Dihydrofolate Synthetase Research Articles

Effect and Mechanism of Cotrimoxazole Against Talaromyces marneffei in vitro.

Talaromyces marneffei (formerly Penicillium marneffei) is an important thermally dimorphic fungus endemic which is characterized by one of the most frequent opportunistic infections in HIV/AIDS patients, mainly prevalent in Southeast Asia, southern China, and northeastern India. Cotrimoxazole(CTX) inhibits folic acid synthesis which is important for the survival of many bacteria, protozoa, and fungi, thereby commonly used to prevent several opportunistic infections among HIV/AIDS patients. In addition to preventing other HIV-associated opportunistic infections, CTX prophylaxis are considered to have the potential to prevent T. marneffei infection in HIV/AIDS patients receiving antiretroviral therapy (ART). However, the effect of cotrimoxazole towards T. marneffei fungus in vitro remains unclear. Human THP-1 macrophages were used as cell model in vitro to explore the effect and mechanism of cotrimoxazole resistance towards T. marneffei. Cell viability assay and drug sensitivity colony forming units (CFU) experiments were conducted to determine the minimum inhibitory concentration (MIC) of cotrimoxazole inside and outside THP-1 macrophages respectively. Enzyme-linked immunosorbent assay (Elisa) was used to measure the concentration of Dihydropteroic acid synthetase (DHPS), Dihydrofolate synthetase (DHFS) and Dihydrofolate reductase (DHFR) between T. marneffei adding TMP/SMX and without adding TMP/SMX group respectively. Real-time fluorescence quantitative PCR(qPCR) was performed to detect the mRNA expression levels in Dectin-1 mediated signaling pathway and downstream inflammatory cytokines including IL-6, IL-10, IL-23A, CXCL8 and TNF-α released by T. marneffei-infected macrophages between adding TMP/SMX and without adding TMP/SMX group respectively. Cotrimoxazole can inhibit the proliferation of T. marneffei within safe concentration inside and outside THP-1 macrophages. Drug susceptibility results showed the minimal inhibit concentration(MIC) of 1:5 TMP/SMX was ranging from 14/70 to 68/340μg/ml. The MIC of SMX was ranging from 100 to 360μg/ml. The MIC of TMP was ranging from 240 to 400μg/ml outside macrophages. The MIC of TMP/SMX was ranging from 36/180 to 68/340μg/ml. The MIC of SMX was ranging from 340 to 360μg/ml. The MIC of TMP was ranging from 320 to 400μg/ml inside macrophages. The synergistic interaction of 1:5 TMP/SMX was more effective in inhibiting T. marneffei than separate SMX and TMP. DHPS, DHFS and DHFR can be inhibited by cotrimoxazole within safe and effective concentration. Dectin-1 expression is increased following T. marneffei infection, leading to the increase of IL-6, IL-10, IL-23A and the decrease of CXCL8 and TNF-α. Conversely, cotrimoxazole decrease the levels of Dectin-1, IL-6, IL-10, IL-23A and increase the levels of CXCL8 and TNF-α, thereby enhancing the intracellular killing-T. marneffei capacity of macrophages. Our findings indicated that cotrimoxazole directly inhibited T. marneffei growth by blocking DHPS, DHFS and DHFR and indirectly inhibited T. marneffei growth perhaps by regulating the Dectin-1 signaling pathway, which may effectively interfere with the defense ability of the host against T. marneffei infection.

