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PubMed2006

A murine model for human sepiapterin-reductase deficiency.

Tetrahydrobiopterin (BH(4)) is an essential cofactor for several enzymes, including all three forms of nitric oxide synthases, the three aromatic hydroxylases, and glyceryl-ether mono-oxygenase. A proper level of BH(4) is, therefore, necessary for the metabolism of phenylalanine and the production of nitric oxide, catecholamines, and serotonin. BH(4) deficiency has been shown to be closely associated with diverse neurological psychiatric disorders. Sepiapterin reductase (SPR) is an enzyme that catalyzes the final step of BH(4) biosynthesis. Whereas the number of cases of neuropsychological disorders resulting from deficiencies of other catalytic enzymes involved in BH(4) biosynthesis and metabolism has been increasing, only a handful of cases of SPR deficiency have been reported, and the role of SPR in BH(4) biosynthesis in vivo has been poorly understood. Here, we report that mice deficient in the Spr gene (Spr(-/-)) display disturbed pterin profiles and greatly diminished levels of dopamine, norepinephrine, and serotonin, indicating that SPR is essential for homeostasis of BH(4) and for the normal functions of BH(4)-dependent enzymes. The Spr(-/-) mice exhibit phenylketonuria, dwarfism, and impaired body movement. Oral supplementation of BH(4) and neurotransmitter precursors completely rescued dwarfism and phenylalanine metabolism. The biochemical and behavioral characteristics of Spr(-/-) mice share striking similarities with the symptoms observed in SPR-deficient patients. This Spr mutant strain of mice will be an invaluable resource to elucidate many important issues regarding SPR and BH(4) deficiencies.

Alcohol Oxidoreductases↗

PubMed1999

N-terminal domain swapping and metal ion binding in nitric oxide synthase dimerization.

Nitric oxide synthase oxygenase domains (NOS(ox)) must bind tetrahydrobiopterin and dimerize to be active. New crystallographic structures of inducible NOS(ox) reveal that conformational changes in a switch region (residues 103-111) preceding a pterin-binding segment exchange N-terminal beta-hairpin hooks between subunits of the dimer. N-terminal hooks interact primarily with their own subunits in the 'unswapped' structure, and two switch region cysteines (104 and 109) from each subunit ligate a single zinc ion at the dimer interface. N-terminal hooks rearrange from intra- to intersubunit interactions in the 'swapped structure', and Cys109 forms a self-symmetric disulfide bond across the dimer interface. Subunit association and activity are adversely affected by mutations in the N-terminal hook that disrupt interactions across the dimer interface only in the swapped structure. Residue conservation and electrostatic potential at the NOS(ox) molecular surface suggest likely interfaces outside the switch region for electron transfer from the NOS reductase domain. The correlation between three-dimensional domain swapping of the N-terminal hook and metal ion release with disulfide formation may impact inducible nitric oxide synthase (i)NOS stability and regulation in vivo.

Amino Acid Sequence↗

PubMed1999

Increased transport of pteridines compensates for mutations in the high affinity folate transporter and contributes to methotrexate resistance in the protozoan parasite Leishmania tarentolae.

Functional cloning led to the isolation of a novel methotrexate (MTX) resistance gene in the protozoan parasite Leishmania. The gene corresponds to orfG, an open reading frame (ORF) of the LD1/CD1 genomic locus that is frequently amplified in several Leishmania stocks. A functional ORF G-green fluorescence protein fusion was localized to the plasma membrane. Transport studies indicated that ORF G is a high affinity biopterin transporter. ORF G also transports folic acid, with a lower affinity, but does not transport the drug analog MTX. Disruption of both alleles of orfG led to a mutant strain that became hypersensitive to MTX and had no measurable biopterin transport. Leishmania tarentolae MTX-resistant cells without their high affinity folate transporters have a rearranged orfG gene and increased orfG RNA levels. Overexpression of orfG leads to increased biopterin uptake and, in folate-rich medium, to increased folate uptake. MTX-resistant cells compensate for mutations in their high affinity folate/MTX transporter by overexpressing ORF G, which increases the uptake of pterins and selectively increases the uptake of folic acid, but not MTX.

