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PubMed1979

Purification and properties of the enzymes from Drosophila melanogaster that catalyze the synthesis of sepiapterin from dihydroneopterin triphosphate.

Sepiapterin synthase, the enzyme system responsible for the synthesis of sepiapterin from dihydroneopterin triphosphate, has been partially purified from extracts of the heads of young adult fruit flies (Drosophila melanogaster). The sepiapterin synthase system consists of two components, termed "enzyme A" (MW 82,000) and "enzyme B" (MW 36,000). Some of the properties of the enzyme system are as follows: NADPH and a divalent cation, supplied most effectively as MgCl2, are required for activity; optimal activity occurs are pH 7.4 and 30 C; the Km for dihydroneopterin triphosphate is 10 microM; and a number of unconjugated pterins, including biopterin and sepiapterin, are inhibitory. Dihydroneopterin cannot be used as substrate in place of dihydroneopterin triphosphate. Evidence is presented in support of a proposed reaction mechanism for the enzymatic conversion of dihydroneopterin triphosphate to sepiapterin in which enzyme A catalyzes the production of a labile intermediate by nonhydrolytic elimination of the phosphates of dihydroneopterin triphosphate, and enzyme B catalyzes the conversion of this intermediate, in the presence of NADPH, to sepiapterin. An analysis of the activity of sepiapterin synthase during development in Drosophila revealed the presence of a small amount of activity in eggs and young larvae and a much larger amount in late pupae and young adults. Sepiapterin synthase activity during development corresponds with the appearance of sepiapterin in the flies. Of a variety of eye color mutants of Drosophila melanogaster tested for sepiapterin synthase activity, only purple (pr) flies contained activity that was significantly lower than that found in the wild-type flies (22% of the wild-type activity). Further studies indicated that the amount of enzyme A activity is low in purple flies, whereas the amount of enzyme B activity is equal to that present in wild-type flies.

Alcohol Oxidoreductases↗
PubMed1991

Studies on the partially uncoupled oxidation of tetrahydropterins by phenylalanine hydroxylase.

The uncoupled portion of the partially uncoupled oxidation of tetrahydropterins by phenylalanine hydroxylase can be described by the same model as we have recently derived for the fully uncoupled reaction (Davis, M.D. and Kaufman, S. (1989) J. Biol. Chem. 264, 8585-8596). Although essentially no hydrogen peroxide is formed during the fully coupled oxidation of tetrahydrobiopterin or 6-methyltetrahydropterin by phenylalanine hydroxylase when phenylalanine is the amino acid substrate, significant amounts of hydrogen peroxide are formed during the partially uncoupled oxidation of 6-methyltetrahydropterin when para-fluorophenylalanine or para-chlorophenylalanine are used in place of phenylalanine. Similarly, during the partially uncoupled oxidation of the unsubstituted pterin, tetrahydropterin, even in the presence of phenylalanine, hydrogen peroxide formation is detected. The 4a-carbinolamine tetrahydropterin intermediate has been observed during the fully uncoupled tyrosine-dependent oxidations of tetrahydropterin and 6-methyltetrahydropterin by lysolecithin-activated phenylalanine hydroxylase, suggesting that this species is also a common intermediate for uncoupled oxidations by this enzyme.

Animals↗
PubMed2003

Molecular and biochemical characterization of two tungsten- and selenium-containing formate dehydrogenases from Eubacterium acidaminophilum that are associated with components of an iron-only hydrogenase.