Characterization of newly established Pralatrexate-resistant cell lines and the mechanisms of resistance

BackgroundPralatrexate (PDX) is a novel antifolate approved for the treatment of patients with relapsed/refractory peripheral T-cell lymphoma, but some patients exhibit intrinsic resistance or develop acquired resistance. Here, we evaluated the mechanisms underlying acquired resistance to PDX and explored potential therapeutic strategies to overcome PDX resistance.MethodsTo investigate PDX resistance, we established two PDX-resistant T-lymphoblastic leukemia cell lines (CEM and MOLT4) through continuous exposure to increasing doses of PDX. The resistance mechanisms were evaluated by measuring PDX uptake, apoptosis induction and folate metabolism-related protein expression. We also applied gene expression analysis and methylation profiling to identify the mechanisms of resistance. We then explored rational drug combinations using a spheroid (3D)-culture assay.ResultsCompared with their parental cells, PDX-resistant cells exhibited a 30-fold increase in half-maximal inhibitory concentration values. Induction of apoptosis by PDX was significantly decreased in both PDX-resistant cell lines. Intracellular uptake of [14C]-PDX decreased in PDX-resistant CEM cells but not in PDX-resistant MOLT4 cells. There was no significant change in expression of dihydrofolate reductase (DHFR) or folylpolyglutamate synthetase (FPGS). Gene expression array analysis revealed that DNA-methyltransferase 3β (DNMT3B) expression was significantly elevated in both cell lines. Gene set enrichment analysis revealed that adipogenesis and mTORC1 signaling pathways were commonly upregulated in both resistant cell lines. Moreover, CpG island hypermethylation was observed in both PDX resistant cells lines. In the 3D-culture assay, decitabine (DAC) plus PDX showed synergistic effects in PDX-resistant cell lines compared with parental lines.ConclusionsThe resistance mechanisms of PDX were associated with reduced cellular uptake of PDX and/or overexpression of DNMT3B. Epigenetic alterations were also considered to play a role in the resistance mechanism. The combination of DAC and PDX exhibited synergistic activity, and thus, this approach might improve the clinical efficacy of PDX.

A facile reaction to access novel structural sulfonyl-hybridized imidazolyl ethanols as potential DNA-targeting antibacterial agents

A novel type of sulfonyl-hybridized imidazolyl ethanols as potential DNA-targeting antibacterial agents was constructed via the unique ring-opened reaction of oxiranes by imidazoles for the first time. Some developed target hybrids showed potential antimicrobial potency against the tested microbes. Especially, imidazole derivative 5f could strongly suppressed the growth of MRSA (MIC = 4 μg/mL), which was 2-fold and 16-fold more potent than the positive control sulfathiazole and norfloxacin. This compound exhibited quite low propensity to induce bacterial resistance. Antibacterial mechanism exploration indicated that compound 5f could embed in MRSA DNA to form steady 5f-DNA complex, which possibly hinder DNA replication to exert antimicrobial behavior. Molecular docking showed that molecule 5f could bind with dihydrofolate synthetase through hydrogen bonds. These results implied that imidazole derivative 5f could be served as a promising molecule for the exploration of novel antibacterial candidates.

Synthesis, Biological Evaluation and in Silico Studies of Several Substituted Benzene Sulfonamides as Potential Antibacterial Agents

Four substituted benzene sulfonamides were synthesized to obtain antibacterial agents. The antibacterial activities were evaluated by the cup plate method. Compound 2d showed the most obvious inhibitory effect on seven kinds of strains with MIC ranging from 16 to 64µg/mL. The inhibitory effect of compound 2d on A. niger, C. albicans, B. subtilis, and S. aureus was better than that of the control drug SAN. Moreover, the cytotoxicity was evaluated by MTT assay. Finally, the binding patterns of the compounds with dihydrofolate synthetase and the prediction of oral bioavailability were preliminarily studied by computer simulation. The molecular docking result revealed that compound 2d had a stronger interaction with dihydrofolate synthetase than SAN. The oral bioavailability prediction by Molinspiration online program proved that these compounds exhibited drug-like properties. In conclusion, compound 2d is promising as a new antibacterial agent.

