Isolation and characterization of a dihydropyrimidine dehydrogenase mutant of Pseudomonas chlororaphis

A dihydropyrimidine dehydrogenase mutant of Pseudomonas chlororaphis ATCC 17414 was isolated and characterized in this study. Initially, reductive catabolism of uracil was confirmed to be active in ATCC 17414 cells. Following chemical mutagenesis and d-cycloserine counterselection, a mutant strain unable to utilize uracil as a nitrogen source was identified. It was also unable to utilize thymine as a nitrogen source but could use either dihydrouracil or dihydrothymine as a sole source of nitrogen. Subsequently, it was determined that the mutant strain was deficient for the initial enzyme in the reductive pathway dihydropyrimidine dehydrogenase. The lack of dehydrogenase activity did not seem to have an adverse effect upon the activity of the second reductive pathway enzyme dihydropyrimidinase activity. It was shown that both dihydropyrimidine dehydrogenase and dihydropyrimidinase levels were affected by the nitrogen source present in the growth medium. Dihydropyrimidine dehydrogenase and dihydropyrimidinase activities were elevated after growth on uracil, thymine, dihydrouracil or dihydrothymine as a source of nitrogen.     

Pyrimidine catabolism in Pseudomonas aeruginosa

Pyrimidine catabolism in Pseudomonas aeruginosa was investigated. It was found that the pyrimidine bases uracil and thymidine as well as their respective reductive catabolic products could be utilized as sole sources of nitrogen. Reductive degradation of the pyrimidine bases was noted. The reductive catabolic pathway enzymes dihydropyrimidine dehydrogenase, dihydropyrimidinase and N-carbamoyl-beta-alanine amidohydrolase were all detected in minimal medium grown cells. Induction of pyrimidine catabolism by uracil was observed in this pseudomonad. Pyrimidine degradation in P. aeruginosa was not subject to catabolite repression.     

Pyrimidine base catabolism in Pseudomonas putida biotype B

Reductive catabolism of the pyrimidine bases uracil and thymine was found to occur in Pseudomonas putida biotype B. The pyrimidine reductive catabolic pathway enzymes dihydropyrimidine dehydrogenase, dihydropyrimidinase and N-carbamoyl--alanine amidohydrolase activities were detected in this pseudomonad. The initial reductive pathway enzyme dihydropyrimidine dehydrogenase utilized NADH or NADPH as its nicotinamide cofactor. The source of nitrogen in the culture medium influenced the reductive pathway enzyme activities and, in particular, dihydropyrimidinase activity was highly affected by nitrogen source. The reductive pathway enzyme activities in succinate-grown P. putida biotype B cells were induced when uracil served as the nitrogen source.     

Pyrimidine ribonucleoside catabolic enzyme activities ofPseudomonas pickettii

Pyrimidine ribonucleoside catabolic enzyme activities of the opportunistic pathogenPseudomonas pickettii were examined. Of the pyrimidine and related compounds tested, only dihydrouracil (nitrogen source) and ribose (carbon source) supported growth. Thin-layer chromatographic separation of the uridine and cytidine catabolities produced byP. pickettii extracts indicated that this pseudomonad contained nucleoside hydrolase activity. Its presence was confirmed by enzyme assay. Hydrolase activity was elevated in both glucose- and ribose-grown cells relative to succinate-grown cells. Nucleoside hydrolase activity was depressed when dihydrouracil served as a nitrogen source. Cytosine deaminase activity was present in extracts prepared from succinate-, glucose- or ribose-grown cells when (NH₄)₂SO₄ served as the nitrogen source although cells grown on glucose or ribose exhibited a higher enzyme activity. Cytosine deaminase activity was not detected in extracts prepared from cells grown on dihydrouracil as a nitrogen source. Both dihydropyrimidine dehydrogenase and dihydropyrimidinase activities were measurable inP. pickettii. The dehydrogenase activity was higher with NADH than with NADPH as its nicotinamide cofactor when uracil served as its substrate. Carbon source did not affect dehydrogenase or dihydropyrimidinase activity greatly but both activities were diminished in cells grown on the nitrogen source dihydrouracil.     

Degradation of the pyrimidine bases uracil and thymine by Escherichia coli B

Degradation of the pyrimidine bases uracil and thymine by Escherichia coli B was investigated. The known products of the reductive pathway of pyrimidine base catabolism were tested to determine if they could support the growth of E. coli B cells as sole sources of nitrogen or carbon. As might be expected if the reductive pathway was present, it was found that dihydrouracil, N-carbamoyl-beta-alanine, beta-alanine, dihydrothymine and beta-aminoisobutyric acid could sustain the growth of the bacterial cells as sole nitrogen sources by at least a fourteen-fold greater level than that observed if they were included as sole carbon sources. The existence of the reductive pathway of pyrimidine base degradation was confirmed in this micro-organism, since dihydrouracil, N-carbamoyl-beta-alanine and beta-alanine were detected following thin-layer chromatographic separation of the catabolic products of uracil and dihydrouracil.     