Two gene clusters encoding similar formate dehydrogenases (FDH) were identified in Eubacterium acidaminophilum. Each cluster is composed of one gene coding for a catalytic subunit ( fdhA-I, fdhA-II) and one for an electron-transferring subunit ( fdhB-I, fdhB-II). Both fdhA genes contain a TGA codon for selenocysteine incorporation and the encoded proteins harbor five putative iron-sulfur clusters in their N-terminal region. Both FdhB subunits resemble the N-terminal region of FdhA on the amino acid level and contain five putative iron-sulfur clusters. Four genes thought to encode the subunits of an iron-only hydrogenase are located upstream of the FDH gene cluster I. By sequence comparison, HymA and HymB are predicted to contain one and four iron-sulfur clusters, respectively, the latter protein also binding sites for FMN and NAD(P). Thus, HymA and HymB seem to represent electron-transferring subunits, and HymC the putative catalytic subunit containing motifs for four iron-sulfur clusters and one H-cluster specific for Fe-only hydrogenases. HymD has six predicted transmembrane helices and might be an integral membrane protein. Viologen-dependent FDH activity was purified from serine-grown cells of E. acidaminophilum and the purified protein complex contained four subunits, FdhA and FdhB, encoded by FDH gene cluster II, and HymA and HymB, identified after determination of their N-terminal sequences. Thus, this complex might represent the most simple type of a formate hydrogen lyase. The purified formate dehydrogenase fraction contained iron, tungsten, a pterin cofactor, and zinc, but no molybdenum. FDH-II had a two-fold higher K(m) for formate (0.37 mM) than FDH-I and also catalyzed CO(2) reduction to formate. Reverse transcription (RT)-PCR pointed to increased expression of FDH-II in serine-grown cells, supporting the isolation of this FDH isoform. The fdhA-I gene was expressed as inactive protein in Escherichia coli. The in-frame UGA codon for selenocysteine incorporation was read in the heterologous system only as stop codon, although its potential SECIS element exhibited a quite high similarity to that of E. coli FDH.

Cloning, Molecular↗
PubMed2004

Tetrahydrofolate-specific enzymes in Methanosarcina barkeri and growth dependence of this methanogenic archaeon on folic acid or p-aminobenzoic acid.

Methanogenic archaea are generally thought to use tetrahydromethanopterin or tetrahydrosarcinapterin (H4SPT) rather than tetrahydrofolate (H4F) as a pterin C1 carrier. However, the genome sequence of Methanosarcina species recently revealed a cluster of genes, purN, folD, glyA and metF, that are predicted to encode for H4F-specific enzymes. We show here for folD and glyA from M. barkeri that this prediction is correct: FolD (bifunctional N5,N10-methylene-H4F dehydrogenase/N5,N10-methenyl-H4F cyclohydrolase) and GlyA (serine:H4F hydroxymethyltransferase) were heterologously overproduced in Escherichia coli, purified and found to be specific for methylene-H4F and H4F, respectively (apparent Km below 5 microM). Western blot analyses and enzyme activity measurements revealed that both enzymes were synthesized in M. barkeri. The results thus indicate that M. barkeri should contain H4F, which was supported by the finding that growth of M. barkeri was dependent on folic acid and that the vitamin could be substituted by p-aminobenzoic acid, a biosynthetic precursor of H4F. From the p-aminobenzoic acid requirement, an intracellular H4F concentration of approximately 5 M was estimated. Evidence is presented that the p-aminobenzoic acid taken up by the growing cells was not required for the biosynthesis of H4SPT, which was found to be present in the cells at a concentration above 3 mM. The presence of both H4SPT and H4F in M. barkeri is in agreement with earlier isotope labeling studies indicating that there are two separate C1 pools in these methanogens.

4-Aminobenzoic Acid↗
PubMed2003

Redox reactions of the pyranopterin system of the molybdenum cofactor.