Structural Analyses of Helicobacter Pylori FolC Conducting Glutamation in Folate Metabolism

FolC plays important roles in the folate metabolism of cells by attaching l-Glu to dihydropteroate (DHP) and folate, which are known activities of dihydrofolate synthetase (DHFS) and folylpolyglutamate synthetase (FPGS), respectively. Here, we determined the crystal structure of Helicobacter pylori FolC (HpFolC) at 1.95 Å resolution using the single-wavelength anomalous diffraction method. HpFolC has globular N- and C-terminal domains connected by a single loop, and a binding site for ATP is located between the two domains. Apo-HpFolC was crystallized in the presence of citrate in a crystallization solution, which was held in the ATP-binding site. Structural motifs such as the P-loop and Ω-loop of HpFolC for binding of ATP and two magnesium ions are well conserved in spite of the low overall sequence similarity to other FolC/FPGSs. The Ω-loop would also recognize a folate molecule, and the DHP-binding loop of HpFolC is expected to exhibit a unique recognition mode on DHP, compared with other FolCs. Because human FolC is known to only exhibit FPGS activity, the DHFS activity of bacterial FolC is an attractive target for the eradication of pathogenic bacteria. Consequently, our structural analyses of HpFolC provide a valuable foundation for a universal antibacterial strategy against H. pylori as well as other pathogenic bacteria.

Prevention of Malaria in Pregnancy

Malaria remains one of the most preventable causes of adverse birth outcomes. Intermittent preventive treatment in pregnancy (IPTp) with sulfadoxine-pyrimethamine is used to prevent malaria, but resistance to this drug combination has decreased its efficacy and new alternatives are needed. In Africa, a meta-analysis showed three-course or monthly IPTp with sulfadoxine-pyrimethamine to be safe and more effective than the original two-course sulfadoxine-pyrimethamine strategy, prompting WHO to update its policy in 2012. Although resistance to sulfadoxine-pyrimethamine reduces the parasitological efficacy of IPTp, this drug combination remains associated with reduced incidence of low birthweight in areas where prevalence of parasites with quintuple Plasmodium falciparum dihydrofolate reductase (Pfdhfr) and dihydropteroate synthetase (Pfdhps) mutations is greater than 90%. Nevertheless, its effectiveness is compromised in women infected with sextuple mutant parasites. Six trials of IPTp showed that neither amodiaquine, mefloquine, nor chloroquine-azithromycin are suitable replacements for sulfadoxine-pyrimethamine because of poor tolerability. Furthermore, four trials showed that intermittent screening and treatment with the current generation of malaria rapid diagnostic tests was not a suitable alternative strategy to IPTp with sulfadoxine-pyrimethamine, even in areas with high prevalence of quintuple mutations. Two trials showed that IPTp with dihydroartemisinin-piperaquine was well tolerated, effective, and acceptable for IPTp, with monthly regimens being the most effective. Coverage of IPTp and insecticide-treated nets continues to lag behind targets. The key barriers to uptake are well documented, and many are open to intervention. Outside of Africa, a single trial suggests a potential role for integrated approaches that combine sulfadoxine-pyrimethamine with azithromycin for IPTp in areas of Papua New Guinea where malaria transmission is high. Modelling analysis suggests the importance of the prevention of malaria early in pregnancy and the need to protect pregnant women declines more slowly than the rate at which transmission declines. Improved funding has led to an increase in the number of prevention trials in the past decade, showing the value of more sustained protection with monthly IPTp regimens. There is a need for confirmatory trials of the safety, efficacy, and feasibility of IPTp with dihydroartemisinin-piperaquine, for studies of intermittent screening and treatment with more sensitive rapid diagnostic tests, for studies of integrated strategies for malaria and other co-infections, and for studies of prevention strategies for malaria in pregnant women who are HIV-positive and living outside of Africa. Additional research is required on how to improve uptake of WHO's updated policy on IPTp with sulfadoxine-pyrimethamine and insecticide-treated nets.

Approaches to Controlled Co-Amplification of Genes for Production of Biopharmaceuticals: Study of the Insertion and Amplification Dynamics of Genetic Cassettes in the Genome of Chinese Hamster Ovary Cells during Co-Expression of Compatible Pair of Plasmids.

