Susceptibilities of Mycobacterium tuberculosis and Mycobacterium avium complex to lipophilic deazapteridine derivatives, inhibitors of dihydrofolate reductase. (1/566)

Twelve lipophilic 2,4-diamino-5-methyl-5-deazapteridine derivatives and trimethoprim were evaluated for activity against Mycobacterium tuberculosis and Mycobacterium avium in vitro. Six of the compounds had MICs of < or =12.8 mg/L and < or =1.28 mg/L against M. tuberculosis and M. avium, respectively; trimethoprim MICs were >128 mg/L and >12.8 but < or =128 mg/L, respectively. Two compounds, with either a 2-methyl-5-methoxy phenyl or 2-methoxy-5-trifluoromethyl phenyl linked at the 6-position of the deazapteridine moiety by a CH2NH bridge, had MICs of < or =0.13 mg/L against M. avium; the two compounds also had apparent I50 values for dihydrofolate reductase of 2 and 8 nM, respectively, compared with an I50 of 400 nM with trimethoprim. Four of the compounds were selectively toxic to mycobacteria as compared with Vero cells. These results demonstrated that lipophilic antifolates can be synthesized which are more active against mycobacteria than trimethoprim and which possess selective toxicity.  (+info)

Human molybdopterin synthase gene: identification of a bicistronic transcript with overlapping reading frames. (2/566)

A universal molybdenum-containing cofactor (MoCo) is essential for the activity of all human molybdoenzymes, including sulphite oxidase. The free cofactor is highly unstable, and all organisms share a similar biosynthetic pathway. The involved enzymes exhibit homologies, even between bacteria and humans. We have exploited these homologies to isolate a cDNA for the heterodimeric molybdopterin (MPT)-synthase. This enzyme is necessary for the conversion of an unstable precursor into molybdopterin, the organic moiety of MoCo. The corresponding transcript shows a bicistronic structure, encoding the small and large subunits of the MPT-synthase in two different open reading frames (ORFs) that overlap by 77 nucleotides. In various human tissues, only one size of mRNA coinciding with the bicistronic transcript was detected. In vitro translation and mutagenesis experiments demonstrated that each ORF is translated independently, leading to the synthesis of a 10-kDa protein and a 21-kDa protein for the small and large subunits, respectively, and indicated that the 3'-proximal ORF of the bicistronic transcript is translated by leaky scanning.  (+info)

Human molybdopterin synthase gene: genomic structure and mutations in molybdenum cofactor deficiency type B. (3/566)

Biosynthesis of the molybdenum cofactor (MoCo) can be divided into (1) the formation of a precursor and (2) the latter's subsequent conversion, by molybdopterin synthase, into the organic moiety of MoCo. These two steps are reflected by the complementation groups A and B and the two formally distinguished types of MoCo deficiency that have an identical phenotype. Both types of MoCo deficiency result in a pleiotropic loss of all molybdoenzyme activities and cause severe neurological damage. MOCS1 is defective in patients with group A deficiency and has been shown to encode two enzymes for early synthesis via a bicistronic transcript with two consecutive open reading frames (ORFs). MOCS2 encodes the small and large subunits of molybdopterin synthase via a single transcript with two overlapping reading frames. This gene was mapped to 5q and comprises seven exons. The coding sequence and all splice site-junction sequences were screened for mutations, in MoCo-deficient patients in whom a previous search for MOCS1 mutations had been negative. In seven of the eight patients whom we investigated, we identified MOCS2 mutations that, by their nature, are most likely responsible for the deficiency. Three different frameshift mutations were observed, with one of them found on 7 of 14 identified alleles. Furthermore, a start-codon mutation and a missense mutation of a highly conserved amino acid residue were found. The locations of the mutations confirm the functional role of both ORFs. One of the patients with identified MOCS2 mutations had been classified as type B, in complementation studies. These findings support the hypothetical mechanism, for both forms of MoCo deficiency, that formerly had been established by cell-culture experiments.  (+info)

Characterization of a novel unconjugated pteridine glycoside, cyanopterin, in Synechocystis sp. PCC 6803. (4/566)

A new pteridine glycoside, called cyanopterin, was isolated from Synechocystis sp. PCC 6803 and its structure was elucidated as 6-[1-(4-O-methyl-(alpha-d-glucuronyl)-(1, 6)-(beta-d-galactosyloxy]methylpterin by chemical degradation and 1H- and 13C-NMR spectroscopic means. Cyanopterin is constitutively synthesized at a relatively high intracellular concentration that is comparable to that of chlorophyll a in a molar ratio of approximately 1 to 1.6. The in vivo oxidation state of cyanopterin is primarily the fully reduced 5,6,7,8-tetrahydro form. The cellular function is unknown at present. The findings have established a model system, using Synechocystis sp. PCC 6803, for studies of the physiological functions of unconjugated pteridine glycosides found mostly in cyanobacteria.  (+info)

Pteridines as inhibitors of xanthine oxidase: structural requirements. (5/566)

