Occurrence of GTP cyclohydrolase I in Bacillus stearothermophilus

A GTP cyclohydrolase which catalyzes the removal of carbon 8 of GTP as formic acid to yield a single pteridine compound occurs in an obligate thermophile Bacillus stearothermophilus ATCC 8005. The enzyme was purified 5.5-fold. Its molecular weight and Stoke's radius were estimated as 105,000 and 45.3 A, respectively. The Km for GTP was 0.98 microM. The temperature and pH optima for activity were 60-65 degrees C and 8.0-8.4, respectively. No divalent cation was required for the reaction. The pteridine product was 3'-triphosphate of 2-amino-4-hydroxy-6-(D-erythro-1',2',3'-trihydroxypropyl)-7,8-dihydropteridine (dihydroneopterin triphosphate), identified by isolating its immediate derivative, 2',3'-cyclic phosphate of 2-amino-4-hydroxy-6-(D-erythro-1',2',3'-trihydroxypropyl)pteridine (neopterin cyclic phosphate). The radioactive product from [8-14C]GTP agreed with 14C-formate. Molar ratio of formate release to pteridine formation was 1.0.     

Examination of intrinsic sulfonamide resistance in Bacillus anthracis: a novel assay for dihydropteroate synthase

Dihydropteroate synthase (DHPS) catalyzes the formation of dihydropteroate and Mg-pyrophosphate from 6-hydroxymethyl-7,8-dihydropterin diphosphate and para-aminobenzoic acid. The Bacillus anthracis DHPS is intrinsically resistant to sulfonamides. However, using a radioassay that monitors the dihydropteroate product, the enzyme was inhibited by the same sulfonamides. A continuous spectrophotometric assay for measuring the enzymatic activity of DHPS was developed and used to examine the effects of sulfonamides on the enzyme. The new assay couples the production of MgPPi to the pyrophosphate-dependent phosphofructokinase/aldolase/triose isomerase/alpha-glycerophosphate dehydrogenase reactions and monitors the disappearance of NADH at 340nm. The coupled enzyme assay demonstrates that resistance of the B. anthracis DHPS results in part from the use of the sulfonamides as alternative substrates, resulting in the formation of sulfonamide-pterin adducts, and not necessarily due to an inability to bind them.     

Examination of intrinsic sulfonamide resistance in Bacillus anthracis: A novel assay for dihydropteroate synthase

Dihydropteroate synthase (DHPS) catalyzes the formation of dihydropteroate and Mg-pyrophosphate from 6-hydroxymethyl-7,8-dihydropterin diphosphate and para-aminobenzoic acid. The Bacillus anthracis DHPS is intrinsically resistant to sulfonamides. However, using a radioassay that monitors the dihydropteroate product, the enzyme was inhibited by the same sulfonamides. A continuous spectrophotometric assay for measuring the enzymatic activity of DHPS was developed and used to examine the effects of sulfonamides on the enzyme. The new assay couples the production of MgPP_i to the pyrophosphate-dependent phosphofructokinase/aldolase/triose isomerase/α-glycerophosphate dehydrogenase reactions and monitors the disappearance of NADH at 340nm. The coupled enzyme assay demonstrates that resistance of the B. anthracis DHPS results in part from the use of the sulfonamides as alternative substrates, resulting in the formation of sulfonamide-pterin adducts, and not necessarily due to an inability to bind them.     

Isolation of two new natural pteridines from photosynthetic bacteria, Rhodopseudomonas sphaeroides

Two new natural pteridines have been isolated from the cultured medium of Rhodopseudomonas sphaeroides GM-1. The compounds are tentatively identified as 2-amino-4-hydroxy-6-hydroxy-6-(1,2, 3,4-tetrahydroxybutyl)pteridine and 2-amino-4-hydroxy-6-(3-hydroxy-4-phosphonoxy-1-butenyl)pteridine by degradative experiments and by electrophoretic and paper chromatographic comparison with authentic materials.     

Activation of snail (Helix pomatia) nervous tissue tyrosine monooxygenase by calcium in vitro.