Plasmid vector family p1.1 based on non-coding regions of Chinese hamster housekeeping gene EEF1A and concatemer of Epstein-Barr virus terminal repeat increases the frequency of genome integration and provides rapid amplification of the target genes in the genome. For a pair of fluorescent proteins eGFP and mCherry it was shown that p1.1 vectors bearing dihydrofolate reductase and glutamine synthetase selection markers upon co-transfection into CHO DG44 cell line allow obtaining a polyclonal cell population in which ~70% of cells express both genes. The subsequent one-step gene amplification of the genome-integrated genetic cassettes under the selective pressure of increased concentrations of methotrexate can increase the expression of both integrated genes up to 8.2% eGFP and 9.9% mCherry of total protein. This approach can be used for the development of cell lines for the production of functional heterodimeric proteins, e.g. polypeptide hormones and therapeutic antibodies.

Tetrahydrofolate Modulates Floral Transition through Epigenetic Silencing.

Folates, termed from tetrahydrofolate (THF) and its derivatives, function as coenzymes in one-carbon transfer reactions and play a central role in synthesis of nucleotides and amino acids. Dysfunction of cellular folate metabolism leads to serious defects in plant development; however, the molecular mechanisms of folate-mediated cellular modifications and physiological responses in plants are still largely unclear. Here, we reported that THF controls flowering time by adjusting DNA methylation-regulated gene expression in Arabidopsis (Arabidopsis thaliana). Wild-type seedlings supplied with THF as well as the high endogenous THF content mutant dihydrofolate synthetase folypoly-Glu synthetase homolog B exhibited significant up-regulation of the flowering repressor of Flowering Wageningen and thereby delaying floral transition in a dose-dependent manner. Genome-wide transcripts and DNA methylation profiling revealed that THF reduces DNA methylation so as to manipulate gene expression activity. Moreover, in accompaniment with elevated cellular ratios between monoglutamylated and polyglutamylated folates under increased THF levels, the content of S-adenosylhomo-Cys, a competitive inhibitor of methyltransferases, was obviously higher, indicating that enhanced THF accumulation may disturb cellular homeostasis of the concerted reactions between folate polyglutamylation and folate-dependent DNA methylation. In addition, we found that the loss-of-function mutant of CG DNA methyltransferase MET1 displayed much less responsiveness to THF-associated flowering time alteration. Taken together, our studies revealed a novel regulatory role of THF on epigenetic silencing, which will shed lights on the understanding of interrelations in folate homeostasis, epigenetic variation, and flowering control in plants.

Antibody-membrane switch (AMS) technology for facile cell line development.

Generation of high-productivity cell lines remains a major bottleneck in therapeutic antibody development. Conventional cell line development often depends on gene amplification methodologies using dihydrofolate reductase or glutamine synthetase. Higher productivity is associated with an increased gene copy number. However, lack of selection pressure under the conditions of large-scale manufacturing leads to clonal instability. We have developed a novel method for cell line development, antibody-membrane switch (AMS) technology, that does not rely on gene amplification. This fluorescence-activated cell sorting (FACS)-based, high-throughput method is facilitated by cell-surface antibody expression to rapidly and efficiently isolate high-producing cells. The switch between membrane expression and secretion is achieved by alternative splicing and specific DNA recombination. The antibody of interest is initially displayed on the cell surface to facilitate FACS. Isolated high-producing cells are then seamlessly transformed into production cells after removing the membrane-anchoring domain sequence with a DNA recombinase. AMS technology has been applied in a number of antibody cell line development projects, which typically last 2-3 months. The top production cell lines exhibit very high specific productivity of 40-60 pg/cell/day resulting in production titers of 2-4 g/l in 10-day batch culture.

Role of Dihydroquinonoid Formation in Substrate Specificity of Escherichia coli Dihydrofolate Synthetase‐ Folylpolyglutamate Synthetase.