Different pteridine derivatives were investigated for their inhibitory action on xanthine oxidase. From 27 investigated compounds, 13 showed concentration-dependent inhibition of the enzyme. Concentrations necessary for 50% inhibition ranged from <0.1 up to >100 microM. Different types of inhibition were found concerning xanthine and pterin as substrates: competitive, noncompetitive and mixed type. Out of 18 aromatic compounds tested, 12 were inhibitors. Only one out of nine reduced derivatives served as inhibitor. A simple regression model was used to specify the structural requirements for a pteridine to be an inhibitor. The most characteristic features of an inhibitor are aromaticity and no substitution at position 7 of the pteridine ring.  (+info)

Re-design of Rhodobacter sphaeroides dimethyl sulfoxide reductase. Enhancement of adenosine N1-oxide reductase activity. (6/566)

The periplasmic DMSO reductase from Rhodobacter sphaeroides f. sp. denitrificans has been expressed in Escherichia coli BL21(DE3) cells in its mature form and with the R. sphaeroides or E. coli N-terminal signal sequence. Whereas the R. sphaeroides signal sequence prevents formation of active enzyme, addition of a 6x His-tag at the N terminus of the mature peptide maximizes production of active enzyme and allows for affinity purification. The recombinant protein contains 1.7-1.9 guanines and greater than 0.7 molybdenum atoms per molecule and has a DMSO reductase activity of 3.4-3.7 units/nmol molybdenum, compared with 3.7 units/nmol molybdenum for enzyme purified from R. sphaeroides. The recombinant enzyme differs from the native enzyme in its color and spectrum but is indistinguishable from the native protein after redox cycling with reduced methyl viologen and Me2SO. Substitution of Cys for the molybdenum-ligating Ser-147 produced a protein with DMSO reductase activity of 1.4-1.5 units/nmol molybdenum. The mutant protein differs from wild type in its color and absorption spectrum in both the oxidized and reduced states. This substitution leads to losses of 61-99% of activity toward five substrates, but the adenosine N1-oxide reductase activity increases by over 400%.  (+info)

Biopterin derivatives in normal and phenylketonuric patients after oral loads of L-phenylalanine, L-tyrosine, and L-tryptophan. (7/566)

Plasma biopterin derivatives studied in 10 normal and 21 phenylketonuric children showed a significantly high concentration in the latter group. Biopterin derivatives correlated with plasma phenylalanine concentration, but in normal adults given an oral phenylalanine load the rate of increase with phenylalanine differed from that in phenylketonuric patients. A patient with hyperphenylalaninaemia, not due to phenylketonuria, had an abnormal biopterin derivatives response to phenylalanine distinct from that of patients with classical phenylketonuria. This may be a useful investigation to differentiate some variants of phenylketonuria.  (+info)

The strict molybdate-dependence of glucose-degradation by the thermoacidophile Sulfolobus acidocaldarius reveals the first crenarchaeotic molybdenum containing enzyme--an aldehyde oxidoreductase. (8/566)

In order to investigate the effects of trace elements on different metabolic pathways, the thermoacidophilic Crenarchaeon Sulfolobus acidocaldarius (DSM 639) has been cultivated on various carbon substrates in the presence and absence of molybdate. When grown on glucose (but neither on glutamate nor casein hydrolysate) as sole carbon source, the lack of molybdate results in serious growth inhibition. By analysing cytosolic fractions of glucose adapted cells for molybdenum containing compounds, an aldehyde oxidoreductase was detected that is present in the cytosol to at least 0.4% of the soluble protein. With Cl2Ind (2,6-dichlorophenolindophenol) as artificial electron acceptor, the enzyme exhibits oxidizing activity towards glyceraldehyde, glyceraldehyde-3-phosphate, isobutyraldehyde, formaldehyde, acetaldehyde and propionaldehyde. At its pH-optimum (6.7), close to the intracellular pH of Sulfolobus, the glyceraldehyde-oxidizing activity is predominant. The protein has an apparent molecular mass of 177 kDa and consists of three subunits of 80.5 kDa (alpha), 32 kDa (beta) and 19.5 kDa (gamma). It contains close to one Mo, four Fe, four acid-labile sulphides and four phosphates per protein molecule. Methanol extraction revealed the existence of 1 FAD per molecule and 1 molybdopterin per molecule, which was identified as molybdopterin guanine dinucleotide on the basis of perchloric acid cleavage and thin layer chromatography. EPR-spectra of the aerobically prepared enzyme exhibit the so-called 'desulpho-inhibited'-signal, known from chemically modified forms of molybdenum containing proteins. Anaerobically prepared samples show both, the signals arising from the active molybdenum-cofactor as well as from the two [2Fe-2S]-clusters. According to metal-, cofactor-, and subunit-composition, the enzyme resembles the members of the xanthine oxidase family. Nevertheless, the melting point and long-term thermostability of the protein are outstanding and perfectly in tune with the growth temperature of S. acidocaldarius (80 degrees C). The findings suggest the enzyme to function as a glyceraldehyde oxidoreductase in the course of the nonphosphorylated Entner-Doudoroff pathway and thereby may attribute a new physiological role to this class of enzyme.  (+info)

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