Addition of calcium chloride to soluble preparations of tyrosine monooxygenase from snail brain appears to produce an activation of the enzyme when assayed with subsaturating concentrations of the pteridine cofactor 6 MPH4 (2-amino-4-hydroxy-6-methyltetrahydropteridine). While some increase in the activity occurs with calcium chloride at a concentration of 0.01 mM, activation is increased by about 100% at 1mM and reaches a maximum at 5mM (144%) where it remains more or less constant up to 10mM. Barium chloride also produces an activating effect although it is much less pronounced while magnesium chloride is without effect. EGTA has no direct effect on the enzyme but antagonises the activation produced by calcium chloride. The activation of tyrosine monooxygenase by calcium is reflected in changes in the kinetic properties of the enzyme, decreasing the Km from 43 muM to 19 muM for tyrosine and from 670muM to 230muM for the pteridine cofactor. No change was observed with V values for either tyrosine or pteridine cofactor. It is suggested that calcium, which enters the nerve terminal during nerve stimulation, regulates the transmitter dopamine by activating the rate-limiting enzyme tyrosine monooxygenase.     

Occurrence of Crithidia Factors and Folic Acid in Various Bacteria

Crithidia factors and folic acid were found to be widely distributed in culture fluids and in cells of 27 species of bacteria, when cultured under aerobic conditions into the stationary phase. Most bacteria excreted more Crithidia factors and folic acid than they retained in their cells. One Crithidia factor produced by Serratia indica and one produced by Bacillus cereus differed from biopterin in their chromatographic behavior. The factor excreted by S. indica appeared to be a 2-amino-4-hydroxy-6-substituted pteridine on the basis of KMnO(4) oxidation and ultraviolet absorption spectra. One of the folate compounds excreted by this organism was shown to be identical to 5,10-methylidynetetrahydrofolic acid by bioautography.     

Sulfonamide resistance in Streptococcus pneumoniae: DNA sequence of the gene encoding dihydropteroate synthase and characterization of the enzyme.

A chromosomal gene of Streptococcus pneumoniae carrying a spontaneous mutation to sulfonamide resistance was identified. Comparison of its DNA sequence with the wild-type sequence showed that the mutation, sul-d, consisted of an insert of 6 base pairs, a repeat of an adjacent 6-base-pair segment. The gene encoded a 34-kilodalton polypeptide, SulA, which as a dimer or trimer constituted the enzyme dihydropteroate synthase. This was shown by enzyme activity measurements, expression in minicells of Bacillus subtilis, and the amino-terminal sequence of the polypeptide product. Subcloning of the gene in an Escherichia coli expression vector allowed purification of the enzyme to 80% homogeneity in a single step and at high yield. Although a deleted plasmid, pLS83, produced the mutant dihydropteroate synthase, it did not confer sulfonamide resistance in vivo. It is suggested that the SulA polypeptide is also a component of an enzyme that acts in another step of folate biosynthesis and that this step is inhibited in vivo by either free or conjugated sulfonamides.     

Immunoaffinity purification and characterization of thromboxane synthase from porcine lung

Thromboxane synthase has been purified 620-fold from porcine lung microsomes by a three-step purification procedure including Lubrol-PX solubilization, reactive blue-agarose chromatography, and immunoaffinity chromatography. The purified enzyme exhibited a single protein band (53,000 daltons) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Rabbit antiserum raised against the purified enzyme immunoprecipitated thromboxane synthase activity from crude enzyme preparations of porcine lung, cow lung, and human platelets, indicating the existence of structural homology of the enzyme in these species. Immunoblotting experiment identified the same polypeptide (53,000 daltons) in porcine lung and a polypeptide of 50,000 daltons in human platelets, confirming the identity of the enzyme and the specificity of the antiserum. Purified thromboxane synthase is a hemoprotein with a Soret-like absorption peak at 418 nm. The enzyme reaction has a Km for 15-hydroxy-9 alpha, 11 alpha-peroxidoprosta-5, 13-dienoic acid of 12 microM, an optimal pH of 7.5, and an optimal temperature of reaction at 30 degrees C. Purified thromboxane synthase catalyzed the formation of both thromboxane B2 and 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT). The ratios of HHT to thromboxane B2 varied from 1.6 to 2.1 dependent on the reaction conditions. Except that HHT was formed at a greater rate, the formation of HHT and that of thromboxane responded identically to pH, temperature, substrate concentration, kinetics of formation, metal ions, and inhibitors suggesting that the two products are probably formed at the same active site via a common intermediate. Thromboxane synthase was irreversibly inactivated by 15-hydroxy-9 alpha, 11 alpha-peroxidoprosta-5,13-dienoic acid during catalysis and by treatment of 15-hydroperoxyeicosatetraenoic acid. The irreversible inactivation, however, could be protected by reversible inhibitors such as sodium (E)-3-[4-(1-imidazolylmethyl)phenyl]-2-propenoate and 15-hydroxy-11 alpha,9 alpha-(epoxymethano)-prosta-5,13-dienoic acid, suggesting that the inactivation occurred at the active site of the enzyme. The catalytic inactivation of thromboxane synthase and the greater rate of formation of HHT in thromboxane-synthesizing system may probably play important regulatory roles in the control of thromboxane synthesis.     