Animals↗

PubMed1994

The Drosophila molybdenum cofactor gene cinnamon is homologous to three Escherichia coli cofactor proteins and to the rat protein gephyrin.

Essentially all organisms depend upon molybdenum oxidoreductases which require a molybdopterin cofactor for catalytic activity. Mutations resulting in a lack of the cofactor show a pleiotropic loss of molybdoenzyme activities and thereby define genes involved in cofactor biosynthesis or utilization. In prokaryotes, two operons are directly associated with biosynthesis of the pterin moiety and its side chain while additional loci play a role in the acquisition of molybdenum and/or activation of the cofactor. Here we report the cloning of cinnamon, a Drosophila molybdenum cofactor gene encoding a protein with sequence similarity to three of the prokaryotic cofactor proteins. In addition, the Drosophila cinnamon protein is homologous to gephyrin, a protein isolated from the rat central nervous system. Our results suggest that some portions of the prokaryotic cofactor biosynthetic pathway composed of monofunctional proteins have evolved into a multifunctional protein in higher eukaryotes.

Amino Acid Sequence↗

PubMed2004

Rescue of lethal molybdenum cofactor deficiency by a biosynthetic precursor from Escherichia coli.

Substitution therapies for orphan genetic diseases, including enzyme replacement methods, are frequently hampered by the limited availability of the required therapeutic substance. We describe the isolation of a pterin intermediate from bacteria that was successfully used for the therapy of a hitherto incurable and lethal disease. Molybdenum cofactor (Moco) deficiency is a pleiotropic genetic disorder characterized by the loss of the molybdenum-dependent enzymes sulphite oxidase, xanthine oxidoreductase and aldehyde oxidase due to mutations in Moco biosynthesis genes. An intermediate of this pathway-'precursor Z'-is more stable than the cofactor itself and has an identical structure in all phyla. Thus, it was overproduced in the bacterium Escherichia coli, purified and used to inject precursor Z-deficient knockout mice that display a phenotype which resembles that of the human deficiency state. Precursor Z-substituted mice reach adulthood and fertility. Biochemical analyses further suggest that the described treatment can lead to the alleviation of most symptoms associated with human Moco deficiency.

Animals↗

PubMed2007

Role of ABC transporter MRPA, gamma-glutamylcysteine synthetase and ornithine decarboxylase in natural antimony-resistant isolates of Leishmania donovani.

OBJECTIVES: The resistance of clinical isolates of Leishmania donovani to sodium antimony gluconate (SAG), the mainstay of treatment in Indian visceral leishmaniasis, has become a critical issue in India. The present work investigates the mechanism of resistance to SAG in parasites isolated from patients who are unresponsive to SAG. METHODS AND RESULTS: Susceptibility to SAG as determined in vitro with intracellular amastigotes correlated well with the clinical response. The ABC transporter gene MRPA was amplified in resistant field isolates as part of an extrachromosomal circle. Co-amplification of the pterin reductase gene (PTR1) and MRPA suggests amplification of the H locus in SAG-resistant isolates. Amplification of MRPA was correlated to increased RNA as determined by real-time PCR. MRPA is an ABC-thiol transporter, and cysteine and glutathione were increased in the resistant isolates. Ornithine decarboxylase (a rate limiting enzyme in polyamine biosynthesis), and gamma-glutamylcysteine synthetase (a rate limiting enzyme in glutathione biosynthesis), the two building blocks of the main cellular thiol trypanothione, were overexpressed in some of the resistant isolates. CONCLUSIONS: A variety of resistance mechanisms to SAG, most of them consistent with a model based on the study of resistance in vitro, were present in clinical isolates from the same geographical region.