This work provides the first extensive study of the redox reactivity of the pyranopterin system that is a component of the catalytic site of all molybdenum and tungsten enzymes possessing molybdopterin. The pyranopterin system possesses certain characteristics typical of tetrahydropterins, such as a reduced pyrazine ring; however, it behaves as a dihydropterin in redox reactions with oxidants. Titrations using ferricyanide and dichloroindophenol (DCIP) prove a 2e(-)/2H(+) stoichiometry for pyranopterin oxidations. Oxidations of pyranopterin by Fe(CN)(6)(3-) or DCIP are slower than tetrahydropterin oxidation under a variety of conditions, but are considerably faster than observed for oxidations of dihydropterin. The rate of pyranopterin oxidation by DCIP was studied in a variety of media. In aqueous buffered solution the pyranopterin oxidation rate has minimal pH dependence, whereas the rate of tetrahydropterin oxidation decreases 100-fold over the pH range 7.4-8.5. Although pyranopterin reacts as a dihydropterin with oxidants, it resists further reduction to a tetrahydropterin. No reduction was achieved by catalytic hydrogenation, even after several days. The reducing ability of the commonly used biological reductants dithionite and methyl viologen radical cation was investigated, but experiments showed no evidence of pyranopterin reduction by any of these reducing agents. This study illustrates the dual personalities of pyranopterin and underscores the unique place that the pyranopterin system holds in the spectrum of pterin redox reactions. The work presented here has important implications for understanding the biosynthesis and reaction chemistry of the pyranopterin cofactor in molybdenum and tungsten enzymes.

2,6-Dichloroindophenol↗
PubMed1985

Biosynthesis of tetrahydrobiopterin: conversion of dihydroneopterin triphosphate to tetrahydropterin intermediates.

It is known that the first step in the de novo synthesis of tetrahydrobiopterin from GTP is the conversion of GTP to dihydroneopterin triphosphate. Recent evidence supports the conclusion that beyond this first step, the pterin intermediates in the pathway are all at the tetrahydro level of reduction. We have now shown that partially purified fractions from rat liver, rat brain and bovine adrenal medulla catalyze the conversion of dihydroneopterin triphosphate to tetrahydrobiopterin, as well as to the putative intermediates in the pathway, 6-pyruvoyl-tetrahydropterin and 6-lactoyl-tetrahydropterin. Results of both enzymatic and chemical studies support the assigned structures for the latter two tetrahydropterins. We have also purified extensively from brain an enzyme, distinct from sepiapterin reductase, that catalyzes the TPNH-dependent reduction of 6-pyruvoyl-tetrahydropterin to 6-lactoyl-tetrahydropterin. The role of this reductase in tetrahydrobiopterin synthesis has not yet been established.

Adrenal Medulla↗
PubMed1977

Dihydroxanthopterinuria in phenylketonuria and lethal hyperphenylalaninemia patients.

7,8-Dihydroxanthopterin in identified in urine from phenylketonuria and lethal hyperphenylalaninemia patients. Because 7,8-dihyroxanthopterin is readily oxidised to xanthopterin, most of the characterisation was performed on xanthopterin. This finding indicates a gross disturbance of tetrahydrobiopterin metabolism in these patients. 7,8-dihydroxanthopterin or a related pterin may play a role in the pathogenesis of neurological symptoms in phenylketonuria and lethal hyperphenylalaninemia.

Amino Acid Metabolism, Inborn Errors↗
PubMed1989

De novo and salvage biosynthesis of pteroylpentaglutamates in the human malaria parasite, Plasmodium falciparum.

Plasmodium falciparum was shown to synthesize pteroylpolyglutamate de novo from guanosine 5'-triphosphate (GTP), p-aminobenzoate (PABA), and L-glutamate (L-Glu). The parasite also had the capacity to synthesize pteroylpolyglutamate from both intact and degradation moieties (p-aminobenzoylglutamate and pterin-aldehyde) of exogenous folate added into the growth medium. The major product was identified as 5-methyl-tetrahydroteroylpentaglutamate following exposure to pteroylpolyglutamate hydrolase and oxidative degradation of the C9-N10 bond in the molecule and identification of products by reversed-phase high performance liquid chromatography. Inhibition of pteroylpentaglutamate synthesis from the radiolabelled metabolic precursors (GTP, PABA, L-Glu) and folate by the antifolate antimalarials, pyrimethamine and sulfadoxine at therapeutic concentrations, may suggest the existence of a unique biosynthetic pathway in the malaria parasite.

Animals↗
PubMed2005

Tetrahydrobiopterin depletion and ubiquitylation of neuronal nitric oxide synthase.