Cloning and Expression of Mycobacterium tuberculosis and Mycobacterium leprae Dihydropteroate Synthase in Escherichia coli

The genes for dihydropteroate synthase of Mycobacterium tuberculosis and Mycobacterium leprae were isolated by hybridization with probes amplified from the genomic DNA libraries. DNA sequencing revealed an open reading frame of 840 bp encoding a protein of 280 amino acids for M. tuberculosis dihydropteroate synthase and an open reading frame of 852 bp encoding a protein of 284 amino acids for M. leprae dihydropteroate synthase. The dihydropteroate synthases were expressed under control of the T5 promoter in a dihydropteroate synthase-deficient strain of Escherichia coli. Using three chromatography steps, we purified both M. tuberculosis and M. leprae dihydropteroate synthases to >98% homogeneity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed molecular masses of 29 kDa for M. tuberculosis dihydropteroate synthase and 30 kDa for M. leprae dihydropteroate synthase. Gel filtration of both enzymes showed a molecular mass of ca. 60 kDa, indicating that the native enzymes exist as dimers of two identical subunits. Steady-state kinetic parameters for dihydropteroate synthases from both M. tuberculosis and M. leprae were determined. Representative sulfonamides and dapsone were potent inhibitors of the mycobacterial dihydropteroate synthases, but the antimycobacterial agent p-aminosalicylate, a putative dihydropteroate synthase inhibitor, was a poor inhibitor of the enzymes.     

Cloning, sequencing, and enhanced expression of the dihydropteroate synthase gene of Escherichia coli MC4100.

The Escherichia coli gene coding for dihydropteroate synthase (DHPS) has been cloned and sequenced. The protein has 282 amino acids and a compositional molecular mass of 30,314 daltons. Increased expression of the enzyme was realized by using a T7 expression system. The enzyme was purified and crystallized. A temperature-sensitive mutant was isolated and found to express a DHPS with a lower specific activity and lower affinities for para-aminobenzoic acid and sulfathiazole. The allele had a point mutation that changed a phenylalanine codon to a leucine codon, and the mutation was in a codon that is conserved among published DHPS sequences.     

Isolation and Partial Characterization of a Temperature-Sensitive Escherichia coli Mutant with Altered Glutaminyl-Transfer Ribonucleic Acid Synthetase

A temperature-sensitive mutant of Escherichia coli has been found in which the conditional growth is a result of a thermosensitive glutaminyl-transfer ribonucleic acid synthetase. The corresponding genetic locus glnS is cotransduced with lip. In a strain containing the mutationally altered glutaminyl-transfer ribonucleic acid synthetase, no derepression of the enzyme itself nor of glutamine synthetase was observed.     

Localization of the thermosensitive X-prolyl dipeptidyl aminopeptidase in the vacuolar membrane of Saccharomyces cerevisiae

Most of the X-prolyl dipeptidyl aminopeptidase activity of Saccharomyces cerevisiae was found to be associated with purified vacuolar membranes (specific activity approx. 75-times higher than in the protoplast lysate). The tonoplast-bound enzyme is thermosensitive. Another heat-resistant enzyme was found in the protoplast lysate. The tonoplast-bound thermosensitive enzyme shows an apparent Km of 0.06 mM against L-alanyl-L-prolyl-p-nitroanilide while the heat-resistant enzyme shows an apparent Km of 0.4 mM against the same substrate.     

Identification by affinity labeling of potential sites in 23-S rRNA interacting with the 3'' end of tRNA

Tyrosine 3-monooxygenase was purified to homogeneity, as judged by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, from rat adrenal. The specific activity of the final preparation was approximately 1,600 nmol min-1 mg protein-1, which was much higher than the highest yet reported. The enzyme was markedly stabilized in the presence of glycerol, Tween 80 and EDTA. As judged by gel filtration on Ultrogel AcA 34, sodium dodecyl sulfate/polyacrylamide gel electrophoresis and cross-linking studies, the enzyme appeared to be composed of four identical subunits, each possessing a molecular weight of 59,000. The isoelectric point of the enzyme was estimated to be 6.7 in the presence of 8 M urea and 6.6 in its absence. Amino acid analysis of the enzyme revealed a fairly high content of serine residues in this protein. Purification of the enzyme caused changes in the kinetic properties of the enzyme. The Km for 2-amino-4-hydroxy-6-methyl-5,6,7,8-tetrahydropteridine decreased from 220 microM to 58 microM. The pH profile for the enzyme activity became more broad and the pH optimum was changed from an acid pH to a neutral pH. Although polyanions, such as heparin and dextran sulfate, markedly stimulated the activity of crude enzyme by increasing the V, they were much less effective in the activation of purified enzyme. A marked stimulation of the enzyme activity by phospholipids, such as phosphatidylserine, phosphatidylinositol, phosphatidylcholine and phosphatidylethanolamine, were not observed in both pure and crude preparations even at low concentrations of the pterin cofactor.     