Pyrimidine-degrading enzymes. Purification and properties of beta-ureidopropionase of Euglena gracilis.

In photoorganotrophically grown, mid-log phase cells of Euglena gracilis, enzymes of pyrimidine degradation including uracil reductase, dihydrouracil dehydrogenase, dihydropyrimidinase, and beta-ureidopropionase, were detected in a crude extract. beta-Ureidopropionase (N-carbamoyl-beta-alanine amidohydrolase, EC 3.5.1.6) was purified 100-fold by heat treatment, ammonium sulphate fractionation and chromatography using Sepharose 6B and DEAE-Sephadex A-25. The enzyme follows Michaelis-Menten kinetics (Km of beta-ureidopropionase for beta-ureidopropionate 3.8 . 10(-5) M, Hill coefficient n = 1). Other enzyme properties are: pH optimum 6.25, temperature optimum 60 degrees C, stimulation by Mg2+, inhibition by Cu2+, Mr approximately 1.5--2 . 10(6). beta-Ureidoisobutyrate, the intermediate of thymine degradation, and beta-ureidopropionate are competing substrates of beta-ureidopropionase (Ki = Km of beta-ureidopropionase for beta-ureidoisobutyrate 1.8 . 10(-5) M). Structural analogues of beta-ureidopropionate, isobutyrate and propionate are competitive inhibitors (Ki of beta-ureidopropionase 0.3 and 0.16 mM, respectively). There were no indications of regulatory function of beta-ureidopropionase in pyrimidine degradation.     

Reductive catabolism of uracil and thymine by Burkholderia cepacia

Catabolism of uracil and thymine in Burkholderia cepacia ATCC 25416 was shown to occur using a reductive pathway. The first pathway enzyme, dihydropyrimidine dehydrogenase, was shown to utilize NADPH as its nicotinamide cofactor. Growth of B. cepacia on pyrimidine bases as the nitrogen source instead of on ammonium sulfate increased dehydrogenase activity at least 32-fold. The second and third reductive pathway enzymes, dihydropyrimidinase and N-carbamoyl-β-alanine amidohydrolase, respectively, exhibited activities elevated more than 21-fold when pyrimidine or dihydropyrimidine bases served as the nitrogen source rather than ammonium sulfate. The pathway enzyme activities were induced after growth on 5-methylcytosine. Received: 17 January 1997 / Accepted: 5 May 1997     

A Salmonella typhimurium strain defective in uracil catabolism and beta-alanine synthesis

A selection procedure for uracil catabolism mutant strains involving indicator dye plates was developed. Using this method, a strain defective in uracil catabolism has been isolated in Salmonella typhimurium that was temperature-sensitive at 42 degrees C where it required low concentrations of N-carbamoyl-beta-alanine, beta-alanine or pantothenic acid for growth. An extract of the mutant strain degraded uracil at 37 degrees C at a significantly diminished rate compared to that observed for the wild-type strain under the same growth conditions. The conversion of dihydrouracil to N-carbamoyl-beta-alanine was blocked at all temperatures examined in the mutant strain. By means of genetic analysis, the mutant strain was determined to be defective at two genetic loci. Transduction studies with bacteriophage P22 indicated that the panD gene is mutated in this strain, accounting for its beta-alanine requirement. Episomal transfers between Escherichia coli and the mutant strain provided evidence that the defect in uracil catabolism was located in another region of the S. typhimurium chromosome.     

Pyrimidine base and ribonucleoside utilization by thePseudomonas alcaligenes group

Pyrimidine base and ribonucleoside utilization was investigated in the two type strains of thePseudomonas alcaligenes group. As sole sources of nitrogen, the pyrimidine bases uracil, thymine and cytosine as well as the dihydropyrimidine bases dihydrouracil and dihydrothymine supported the growth ofPseudomonas pseudoalcaligenes ATCC 17440 but neither these bases nor pyrimidine nucleosides supportedPseudomonas alcaligenes ATCC 14909 growth. Ribose, deoxyribose, pyrimidine and dihydropyrimidine bases as well as pyrimidine nucleosides failed to be utilized by eitherP. pseudoalcaligenes orP. alcaligenes as sole carbon sources. The activities of the pyrimidine salvage enzymes nucleoside hydrolase, cytosine deaminase, dihydropyrimidine dehydrogenase and dihydropyrimidinase were detected in cell-free extracts ofP. pseudoalcaligenes andP. alcaligenes. InP. pseudoalcaligenes, the levels of cytosine deaminase, dihydropyrimidine dehydrogenase and dihydropyrimidinase could be affected by the nitrogen source present in the culture medium.     