Tetrahydrobiopterin is a necessary cofactor for the synthesis of nitric oxide by the hemeprotein enzyme, NO-synthase (NOS). It is widely thought that inadequate levels of tetrahydrobiopterin lead to tissue injury and organ dysfunction due, in part, to formation of superoxide from pterin-deficient NOS. In the course of studies on the ubiquitylation of neuronal NOS (nNOS), we have found that certain substrate analogs, such as N(G)-nitro-L-arginine, stabilize the dimeric form of nNOS and protect the enzyme from ubiquitylation. Since tetrahydrobiopterin is known to bind near heme and confers stability to the active dimeric structure of nNOS, we wondered if the loss of tetrahydrobiopterin could be an endogenous signal for nNOS ubiquitylation and degradation. We show here in HEK293 cells stably transfected with nNOS that depletion of tetrahydrobiopterin by treatment with 2,4-diamino-6-hydroxypyrimidine leads to destabilization of the dimeric form and enhances ubiquitylation of nNOS. Sepiapterin, a precursor to tetrahydrobiopterin in the salvage pathway, completely reverses the effect of 2,4-diamino-6-hydroxypyrimidine on nNOS ubiquitylation. Consistent with that found in cells, the in vitro ubiquitylation of nNOS by reticulocyte proteins decreases when tetrahydrobiopterin is present. Thus, inadequate amounts of tetrahydrobiopterin may lead to a sustained decrease in the steady state level of nNOS that is not readily reversed.

Biopterins↗
PubMed2003

Tetrahydrobiopterin is synthesized from 6-pyruvoyl-tetrahydropterin by the human aldo-keto reductase AKR1 family members.

Tetrahydrobiopterin (BH(4)) is a cofactor for aromatic amino acid hydroxylases and nitric oxide synthase. The biosynthesis includes two reduction steps catalyzed by sepiapterin reductase. An intermediate, 6-pyruvoyltetrahydropterin (PPH(4)) is reduced to 1(')-oxo-2(')-hydroxypropyl-tetrahydropterin (1(')-OXPH(4)) or 1(')-hydroxy-2(')-oxopropyl-tetrahydropterin (2(')-OXPH(4)), which is further converted to BH(4). However, patients with sepiapterin reductase deficiency show normal urinary excretion of pterins without hyperphenylalaninemia, suggesting that other enzymes catalyze the two reduction steps. In this study, the reductase activities for the tetrahydropterin intermediates were examined using several human recombinant enzymes belonging to the aldo-keto reductase (AKR) family and short-chain dehydrogenase/reductase (SDR) family. In the reduction of PPH(4) by AKR family enzymes, 2(')-OXPH(4) was formed by 3 alpha-hydroxysteroid dehydrogenase type 2, whereas 1(')-OXPH(4) was produced by aldose reductase, aldehyde reductase, and 20 alpha-hydroxysteroid dehydrogenase, and both 1(')-OXPH(4) and 2(')-OXPH(4) were detected as the major and minor products by 3 alpha-hydroxysteroid dehydrogenases (types 1 and 3). The activities of aldose reductase and 3 alpha-hydroxysteroid dehydrogenase type 2 (106 and 35 nmol/mg/min, respectively) were higher than those of the other enzymes (0.2-4.0 nmol/mg/min). Among the SDR family enzymes, monomeric carbonyl reductase exhibited low 1(')-OXPH(4)-forming activity of 5.0 nmol/mg/min, but L-xylulose reductase and peroxisomal tetrameric carbonyl reductase did not form any reduced product from PPH(4). Aldose reductase reduced 2(')-OXPH(4) to BH(4), but the other enzymes were inactive towards both 2(')-OXPH(4) and 1(')-OXPH(4). These results indicate that the tetrahydropterin intermediates are natural substrates of the human AKR family enzymes and suggest a novel alternative pathway from PPH(4) to BH(4), in which 3 alpha-hydroxysteroid dehydrogenase type 2 and aldose reductase work in concert.

