorum factor-induced reversal of MTX was several orders of magnitude more efficient in the presence of both FdUrd and dThd than in the presence of dThd alone or in the absence of both nucleosides. Likewise, the presence of FdUrd (3 microM) and dThd (5.6 microM) completely prevented the lethality of 0.3 mM MTX to L1210 cells in culture medium supplemented with micromolar concentrations of citrovorum factor. We propose that this protection against the cytotoxic effects of MTX by dThd, hypoxanthine, and FdUrd have a common biochemical mechanism--namely, inhibition of the de novo synthesis of thymidylate by either a direct [FdUrd; inhibition of thymidylate synthetase (thymidylate synthase; 5,10-methylenetetrahydrofolate:dUMP C-methyl-transferase, EC 2.1.1.45)] or indirect (dThd and hypoxanthine; feedback inhibition by anabolites on ribonucleotide reductase and deoxycytidylate deaminase) effect. The resultant decreased rate of loss of reduced folates due to de novo thymidylate synthesis would allow a higher degree of inhibition of dihydrofolate reductase to be endured without damage to the cell.

Animals↗
PubMed1990

Cloning of three human multifunctional de novo purine biosynthetic genes by functional complementation of yeast mutations.

Functional complementation of mutations in the yeast Saccharomyces cerevisiae has been used to clone three multifunctional human genes involved in de novo purine biosynthesis. A HepG2 cDNA library constructed in a yeast expression vector was used to transform yeast strains with mutations in adenine biosynthetic genes. Clones were isolated that complement mutations in the yeast ADE2, ADE3, and ADE8 genes. The cDNA that complemented the ade8 (phosphoribosylglycinamide formyltransferase, GART) mutation, also complemented the ade5 (phosphoribosylglycinamide synthetase) and ade7 [phosphoribosylaminoimidazole synthetase (AIRS; also known as PAIS)] mutations, indicating that it is the human trifunctional GART gene. Supporting data include homology between the AIRS and GART domains of this gene and the published sequence of these domains from other organisms, and localization of the cloned gene to human chromosome 21, where the GART gene has been shown to map. The cDNA that complemented ade2 (phosphoribosylaminoimidazole carboxylase) also complemented ade1 (phosphoribosylaminoimidazole succinocarboxamide synthetase), supporting earlier data suggesting that in some organisms these functions are part of a bifunctional protein. The cDNA that complemented ade3 (formyltetrahydrofolate synthetase) is different from the recently isolated human cDNA encoding this enzyme and instead appears to encode a related mitochondrial enzyme.

Acyltransferases↗
PubMed1992

Structures of apo and complexed Escherichia coli glycinamide ribonucleotide transformylase.

The three-dimensional structure of phosphoribosylglycinamide formyltransferase (10-formyltetrahydrofolate:5'-phosphoribosylglycinamide formyltransferase, EC 2.1.2.2) has been solved both as an apoenzyme at 2.8-A resolution and as a ternary complex with the substrate glycinamide ribonucleotide and a folate inhibitor at 2.5-A resolution. The structure is a modified doubly wound alpha/beta sheet with flexibility in the active site, including a disordered loop in the apo structure, which is ordered in the ternary complex structure. This enzyme is a target for anti-cancer therapy and now for structure-based drug design.

Acyltransferases↗
PubMed1995

Regulation of Saccharomyces cerevisiae flavohemoglobin gene expression.

The Saccharomyces cerevisiae hemoglobin is a flavoprotein of unknown function. It shares extensive sequence homology with the globin of Candida as well as those of several bacterial species. We have studied its gene regulation in order to better understand its purpose in the cell. Transcriptional analyses indicate that, in sharp contrast to the bacterial globins of Vitreoscilla and Alcaligenes eutrophus, the S. cerevisiae globin message is induced during logarithmic growth and under oxygen-replete conditions. Transcription of the S. cerevisiae hemoglobin gene is positively regulated by the transcription factors heme-activated protein (HAP) 1 and HAP2/3/4, which respond to intracellular heme levels. Anaerobically, there is a low level, HAP-independent induction of hemoglobin mRNA. Unlike other systems influenced by the HAP2/3/4 transcription factor complex, no activation of hemoglobin expression by growth in non-fermentable carbon sources is observed. Flavohemoglobin gene disruption does not alter cell viability or growth in a variety of oxygen conditions and carbon sources. Physical and genetic mapping of the S. cerevisiae flavohemoglobin gene places it on chromosome seven near the formyltetrahydrofolate synthase (ADE3) locus. These data indicate that, despite the high degree of homology, the S. cerevisiae globin may have a function distinct from those proposed for bacterial globins.

Base Sequence↗