Amidohydrolases of the reductive pyrimidine catabolic pathway purification, characterization, structure, reaction mechanisms and enzyme deficiency

In the reductive pyrimidine catabolic pathway uracil and thymine are converted to beta-alanine and beta-aminoisobutyrate. The amidohydrolases of this pathway are responsible for both the ring opening of dihydrouracil and dihydrothymine (dihydropyrimidine amidohydrolase) and the hydrolysis of N-carbamyl-beta-alanine and N-carbamyl-beta-aminoisobutyrate (beta-alanine synthase). The review summarizes what is known about the properties, kinetic parameters, three-dimensional structures and reaction mechanisms of these proteins. The two amidohydrolases of the reductive pyrimidine catabolic pathway have unrelated folds, with dihydropyrimidine amidohydrolase belonging to the amidohydrolase superfamily while the beta-alanine synthase from higher eukaryotes belongs to the nitrilase superfamily. beta-Alanine synthase from Saccharomyces kluyveri is an exception to the rule and belongs to the Acyl/M20 family.     

Kinetic mechanism of dihydropyrimidine dehydrogenase from pig liver

Data on initial velocity and isotope exchange at equilibrium suggest a nonclassical ping-pong mechanism for the dihydropyrimidine dehydrogenase from pig liver. Initial velocity patterns in the absence of inhibitors appeared parallel at low reactant concentration, with substrate inhibition by NADPH that is competitive with uracil and with substrate inhibition by uracil that is uncompetitive with NADPH. The Km values for both uracil (1 microM) and NADPH (7 microM) are low. As a result, it was difficult to determine whether the initial velocity pattern in the absence of added inhibitors was parallel. Thus, the pattern was redetermined in the presence of the dead-end inhibitor 2,6-dihydroxypyridine, which binds to both sites. This treatment effectively eliminates the inhibition by both substrates and increases their Km values, giving a strictly parallel pattern. Product and dead-end inhibition patterns are consistent with a mechanism in which NADPH reduces the enzyme at site 1 and electrons are transferred to site 2 to reduce uracil to dihydrouracil. The predicted mechanism is corroborated by exchange between [14C] NADP and NADPH as well as [14C]thymine and dihydrothymine in the absence of the other substrate-product pair.     

Isolation and characterization of an Escherichia coli B mutant strain defective in uracil catabolism

A reductive pathway of uracil catabolism was shown to be functioning in Escherichia coli B ATCC 11303 by virtue of thin-layer chromatographic and enzyme analyses. A mutant defective in uracil catabolism was isolated from this strain and subsequently characterized. The three enzyme activities associated with the reductive pathway of pyrimidine catabolism were detectable in the wild-type E. coli B cells, while the mutant strain was found to be deficient for dihydropyrimidine dehydrogenase activity. The dehydrogenase was shown to utilize NADPH as its nicotinamide cofactor. Growth of ATCC 11303 cells on uracil or glutamic acid instead of ammonium sulfate as a nitrogen source increased the reductive pathway enzyme activities. The mutant strain exhibited increased catabolic enzyme activities after growth on ammonium sulfate or glutamic acid.     

The crystal structures of dihydropyrimidinases reaffirm the close relationship between cyclic amidohydrolases and explain their substrate specificity

In eukaryotes, dihydropyrimidinase catalyzes the second step of the reductive pyrimidine degradation, the reversible hydrolytic ring opening of dihydropyrimidines. Here we describe the three-dimensional structures of dihydropyrimidinase from two eukaryotes, the yeast Saccharomyces kluyveri and the slime mold Dictyostelium discoideum, determined and refined to 2.4 and 2.05 angstroms, respectively. Both enzymes have a (beta/alpha)8-barrel structural core embedding the catalytic di-zinc center, which is accompanied by a smaller beta-sandwich domain. Despite loop-forming insertions in the sequence of the yeast enzyme, the overall structures and architectures of the active sites of the dihydropyrimidinases are strikingly similar to each other, as well as to those of hydantoinases, dihydroorotases, and other members of the amidohydrolase superfamily of enzymes. However, formation of the physiologically relevant tetramer shows subtle but nonetheless significant differences. The extension of one of the sheets of the beta-sandwich domain across a subunit-subunit interface in yeast dihydropyrimidinase underlines its closer evolutionary relationship to hydantoinases, whereas the slime mold enzyme shows higher similarity to the noncatalytic collapsin-response mediator proteins involved in neuron development. Catalysis is expected to follow a dihydroorotase-like mechanism but in the opposite direction and with a different substrate. Complexes with dihydrouracil and N-carbamyl-beta-alanine obtained for the yeast dihydropyrimidinase reveal the mode of substrate and product binding and allow conclusions about what determines substrate specificity, stereoselectivity, and the reaction direction among cyclic amidohydrolases.     