Escherichia coli dihydrofolate synthetase‐folylpolyglutamate synthetase (DHFS‐FPGS) (FolC) catalyzes the ATP‐dependent addition of glutamate to both dihydropteroate and pteroylglutamate substrates. The mechanism used by the enzyme to differentiate between folate and pteroate substrates was investigated using equilibrium binding and rapid‐reaction kinetic techniques, and structural modeling approaches. The absorbance spectrum of dihydropteroate displays a 40 nm red shift upon binding to DHFS‐FPGS· ATP, and the bound dihydropteroate exhibits increased fluorescence. Spectral and steady‐state kinetic data suggests that DHFS‐FPGS·ATP catalyzes the rapid formation of quinonoid dihydropteroate by removal of the N‐3 nitrogen proton and protonation of the C‐6 carbon of dihydropteroate. A similar spectral shift was observed for dihydrofolate when bound to enzyme in the presence of ATP or ADP. Rapid‐reaction kinetic studies demonstrate that the DHFS‐FPGS·ADP catalyzed formation of dihydrofolate quinonoid is associated with a conformational change in the enzyme. The results of this study suggest that the enzyme‐catalyzed formation of the quinonoid structure and the associated conformational change in the enzyme is required for effective DHFS activity but inhibitory for FPGS activity. These studies were supported by NIH R37DK58144 to PJS.

ISOLATION, IDENTIFICATION AND PARTIAL PURIFICATION OF DIHYDROFOLATE SYNTHETASE FROM HYPOTYL STEMS CALLUS OF LETTUSE PLANT ( Lactuca sativa L. )

Callus from hypocotyl explants of lettuce ( Lactuca sativa L.) was initiated on a modified Murashige and Skoog medium containing 1.0 mg/l of benzyladenine (BA) and naphthaleneacetic acid ( NAA ) .Dihydrofolate synthetase (EC.6.3.2.12) was found in lettuce callus and purified about 36 fold by treating with ethanol and chloroform, ammonium sulfate and gel filtration on Sephadex G-200. The molecular weight of dihydrofolate synthetase was found to be around 40 KD. By Sephadex G-200 column chromatography. The optimum pH for the activity was 6.8 . The optimum reaction temperature was 32C°. The Michaelis constants using dihydropetroic acid and adenosinetriphosphate were 0.036x10-3M and 5.263x10-3M respectively.

Mutagenesis of Folylpolyglutamate Synthetase Indicates That Dihydropteroate and Tetrahydrofolate Bind to the Same Site

The folylpolyglutamate synthetase (FPGS) enzyme of Escherichia coli differs from that of Lactobacillus casei in having dihydrofolate synthetase activity, which catalyzes the production of dihydrofolate from dihydropteroate. The present study undertook mutagenesis to identify structural elements that are directly responsible for the functional differences between the two enzymes. The amino terminal domain (residues 1-287) of the E. coli FPGS was found to bind tetrahydrofolate and dihydropteroate with the same affinity as the intact enzyme. The domain-swap chimera proteins between the E. coli and the L. casei enzymes possess both folate or pteroate binding properties and enzymatic activities of their amino terminal portion, suggesting that the N-terminal domain determines the folate substrate specificity. Recent structural studies have identified two unique folate binding sites, the omega loop in L. casei FPGS and the dihydropteroate binding loop in the E. coli enzyme. Mutants with swapped omega loops retained the activities and folate or pteroate binding properties of the rest of the enzyme. Mutating L. casei FPGS to contain an E. coli FPGS dihydropteroate binding loop did not alter its substrate specificity to using dihydropteroate as a substrate. The mutant D154A, a residue specific for the dihydropteroate binding site in E. coli FPGS, and D151A, the corresponding mutant in the L. casei enzyme, were both defective in using tetrahydrofolate as their substrate, suggesting that the binding site corresponding to the E. coli pteroate binding site is also the tetrahydrofolate binding site for both enzymes. Tetrahydrofolate diglutamate was a slightly less effective substrate than the monoglutamate with the wild-type enzyme but was a 40-fold more effective substrate with the D151A mutant. This suggests that the 5,10-methylenetetrahydrofolate binding site identified in the L. casei ternary structure may bind diglutamate and polyglutamate folate derivatives.