Alcohol Oxidoreductases↗
PubMed2003

Phenylalanine hydroxylase gene in psychiatric patients: screening and functional assay of mutations.

BACKGROUND: Reports relating phenylalanine kinetics and metabolism to psychiatric disorders led us to undertake the comprehensive screening of the phenylalanine hydroxylase (PAH) coding region and functional testing of discovered mutations in a sample of psychiatric patients and healthy control subjects. METHODS: Genomic DNA from psychiatric patients and control subjects was assayed for sequence variants in all PAH coding regions and splice junctions. In vivo functional analysis of mutations was conducted by assessing the kinetics and conversion to tyrosine of a standardized phenylalanine dose and by measuring fasting pterin levels. RESULTS: A known missense mutation was observed in a schizoaffective subject, and a novel missense mutation was discovered in four subjects with schizophrenia and one normal subject. The schizoaffective patient heterozygous for the known A403V mutation showed the lowest rate of phenylalanine kinetics and lowest conversion to tyrosine in the patient sample. The four schizophrenic patients heterozygous for the novel K274E mutation showed significantly decreased phenylalanine kinetics, reduced conversion to tyrosine, and increased synthesis of the PAH cofactor tetrahydrobiopterin compared with schizophrenic subjects without the mutation. CONCLUSIONS: The study findings suggest that larger scale studies are warranted to test the relationship of the PAH genotype with a psychiatric phenotype.

Adult↗
PubMed2000

A new route to peroxynitrite: a role for xanthine oxidoreductase.

Peroxynitrite, a potent oxidising, nitrating and hydroxylating agent, results from the reaction of nitric oxide with superoxide. We show that peroxynitrite can be produced by the action of a single enzyme, xanthine oxidoreductase (XOR), in the presence of inorganic nitrite, molecular oxygen and a reducing agent, such as pterin. The effects of oxygen concentration on peroxynitrite production have been examined. The physiologically predominant dehydrogenase form of the enzyme is more effective than the oxidase form under aerobic conditions. It is proposed that XOR-derived peroxynitrite fulfils a bactericidal role in milk and in the digestive tract.

Animals↗
PubMed2003

Tetrahydrofolate and 5-methyltetrahydrofolate are folates with high antioxidant activity. Identification of the antioxidant pharmacophore.

The presumed protective effect of folic acid on the pathogenesis of cardiovascular, hematological and neurological diseases and cancer has been associated with the antioxidant activity of folic acid. Peroxynitrite (PON) scavenging activity and inhibition of lipid peroxidation (LPO) of the physiological forms of folate and of structurally related compounds were tested. It was found that the fully reduced forms of folate, i.e. tetrahydrofolate (THF) and 5-methyltetrahydrofolate (5-MTHF), had the most prominent antioxidant activity. It appeared that their protection against LPO is less pronounced than their PON scavenging activity. The antioxidant activity of these forms of folic acid resides in the pterin core, the antioxidant pharmacophore is 4-hydroxy-2,5,6-triaminopyrimidine. It is suggested that an electron donating effect of the 5-amino group is of major importance for the antioxidant activity of 4-hydroxy-2,5,6-triaminopyrimidine. A similar electron donating effect is probably important for the antioxidant activity of THF and 5-MTHF.

Animals↗
PubMed1999

2.0 A X-ray structure of the ternary complex of 7,8-dihydro-6-hydroxymethylpterinpyrophosphokinase from Escherichia coli with ATP and a substrate analogue.

The X-ray crystal structure of 7,8-dihydro-6-hydroxymethylpterinpyrophosphokinase (PPPK) in a ternary complex with ATP and a pterin analogue has been solved to 2.0 A resolution, giving, for the first time, detailed information of the PPPK/ATP intermolecular interactions and the accompanying conformational change. The first 100 residues of the 158 residue peptide contain a betaalpha betabeta alphabeta motif present in several other proteins including nucleoside diphosphate kinase. Comparative sequence examination of a wide range of prokaryotic and lower eukaryotic species confirms the conservation of the PPPK active site, indicating the value of this de novo folate biosynthesis pathway enzyme as a potential target for the development of novel broad-spectrum anti-infective agents.