On the iron-sulfur clusters in the complex redox enzyme dihydropyrimidine dehydrogenase.

Porcine liver dihydropyrimidine dehydrogenase is a homodimeric iron-sulfur flavoenzyme that catalyses the first and rate-limiting step of pyrimidine catabolism. The enzyme subunit contains 16 atoms each of nonheme iron and acid-labile sulfur, which are most likely arranged into four [4Fe-4S] clusters. However, the presence and role of such Fe-S clusters in dihydropyrimidine dehydrogenase is enigmatic, because they all appeared to be redox-inactive during absorbance-monitored titrations of the enzyme with its physiological substrates. In order to obtain evidence for the presence and properties of the postulated four [4Fe-4S] clusters of dihydropyrimidine dehydrogenase, a series of EPR-monitored redox titrations of the enzyme under a variety of conditions was carried out. No EPR-active species was present in the enzyme 'as isolated'. In full agreement with absorbance-monitored experiments, only a small amount of neutral flavin radical was detected when the enzyme was incubated with excess NADPH or dihydrouracil under anaerobic conditions. Reductive titrations of dihydropyrimidine dehydrogenase with dithionite at pH 9.5 and photochemical reduction at pH 7.5 and 9.5 in the presence of deazaflavin and EDTA led to the conclusion that the enzyme contains two [4Fe-4S]2+,1+ clusters, which both exhibit a midpoint potential of approximately -0.44 V (pH 9.5). The two clusters are most likely close in space, as demonstrated by the EPR signals which are consistent with dipolar interaction of two S = 1/2 species including a half-field signal around g approximately 3.9. Under no circumstances could the other two postulated Fe-S centres be detected by EPR spectroscopy. It is concluded that dihydropyrimidine dehydrogenase contains two [4Fe-4S] clusters, presumably determined by the C-terminal eight-iron ferredoxin-like module of the protein, whose participation in the enzyme-catalysed redox reaction is unlikely in light of the low midpoint potential measured. The presence of two additional [4Fe-4S] clusters in dihydropyrimidine dehydrogenase is proposed based on thorough chemical analyses on various batches of the enzyme and sequence analyses. The N-terminal region of dihydropyrimidine dehydrogenase is similar to the glutamate synthase beta subunit, which has been proposed to contain most, if not all, the cysteinyl ligands that participate in the formation of the [4Fe-4S] clusters of the glutamate synthase holoenzyme. It is proposed that the motif formed by the Cys residues at the N-terminus of the glutamate synthase beta subunit, which are conserved in dihydropyrimidine dehydrogenase and in several beta-subunit-like proteins or protein domains, corresponds to a novel fingerprint that allows the formation of [4Fe-4S] clusters of low to very low midpoint potential.     

Metabolism of dihydrouracil in Rhodosporidium toruloides.

Previous studies, including those done with a similar species, have indicated that dihydrouracil is formed by the breakdown of uracil and is degraded into N-carbamyl-beta-alanine. (Fink et al., J. Biol. Chem. 201:349-355, 1953; S. R. Vilks and M. Y. Vitols, Mikrobiologiya 42:567-583, 1973; O. A. Milstein and M. L. Bekker, J. Bacteriol. 127:1-6, 1976). In the present work the conversion of dihydrouracil to uracil is studied in Rhodosporidium toruloides, and the growth characteristics of mutants that have lost the ability to use dihydrouracil as a source of nitrogen are examined. It is concluded that dihydrouracil must be converted to uracil before catabolism of the pyrimidine ring can take place.     