ATP-Binding Cassette Transporters↗

PubMed2002

Characterization of Arabidopsis photolyase enzymes and analysis of their role in protection from ultraviolet-B radiation.

DNA photolyases are enzymes which mediate the light-dependent repair (photoreactivation) of UV-induced damage products in DNA by direct reversal of base damage rather than via excision repair pathways. Arabidopsis thaliana contains two photolyases specific for photoreactivation of either cyclobutane pyrimidine dimers (CPDs) or pyrimidine (6-4)pyrimidones (6-4PPs), the two major UV-B-induced photoproducts in DNA. Reduced FADH and a reduced pterin were identified as cofactors of the native Arabidopsis CPD photolyase protein. This is the first report of the chromophore composition of any native class II CPD photolyase protein to our knowledge. CPD photolyase protein levels vary between tissues and with leaf age and are highest in flowers and leaves of 3-5-week-old Arabidopsis plants. White light or UV-B irradiation induces CPD photolyase expression in Arabidopsis tissues. This contrasts with the 6-4PP photolyase protein which is constitutively expressed and not regulated by either white or UV-B light. Arabidopsis CPD and 6-4PP photolyase enzymes can remove UV-B-induced photoproducts from DNA in planta even when plants are grown under enhanced levels of UV-B irradiation and at elevated temperatures although the rate of removal of CPDs is slower at high growth temperatures. These studies indicate that Arabidopsis possesses the photorepair capacity to respond effectively to increased UV-B-induced DNA damage under conditions predicted to be representative of increases in UV-B irradiation levels at the Earth's surface and global warming in the twenty-first century.

Adaptation, Physiological↗

PubMed2000

Urinary excretion of folate catabolites responds to changes in folate intake more slowly than plasma folate and homocysteine concentrations and lymphocyte DNA methylation in postmenopausal women.

Folate turnover involves urinary excretion, fecal excretion, and catabolism that involves cleavage of the C9-N10 bond to yield pterins and para-aminobenzoylglutamate (pABG). Little is known about the relationship between the function of folate pools and their rates of catabolism. We report here an investigation of excretion of urinary pABG and its primary excretory form, para-acetamidobenzoylglutamate (ApABG) in samples collected during a previously published study of postmenopausal women. Ten women (49-63 y) were fed a low folate diet (56 microg/d) supplemented with folic acid to yield total folate intakes of 195 microg/d (d 1-5), 56 microg/d (d 6-41), 111 microg/d (d 42-69), 286 microg/d (d 70-80) and 516 microg/d (d 81-91). This caused changes in plasma folate, plasma homocysteine and global methylation of lymphocyte DNA. For each subject, a 7-d pooled urine sample was collected over d 1-7, 36-42, 64-70 and 85-91. ApABG constituted >85% of total catabolite excretion, and folate intake did not significantly influence ApABG or pABG excretion. The molar ratio of total catabolite excretion/folate intake varied significantly, with ratios of 1.0 +/- 0.17 (d 1-7), 3.0 +/- 0.55 (d 36-42), 1.1 +/- 0.18 (d 64-70) and 0. 33 +/- 0.054 (d 85-91). These observations indicate that the rate of folate catabolite excretion is related mainly to masses of slow-turnover folate pools governed by long-term folate intake. Folate pools functioning in some forms of methyl group metabolism respond to dietary changes in folate intake much more rapidly.

DNA Methylation↗

PubMed2002

Consumption of the folate breakdown product para-aminobenzoylglutamate contributes minimally to urinary folate catabolite excretion in humans: investigation using [(13)C(5)]para-aminobenzoylglutamate.

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

Adolescent↗

PubMed2002

Molybdoenzymes and molybdenum cofactor in plants.