Folate Biosynthesis, Reduction, and Polyglutamylation and the Interconversion of Folate Derivatives.

Many microorganisms and plants possess the ability to synthesize folic acid derivatives de novo, initially forming dihydrofolate. All the folic acid derivatives that serve as recipients and donors of one-carbon units are derivatives of tetrahydrofolate, which is formed from dihydrofolate by an NADPH-dependent reduction catalyzed by dihydrofolate reductase (FolA). This review discusses the biosynthesis of dihydrofolate monoglutamate, its reduction to tetrahydrofolate monoglutamate, and the addition of glutamyl residues to form folylpolyglutamates. Escherichia coli and Salmonella, like many microorganisms that can synthesize folate de novo, appear to lack the ability to transport folate into the cell and are thus highly susceptible to inhibitors of folate biosynthesis. The review includes a brief discussion of the inhibition of folate biosynthesis by sulfa drugs. The folate biosynthetic pathway can be divided into two sections. First, the aromatic precursor chorismate is converted to paminobenzoic acid (PABA) by the action of three proteins. Second, the pteridine portion of folate is made from GTP and coupled to PABA to generate dihydropteroate, and the bifunctional protein specified by folC, dihydrofolate synthetase, or folylpolyglutamate synthetase, adds the initial glutamate molecule to form dihydrofolate (H2PteGlu1, or dihydropteroylmonoglutamate). Bacteriophage T4 infection of E. coli has been shown to cause alterations in the metabolism of folate derivatives. Infection is associated with an increase in the chain lengths in folylpolyglutamates and particularly the accumulation of hexaglutamate derivatives.

Island-wide diversity in single nucleotide polymorphisms of the Plasmodium vivax dihydrofolate reductase and dihydropteroate synthetase genes in Sri Lanka

BackgroundSingle nucleotide polymorphisms (SNPs) in the Plasmodium vivax dihydrofolate reductase (Pfdhfr) and dihydropteroate synthetase (Pvdhps) genes cause parasite resistance to the antifolate drug combination, sulphadoxine/pyrimethamine (SP). Monitoring these SNPs provide insights into the level of drug pressure caused by SP use and presumably other antifolate drugs. In Sri Lanka, chloroquine (CQ) with primaquine (PQ) and SP with PQ is used as first and second line treatment, respectively, against uncomplicated Plasmodium falciparum and/or P. vivax infections. CQ/PQ is still efficacious against P. vivax infections, thus SP is rarely used and it is assumed that the prevalence of SNPs related to P. vivax SP resistance is low. However, this has not been assessed in Sri Lanka as in most other parts of Asia. This study describes the prevalence and distribution of SNPs related to P. vivax SP resistance across Sri Lanka.Subjects and methodsP. vivax-positive samples were collected from subjects presenting at government health facilities across nine of the major malaria endemic districts on the island. The samples were analysed for SNPs/haplotypes at codon 57, 58, 61 and 117 of the Pvdhfr gene and 383, 553 and 585 of the Pvdhps gene by applying PCR followed by a hybridization step using sequence specific oligonucleotide probes (SSOPs) in an ELISA format.ResultsIn the study period, the government of Sri Lanka recorded 2,149 P. vivax cases from the nine districts out of which, 454 (21.1%) blood samples were obtained. Pvdhfr haplotypes could be constructed for 373 of these. The FSTS wild-haplotype was represented in 257 samples (68.9%), the double mutant LRTS haplotype was the most frequently observed mutant (24.4%) while the triple mutation (LRTN) was only identified once. Except for two samples of the single mutated Pvdhps GAV haplotype, the remaining samples were wildtype. Geographical differences were apparent, notably a significantly higher frequency of mutant Pvdhfr haplotypes was observed in the Northern districts.ConclusionSince SP is rarely used in Sri Lanka, the high frequency and diversity of Pvdhfr mutations was unexpected indicating the emergence of drug resistant parasites despite a low level of SP drug pressure.