Pteridines produced by Methylococcus capsulatus. Isolation and identification of a neopterin 2′:3′-phosphate

Three pteridines have been isolated from the methane- or methanol-oxidizing bacterium Methylococcus capsulatus. Two of these are known compounds, 2-amino-6-carboxy-4-hydroxypteridine and 2-amino-4-hydroxy-6-methylpteridine. The third is shown by degradative and synthetic experiments to be l-threo-neopterin 2':3'-phosphate. Labelling experiments show that both the pteridine moiety and phosphate residue are derived from a single GTP molecule. The possible metabolic significance of these compounds in methanol oxidation is discussed.     

Photosensitization of singlet oxygen formation by pterins and flavins. Time-resolved studies of oxygen phosphorescence under laser excitation

To elucidate the biochemical roles of singlet molecular oxygen (1(O2)) in the light-dependent reactions photosensitized by biological blue-light photoreceptors, time-resolved measurements of photosensitized 1O2 phosphorescence (1270 nm) were performed in air-saturated aqueous ((D2)O) solutions of pterins (2-amino-4-hydroxy-6,7-dimethylpteridine (DMP) and 2-amino-4-hydroxy-6-tetrahydroxybutyl-(D-arabo)pteridine (TOP)) and flavins (riboflavin and flavin mononucleotide (FMN)) under excitation with nitrogen laser (337.1 nm) pulses. The 1(O2) quantum yields were found to be 0.16, 0.20, 0.50, and 0.50 for DMP, TOP, riboflavin, and FMN, respectively. The data indicate that pterins and flavins are rather efficient photosensitizers of 1(O2) production that might be important for their photobiological functions.     

Sulfonamide resistance gene for plant transformation

The sulfonamide resistance gene from plasmid R46 encodes for a mutated dihydropteroate synthase insensitive to inhibition by sulfonamides. Its coding sequence was fused to the pea ribulose bisphosphate carboxylase/oxygenase transit peptide sequence. Incubation of isolated chloroplasts with the fusion protein synthesised in vitro, showed that the bacterial enzyme was transported to the chloroplast stroma and processed into a mature form. Expression of the gene fusion in transgenic plants resulted in a high level of resistance to sulfonamides. Direct selection of transformed shoots on leaf explants was efficient using sulfonamides as sole selective agents. Transformed shoots rooted normally on sulfonamides at concentrations toxic for untransformed ones. Sulfonamide resistance was transmitted to the progeny of transformed plants as a single Mendelian dominant character. These results demonstrate that this chimeric gene can be used as an efficient and versatile selectable marker for plant transformation.     

Purification and properties of dihydropterin oxidase from Drosophila melanogaster

The enzyme dihydropterin oxidase has been purified to apparent homogeneity from the fruit fly Drosophila melanogaster. This enzyme uses a variety of 2-amino-4-oxo-7,8-dihydropteridine compounds as substrates, including 2-amino-4-oxo-7,8-dihydropteridine (called dihydropterin), Km = 0.11 microM; 6-lactoyl-7,8-dihydropterin, Km = 1.80 microM; and 7,8-dihydrobiopterin, Km = 1.25 microM. The products in each case are the corresponding fully oxidized compounds 2-amino-4-oxopteridine, oxidized 6-lactoyl-7,8-dihydropterin, and 6-L-erythro-dihydroxypropylpterin, respectively. During the reaction, 1 mol of molecular oxygen is consumed per mole of substrate oxidized, and hydrogen peroxide is produced. The molecular weight of the enzyme is approximately 51,500. The enzyme apparently contains two polypeptide chains of identical molecular weight. The prosthetic group of the enzyme has been identified as FAD. From the determination of the occurrence of the enzyme in the various stages of the life cycle of D. melanogaster and from other considerations, the tentative conclusion is reached that the physiological role of dihydropterin oxidase is to convert dihydropterin to 2-amino-4-oxopteridine, a reaction that is believed to be essential in the formation of 2-amino-4-oxo-7-hydroxypterin in D. melanogaster.     