The transition element molybdenum (Mo) is essential for (nearly) all organisms and occurs in more than 40 enzymes catalysing diverse redox reactions, however, only four of them have been found in plants. (1) Nitrate reductase catalyses the key step in inorganic nitrogen assimilation, (2) aldehyde oxidase(s) have been shown to catalyse the last step in the biosynthesis of the phytohormone abscisic acid, (3) xanthine dehydrogenase is involved in purine catabolism and stress reactions, and (4) sulphite oxidase is probably involved in detoxifying excess sulphite. Among Mo-enzymes, the alignment of amino acid sequences permits domains that are well conserved to be defined. With the exception of bacterial nitrogenase, Mo-enzymes share a similar pterin compound at their catalytic sites, the molybdenum cofactor. Mo itself seems to be biologically inactive unless it is complexed by the cofactor. This molybdenum cofactor combines with diverse apoproteins where it is responsible for the correct anchoring and positioning of the Mo-centre within the holo-enzyme so that the Mo-centre can interact with other components of the enzyme's electron transport chain. A model for the three-step biosynthesis of Moco involving the complex interaction of six proteins will be described. A putative Moco-storage protein distributing Moco to the apoproteins of Mo-enzymes will be discussed. After insertion, xanthine dehydrogenase and aldehyde oxidase, but not nitrate reductase and sulphite oxidase, require the addition of a terminal sulphur ligand to their Mo-site, which is catalysed by the sulphur transferase ABA3.

Abscisic Acid↗

PubMed1991

Cloning and functional characterization of a eucaryotic DNA photolyase gene from Neurospora crassa.

We cloned a genomic fragment of a photolyase gene from Neurospora crassa by polymerase chain reaction using synthesized oligonucleotide primers designed from the most conserved amino acid sequences among photolyases of various organisms. Using the cloned fragment as a hybridization probe we isolated a genomic fragment and cDNA clones encoding the complete photolyase gene of this organism. The amino acid sequence of the photolyase deduced from the determined nucleotide sequence indicates a protein consisting of 615 amino acid residues (Mr 69,971), which is most similar to that of Saccharomyces cerevisiae. Like yeast photolyase it contains a protruding amino terminus which is missing in photolyases of bacterial origin. Comparison of amino acids sequences among six photolyases suggests that the Neurospora crassa photolyase is more similar to photolyases of pterin type than those of deazaflavin type.

Amino Acid Sequence↗

PubMed1999

Role of the locus and of the resistance gene on gene amplification frequency in methotrexate resistant Leishmania tarentolae.

The protozoan parasite Leishmania resists the antifolate methotrexate (MTX) by amplifying the R locus dihydrofolate reductase-thymidylate synthase ( dhfr-ts ) gene, the H locus ptr1 pterin reductase gene, and finally by mutation in a common folate/MTX transporter. Amplification of dhfr-ts has never been observed in Leishmania tarentolae MTX resistant mutants while ptr1 amplification is common. We have selected a L.tarentolae ptr1 null mutant for MTX resistance and observed dhfr-ts amplification in this mutant demonstrating that once a preferred resistance mechanism is unavailable, a second one will take over. By introducing the ptr1 gene at the R locus and the dhfr-ts gene at the H locus by gene targeting, we investigated the role of the resistance gene and the locus on the rate of gene amplification. Transfection studies indicated that ptr1 gave higher levels of MTX resistance than dhfr-ts. Consistent with this, when ptr1 was present as part of either the H locus or the R locus it was invariably amplified, while dhfr-ts was only amplified when ptr1 was inactivated. When dhfr-ts was present in a ptr1 null background on both the H locus and the R locus, amplification from the H locus was more frequent suggesting that both the gene and the locus are determining the frequency of gene amplification in Leishmania.

Alleles↗

PubMed1994

Recombinant human tyrosine hydroxylase types 1-4 show regulatory kinetic properties for the natural (6R)-tetrahydrobiopterin cofactor.