Escherichia coli FolC Structure Reveals an Unexpected Dihydrofolate Binding Site Providing an Attractive Target for Anti-microbial Therapy

In some bacteria, such as Escherichia coli, the addition of L-glutamate to dihydropteroate (dihydrofolate synthetase activity) and the subsequent additions of L-glutamate to tetrahydrofolate (folylpolyglutamate synthetase (FPGS) activity) are catalyzed by the same enzyme, FolC. The crystal structure of E. coli FolC is described in this paper. It showed strong similarities to that of the FPGS enzyme of Lactobacillus casei within the ATP binding site and the catalytic site, as do all other members of the Mur synthethase superfamily. FolC structure revealed an unexpected dihydropteroate binding site very different from the folate site identified previously in the FPGS structure. The relevance of this site is exemplified by the presence of phosphorylated dihydropteroate, a reaction intermediate in the DHFS reaction. L. casei FPGS is considered a relevant model for human FPGS. As such, the presence of a folate binding site in E. coli FolC, which is different from the one seen in FPGS enzymes, provides avenues for the design of specific inhibitors of this enzyme in antimicrobial therapy.

Root of the Eukaryota Tree as Inferred from Combined Maximum Likelihood Analyses of Multiple Molecular Sequence Data

Extensive studies aiming to establish the structure and root of the Eukaryota tree by phylogenetic analyses of molecular sequences have thus far not resulted in a generally accepted tree. To re-examine the eukaryotic phylogeny using alternative genes, and to obtain a more robust inference for the root of the tree as well as the relationship among major eukaryotic groups, we sequenced the genes encoding isoleucyl-tRNA and valyl-tRNA synthetases, cytosolic-type heat shock protein 90, and the largest subunit of RNA polymerase II from several protists. Combined maximum likelihood analyses of 22 protein-coding genes including the above four genes clearly demonstrated that Diplomonadida and Parabasala shared a common ancestor in the rooted tree of Eukaryota, but only when the fast-evolving sites were excluded from the original data sets. The combined analyses, together with recent findings on the distribution of a fused dihydrofolate reductase-thymidylate synthetase gene, narrowed the possible position of the root of the Eukaryota tree on the branch leading to Opisthokonta or to the common ancestor of Diplomonadida/Parabasala. However, the analyses did not agree with the position of the root located on the common ancestor of Opisthokonta and Amoebozoa, which was argued by Stechmann and Cavalier-Smith [Curr. Biol. 13:R665-666, 2003] based on the presence or absence of a three-gene fusion of the pyrimidine biosynthetic pathway: carbamoyl-phosphate synthetase II, dihydroorotase, and aspartate carbamoyltransferase. The presence of the three-gene fusion recently found in the Cyanidioschyzon merolae (Rhodophyta) genome sequence data supported our analyses against the Stechmann and Cavalier-Smith-rooting in 2003.

The GLOBULAR ARREST1 gene, which is involved in the biosynthesis of folates, is essential for embryogenesis in Arabidopsis thaliana.

We identified a mutation in Arabidopsis that resulted in defective embryos, and we designated this mutation globular arrest1 (gla1). The predicted amino acid sequence encoded by the GLA1 gene is homologous to the amino acid sequences of folylpolyglutamate synthetase (FPGS) and dihydrofolate synthetase (DHFS), which participate in folate biosynthesis. The defect of gla1 in the formation of calli was rescued by the supplement of 5-formyl tetrahydrofolate. These results indicated that GLA1 is involved in the biosynthesis of tetrahydrofolate. The gla1 embryos developed normally in the early stage of development but did not undergo the transition to the heart stage. Thus, the function of the GLA1 gene in embryogenesis appears to be required after the globular stage. However, when the levels of GLA1 transcripts in transgenic plants were increased by introduction of several copies of a GLA1 transgene (GLA6.8), the gla1 embryos that grew on gla1/gla1 GLA6.8/- plants developed as far as the heart to bent-cotyledon stage. This result suggests that the GLA1 function is provided to embryos by maternal tissues until embryos reach the globular stage.