Adenosine Triphosphate↗
PubMed1998

Tepidopterin, 1-O-(L-threo-biopterin-2'-yl)-beta-N-acetylglucosamine from Chlorobium tepidum.

A novel pterin compound, designated as tepidopterin, was detected from a thermophilic photosynthetic green sulphur bacterium, Chlorobium tepidum. The amount of tepidopterin inside the cell was estimated to be 2-5 micromol g(-1) dry weight of cell. This compound was purified through a high performance liquid chromatography using preparative DeltaPak C18 column. This compound was characterized by chromatographic behavior and by absorption and fluorescence properties. Its structure was determined to be 1-O-(L-threo-biopterin-2'-yl)-beta-N-acetylglucosamine by 1D- and 2D-NMR spectroscopy, mass spectrometry and CD. The relative amount of tetrahydrotepidopterin was estimated to be 96.7% inside the cell, that of dihydrotepidopterin 2.9%, and that of fully oxidized tepidopterin 0.4%. The amount of tepidopterin within the cell increased continuously until the beginning of the stationary phase of the cell growth.

Acetylglucosamine↗
PubMed1998

Methanopterin biosynthesis: methylation of the biosynthetic intermediates.

The basic scheme for the biosynthesis of methanopterin (MPT) in Methanobacterium thermoautotrophicum strain DeltaH, and M. thermoautotrophicum strain Marburg, has been shown to be the same as that recently determined for Methanosarcina thermophila strain TM-1. This scheme has, as one of its unique steps, the condensation of 4-aminobenzoic acid with 5-phospho-alpha-d-ribosyl diphosphate (PRPP) to form 4-(beta-d-ribofuranosyl)aminobenzene 5'-phosphate (beta-RFA-P). Labeling experiments with each of these organisms have established that the sites in the overall sequence of reactions from beta-RFA-P to MPT, where the S-adenosylmethionine-dependent C-9 and C-7 methylations of the pterin-containing intermediates occur, are organism related. In this work, cell extracts of M. thermoautotrophicum strain DeltaH, and M. thermoautotrophicum strain Marburg were found to contain significant amounts of methanopterin lacking the phosphate and 2-hydroxyglutaric acid groups.

Azo Compounds↗
PubMed2001

Reaction of tetrahydrobiopterin with superoxide: EPR-kinetic analysis and characterization of the pteridine radical.

It has been shown that BH(4) ameliorates endothelial dysfunction associated with conditions such as hypertension, cigarette smoking, and diabetes. This effect has been proposed to be due to a superoxide scavenging activity of BH(4). To examine this possibility we determined the rate constant for the reaction between BH(4) and superoxide using electron paramagnetic resonance (EPR) spin trapping competition experiments with 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO). We calculated a rate constant for the reaction between BH(4) and superoxide of 3.9 +/- 0.2 x 10(5) M(-1)s(-1) at pH 7.4 and room temperature. This result suggests that superoxide scavenging by BH(4) is not a major reaction in vivo. HPLC product analysis showed that 7,8-BH(2) and pterin are the stable products generated from the reaction. The formation of BH(4) cation radical (BH(4)(*+)) was demonstrated by direct EPR only under acidic conditions. Isotopic substitution experiments demonstrated that the BH(4)(*+) is mainly delocalized on the pyrazine ring of BH(4). In parallel experiments, we investigated the effect of ascorbate on 7,8-BH(2) reduction and eNOS activity. We demonstrated that ascorbate does not reduce 7,8-BH(2) to BH(4), nor does it stimulate nitric oxide release from eNOS incubated with 7,8-BH(2). In conclusion, it is likely that BH(4)-dependent inhibition of superoxide formation from eNOS is the mechanism that better explains the antioxidant effects of BH(4) in the vasculature.

Ascorbic Acid↗