Human tyrosine hydroxylase (hTH) exists in four isoforms. The four recombinant hTH isoenzymes types 1-4 (hTH1-4) produced in and purified from Escherichia coli showed regulatory kinetic properties for the natural (6R)-L-erythro-tetrahydrobiopterin (RBPH4) as a cofactor. In contrast, the unnatural cofactor (6S)-L-erythro-tetrahydrobiopterin (SBPH4) and a synthetic cofactor (6RS)-methyl-tetrahydropterin (6MPH4) showed usual kinetic characteristics with each of hTH1-4. Substrate inhibition by tyrosine was observed for each of hTH1-4 with natural RBPH4. Two different Km values for pterin cofactor were observed at a high concentration (200 microM) of L-tyrosine only with natural RBPH4, in contrast to a single Km value for unnatural SBPH4 or synthetic 6MPH4. The present results suggest that in the presence of relatively high concentrations (approximately 100 microM) of tyrosine in vivo, RBPH4 cofactor may have a regulatory role for the activity of all four human isoenzymes in vivo. We also found that recombinant hTH1 and 3 were more unstable than hTH2 and 4, suggesting that the 4-amino-acid insertion in hTH2 and 4 may be responsible for the relative stability of hTH2 and 4 isoenzymes.

Base Sequence↗

PubMed1980

Isolation of six unconjugated pteridines from soybean (Glycin max L. Tsurunoko) seeds.

Six unconjugated pteridines stored in soybean seeds were extracted with acid and isolated with a FLorisil column and reversed-phase high-performance liquid chromatography (HPLC). They were identified by HPLC, TLC, and fluorescence spectra to be 6-carboxypterin, erythro-neopterin, threo-neopterin, isoxanthopterin, 6-hydroxymethylpterin, and pterin. They were eluted in that order on HPLC. A much higher density of the pteridines, excepting isoxanthopterin, was found in the axis than in the cotyledon after a 6-imbibition.

Chromatography, High Pressure Liquid↗

PubMed1982

Aldehyde reductase from Crithidia fasciculata: purification and characterization.

Crithidia fasciculata, a pterin requiring protozoa, contained a NADPH-dependent dihydro-6-formylpterin reducing enzyme. The aldehyde reductase was purified and characterized. This enzyme was soluble and constitutive, and had the ability to catalyze the interconversion between aldehydes and alcohols. The optimal pH was 7.0 for the reductase activity and 9.0 for the dehydrogenase activity. The enzyme had a broad substrate specificity and reduced aromatic and aliphatic aldehydes such as dihydro-6-formylpterin, benzaldehyde, pyridine-3-aldehyde, butyraldehyde, DL-glyceraldehyde, and acetaldehyde. The enzyme was inhibited by sulfhydryl reagents and heavy metals, but not inhibited by chelating reagents. Its molecular weight was determined to be 66,000 and 72,000 by gel filtration and sedimentation equilibrium analysis, respectively. The value of 39,000 was obtained by sodium dodecyl sulfate gel electrophoresis indicating a dimeric structure. The aldehyde reductase of Crithidia may be classified as NADP+-dependent aryl-alcohol dehydrogenase [EC 1.1.1.91].

Alcohol Oxidoreductases↗

PubMed1986

Relationship of interferon-gamma and neopterin levels during stimulation with alloantigens in vivo and in vitro.

We have recently shown that interferon-gamma is capable of activating the key enzyme of pterin biosynthesis in macrophages. This leads to excretion of the stable degradation product neopterin. In this article we present experimental evidence suggesting that stimulation of T cells by alloantigens is associated with release of interferon-gamma--which, in the case of rejection, is locally restricted and not always detectable in the bloodstream. Neopterin induced by this lymphokine, however, readily penetrates tissue barriers and is detectable in the serum. This conclusion is based on two different sets of observations: (1) If supernatants of MLCs are compared with sera from patients with documented acute rejection episodes for their interferon-gamma and neopterin levels, a marked gradient is observed to exist between interferon levels measured in vitro and in vivo; this is not the case for neopterin for which comparable levels were seen. (2) Detection of interferon-gamma in sera of allograft recipients invariably precedes an increase of neopterin; on the other hand, increasing neopterin counts are also seen in the absence of detectable interferon-gamma levels in the serum. It thus appears that although interferon-gamma release during allograft rejection is primarily restricted to the tissue, evaluation of certain metabolites of interferon-dependent metabolic pathways enables definition of its endogenous release. Whereas interferon gamma represents a less reliable marker in the monitoring of rejection episodes, it might offer an additional means to differentiate rejection from systemic infections. Such a discrimination can not be achieved with the neopterin marker.