Channeling of 3-hydroxy-4-trans-decenoyl coenzyme A on the bifunctional beta-oxidation enzyme from rat liver peroxisomes and on the large subunit of the fatty acid oxidation complex from Escherichia coli

Rates of the NAD+-dependent oxidation of 2-trans,4-trans-decadienoyl-CoA, a metabolite of trans-omega-6-unsaturated fatty acids, catalyzed by the mitochondrial enoyl-CoA hydratase plus 3-hydroxyacyl-CoA dehydrogenase and by the corresponding enzymes from peroxisomes, as well as Escherichia coli, were compared. The study of the mitochondrial system revealed that the conventional kinetic theory of coupled enzyme reactions cannot be applied to systems in which the primary reaction has a small equilibrium constant, and/or the concentration of coupling enzyme is higher than 0.01 Km for the intermediate and higher than the steady-state concentration of the intermediate. In contrast to the results obtained with the mitochondrial beta-oxidation system of unlinked enzymes, the steady-state velocities of 2-trans,4-trans-decadienoyl-CoA degradation catalyzed by either the peroxisomal bifunctional enzyme or by the E. coli fatty acid oxidation complex were found to be equal to the activities of enoyl-CoA hydratase even though the concentration of coupling enzyme was equal to that of the primary enzyme, and the quotient of Vmax/Km for the dehydration of 3-hydroxy-4-trans-decenoyl-CoA is much larger than the Vmax/Km for its dehydrogenation. The extraordinarily high efficiencies of these two multifunctional proteins in catalyzing the degradation of 2-trans,4-trans-decadienoyl-CoA is best explained by the direct transfer of the 3-hydroxy-4-trans-decenoyl-CoA intermediate from the active site of enoyl-CoA hydratase to that of 3-hydroxyacyl-CoA dehydrogenase. The discovery of an intermediate channeling mechanism on the peroxisomal bifunctional enzyme explains on the molecular level why the peroxisomal beta-oxidation system is well suited for the degradation of trans-fatty acids.     

The origin of unconjugated pteridines in Crithidia fasciculata

Summary By using folic acid-2-¹⁴C in the growth medium as the sole source of pteridine it was found that the kinetoplastid flagellate, Crithidia fasciculata, produced a total of 87 mg of labeled biopterin and 23 g of 2-amino-4-hydroxy-6-hydroxymethylpteridine from 2 mg of folic acid in 2 liters of medium. Eighty percent of the biopterin and 70% of the 6-hydroxymethylpteridine was isolated from the spent medium, the remainder from the cells. These results and the results whereby the total pteridine was supplied as a folic acid analog make it obvious that this flagellate does convert folate to biopterin and is incapable of de novo pteridine synthesis.Supported in part by Research grants AM 01005 and CA 02924 from the National Institutes of Health, United States Public Health Service, and by Deutsche Forschungsgemeinschaft.Dedicated to Professor C. B. Van Niel on the occasion of his 70th birthday.     

Adaptation to sulfonamide resistance in Neisseria meningitidis may have required compensatory changes to retain enzyme function: kinetic analysis of dihydropteroate synthases from N. meningitidis expressed in a knockout mutant of Escherichia coli.

Previously, the effects of three point mutations (at amino acid positions 31, 84, and 194) in the gene coding for a sulfonamide-resistant dihydropteroate synthase of Neisseria meningitidis were analyzed by site-directed mutagenesis. Changes at positions 31 and 194 abolished the phenotypic expression of sulfonamide resistance, while a change at position 84 appeared to be neutral. These studies are here extended to correlate the alterations in phenotype with effects on enzyme kinetics by expressing the cloned meningococcal genes in an Escherichia coli strain that had its dhps gene partially deleted and replaced by a resistance determinant. The most dramatic effects were produced by mutations at position 31. A change from the Leu found in the resistant isolate to a Phe (the residue found in sensitive isolates) led to a 10-fold decrease in the Km and a concomitant drop in the Ki. Changes at position 194 also affected both the Km and Ki but not to the same extent as mutations at position 31. Changing position 84 altered the Km only slightly but significantly. This latter change was interpreted as a compensatory change modulating the function of the enzyme. In another type of resistance gene, 2 amino acid residues, proposed to be an insertion, were deleted, resulting in a sensitive enzyme. However, the resulting Km was raised 10-fold, suggesting that compensatory changes have accumulated in this type of resistance determinant as well. This resistance gene differs by as much as 10% from the sensitive isolates, which makes identification of important mutations difficult.