Tetrahydrofolate biosynthesis in plants: molecular and functional characterization of dihydrofolate synthetase and three isoforms of folylpolyglutamate synthetase in Arabidopsis thaliana.

Tetrahydrofolate coenzymes involved in one-carbon (C1) metabolism are polyglutamylated. In organisms that synthesize tetrahydrofolate de novo, dihydrofolate synthetase (DHFS) and folylpolyglutamate synthetase (FPGS) catalyze the attachment of glutamate residues to the folate molecule. In this study we isolated cDNAs coding a DHFS and three isoforms of FPGS from Arabidopsis thaliana. The function of each enzyme was demonstrated by complementation of yeast mutants deficient in DHFS or FPGS activity, and by measuring in vitro glutamate incorporation into dihydrofolate or tetrahydrofolate. DHFS is present exclusively in the mitochondria, making this compartment the sole site of synthesis of dihydrofolate in the plant cell. In contrast, FPGS is present as distinct isoforms in the mitochondria, the cytosol, and the chloroplast. Each isoform is encoded by a separate gene, a situation that is unique among eukaryotes. The compartmentation of FPGS isoforms is in agreement with the predominance of gamma-glutamyl-conjugated tetrahydrofolate derivatives and the presence of serine hydroxymethyltransferase and C1-tetrahydrofolate interconverting enzymes in the cytosol, the mitochondria, and the plastids. Thus, the combination of FPGS with these folate-mediated reactions can supply each compartment with the polyglutamylated folate coenzymes required for the reactions of C1 metabolism. Also, the multicompartmentation of FPGS in the plant cell suggests that the transported forms of folate are unconjugated.

A bifunctional dihydrofolate synthetase–folylpolyglutamate synthetase in Plasmodium falciparum identified by functional complementation in yeast and bacteria

Folate metabolism in the human malaria parasite Plasmodium falciparum is an essential activity for cell growth and replication, and the target of an important class of therapeutic agents in widespread use. However, resistance to antifolate drugs is a major health problem in the developing world. To date, only two activities in this complex pathway have been targeted by antimalarials. To more fully understand the mechanisms of antifolate resistance and to identify promising targets for new chemotherapies, we have cloned genes encoding as yet uncharacterised enzymes in this pathway. By means of complementation experiments using 1-carbon metabolism mutants of both Escherichia coli and Saccharomyces cerevisiae, we demonstrate here that one of these parasite genes encodes both dihydrofolate synthetase (DHFS) and folylpolyglutamate synthetase (FPGS) activities, which catalyse the synthesis and polyglutamation of folate derivatives, respectively. The malaria parasite is the first known example of a eukaryote encoding both DHFS and FPGS activities in a single gene. DNA sequencing of this gene in antifolate-resistant strains of P. falciparum, as well as drug-inhibition assays performed on yeast and bacteria expressing PfDHFS–FPGS, indicate that current antifolate regimes do not target this enzyme. As PfDHFS–FPGS harbours two activities critical to folate metabolism, one of which has no human counterpart, this gene product offers a novel chemotherapeutic target with the potential to deliver a powerful blockage to parasite growth.

Molecular analysis of the folC gene of Pseudomonas aeruginosa.

We have cloned the Pseudomonas aeruginosa folC gene coding for folylpolyglutamate synthetase-dihydrofolate synthetase, which was located between the trpF and purF loci, and determined the nucleotide sequence of the folC gene and its flanking region. The deduced amino acid sequence of P. aeruginosa FolC was highly homologous to that of Escherichia coli FolC. The cloned gene complemented E. coli folC mutations and was found to encode both folylpolyglutamate synthetase and dihydrofolate synthetase activities. The gene organization around the folC gene in P. aeruginosa was completely conserved with that in E. coli; the accD gene was located upstream of the folC gene, and dedD, cvpA and purF genes followed the folC gene in this order. The gene arrangement and the result of the promoter activity assay suggested that the P. aeruginosa accD and folC genes were co-transcribed.