Acute Disease↗

PubMed2000

Protection against endotoxemia in rats by a novel tetrahydrobiopterin analogue.

We studied the effects of a novel pterin antagonist of NO synthase, the 4-amino analogue of tetrahydrobiopterin (4-ABH4), in a rat model of endotoxic shock and compared its properties with those of N(G)-monomethyl L-arginine (L-NMMA). Treatment with a bolus dose of 4-ABH4 at 2 h after LPS challenge significantly improved the 6-day survival rate, compared with the controls treated with saline. L-NMMA treatment did not significantly influence the survival rate. This bolus treatment, using either compound, had no effect on the plasma nitrite + nitrate or plasma IL-6 levels. The continuous infusion of 4-ABH4 efficiently suppressed the enhanced calcium-dependent/independent NO synthase activities induced by endotoxin in lung homogenates and completely suppressed the increase in plasma nitrite + nitrate caused by endotoxin at 5 h, with no significant difference compared with the L- NMMA treatment. Treatment of RAW264.7 murine macrophages with 4-ABH4 but not with L-NMMA suppressed endotoxin-induced tumor necrosis factor-alpha release by the cells, whereas nitrite in the supernatant decreased in a dose-dependent fashion in both assay systems. Our data show that 4-ABH4, an inhibitor of inducible NO synthase, significantly improves survival in a rat model of endotoxic shock when administered in a bolus dose that does not reduce plasma total nitrite + nitrate levels. Because we observed no overt signs of toxicity and no influence on organ-specific tetrahydrobiopterin levels, we conclude that the novel compound 4-ABH4 is a promising drug candidate for protection against endotoxin-related mortality.

Animals↗

PubMed2002

Peroxynitrite enhances the ability of Salmonella dublin to invade T84 monolayers.

In the intestine, epithelial cells continually produce and secrete low levels of nitric oxide (NO). Salmonella sp. invade epithelium by responding to environmental stimuli. The aims of this study were to determine the effect of reactive nitrogen intermediates (RNIs) on S. dublin and S. typhimurium growth and invasion of T84 epithelial monolayers. Intracellular NO formation was inhibited by 7-nitroindazole (7-NI) or N(G)-monomethyl-L-arginine, monoacetate (L-NMMA); extracellular NO and peroxynitrite were scavenged with ferro-hemoglobin or urate. The effect of authentic peroxynitrite (ONOO-); 3-morpholino-sydnonimine (SIN-1), which releases ONOO- via NO and superoxide; spermine NONOate, which releases only NO; or superoxide generated by xanthine oxidase and pterin on S. dublin and S. typhimurium growth and invasion were examined. Inhibition of NO synthesis and scavenging of extracellular NO or peroxynitrite reduced S. dublin invasion into T84 monolayers and enhanced bacterial growth. Pre-exposure of S. dublin to ONOO- and SIN-1 increased subsequent bacterial invasion into T84 monolayers. Conversely, exposure of bacteria to spermine NONOate or superoxide did not affect S. dublin invasion. In contrast, S. typhimurium invasion was not affected by pre-treatment with NO donors. In conclusion, exposure of S. dublin to ONOO- enhances the ability of the bacteria to invade epithelial cells. These results suggest that luminal ONOO- may have a novel role as an extracellular signal between invasive bacteria and epithelial cells.

Cell Division↗