Pyrimidine base and ribonucleoside catabolic enzyme activities of the Pseudomonas diminuta group

Pyrimidine base and ribonucleoside catabolic enzyme activities of the two type strains of the Pseudomonas diminuta group were investigated for taxonomic classification purposes. The presence of the pyrimidine salvage enzyme nucleoside hydrolase was indicated in both type strains following thin-layer chromatographic analysis. The presence of the hydrolase was also confirmed by enzyme assay. In addition, the activities of the pyrimidine salvage enzymes dihydropyrimidine dehydrogenase and dihydropyrimidinase were measurable in cell-free extracts of both P. diminuta and P. vesicularis. An absence of cytosine deaminase activity was found when assaying extracts of the two type strains. Nucleoside hydrolase and dihydropyrimidine dehydrogenase levels in P. vesicularis were influenced by carbon source while dihydropyrimidinase activity was observed to increase after P. diminuta growth on dihydrothymine as a nitrogen source.     

Inhibition of a Putative Dihydropyrimidinase from Pseudomonas aeruginosa PAO1 by Flavonoids and Substrates of Cyclic Amidohydrolases

Dihydropyrimidinase is a member of the cyclic amidohydrolase family, which also includes allantoinase, dihydroorotase, hydantoinase, and imidase. These metalloenzymes possess very similar active sites and may use a similar mechanism for catalysis. However, whether the substrates and inhibitors of other cyclic amidohydrolases can inhibit dihydropyrimidinase remains unclear. This study investigated the inhibition of dihydropyrimidinase by flavonoids and substrates of other cyclic amidohydrolases. Allantoin, dihydroorotate, 5-hydantoin acetic acid, acetohydroxamate, orotic acid, and 3-amino-1,2,4-triazole could slightly inhibit dihydropyrimidinase, and the IC₅₀ values of these compounds were within the millimolar range. The inhibition of dihydropyrimidinase by flavonoids, such as myricetin, quercetin, kaempferol, galangin, dihydromyricetin, and myricitrin, was also investigated. Some of these compounds are known as inhibitors of allantoinase and dihydroorotase. Although the inhibitory effects of these flavonoids on dihydropyrimidinase were substrate-dependent, dihydromyricetin significantly inhibited dihydropyrimidinase with IC₅₀ values of 48 and 40 μM for the substrates dihydrouracil and 5-propyl-hydantoin, respectively. The results from the Lineweaver−Burk plot indicated that dihydromyricetin was a competitive inhibitor. Results from fluorescence quenching analysis indicated that dihydromyricetin could form a stable complex with dihydropyrimidinase with the K_d value of 22.6 μM. A structural study using PatchDock showed that dihydromyricetin was docked in the active site pocket of dihydropyrimidinase, which was consistent with the findings from kinetic and fluorescence studies. This study was the first to demonstrate that naturally occurring product dihydromyricetin inhibited dihydropyrimidinase, even more than the substrate analogs (>3 orders of magnitude). These flavonols, particularly myricetin, may serve as drug leads and dirty drugs (for multiple targets) for designing compounds that target several cyclic amidohydrolases.     

Rapid gas chromatographic-mass spectrometric diagnosis of dihydropyrimidine dehydrogenase deficiency and dihydropyrimidinase deficiency

A rapid yet reliable chemical diagnosis for dihydropyrimidine dehydrogenase (DHPD) deficiency, and possibly dihydropyrimidinase (DHP) deficiency in cancer patients, prior to therapy with pyrimidine analogues such as 5-fluorouracil, is desired for prevention of severe side-effects by these drugs. We have reported the basic separation and quantitation technology for pyrimidine metabolites using gas chromatography-mass spectrometry. A proposal to use the number (n) of standard deviations (SD) above the normal mean, as the index of the excessive urinary excretion of the metabolites appears not to be commonly used. When used, the values were too small, such as two or three, even in genetic disorders. Here, we applied the method to 11 urine specimens from proven cases including two DHP carriers and proved how specific the method is, because "n"-values were markedly large for thymine (T), uracil (U) and/or dihydrothymine (DHT) and dihydrouracil (DHU). In three cases with DHPD deficiency, two were siblings, one with symptoms and the other without, n was 12 for T and 5.9 for U, and 5-hydroxymethyluracil was distinctly detected. These values indicate that the nature of genetic mutation relates closely to the degree of metabolite accumulation in pyrimidine disorders. In six patients with DHP deficiency, n was 8.4-12 for DHT and 7.2-11 for DHU. Many mutations are known for both genes and the assay of residual enzyme activity may be time-consuming or invasive especially for those with DHP deficiency. Thus, this noninvasive yet comprehensive urinalysis has great value for those without a family history, as the first trial, before DNA or the enzyme assay. Our findings again raise the question whether the metabolic block really causes the symptoms found in pyrimidine disorders.     

Evaluation of pyrimidine- and hydantoin-degrading enzyme activities in aerobic bacteria

Abstract The enzyme activities responsible for the reductive pyrimidine base degradation by aerobic bacteria, which produce hydantoin-degrading enzymes, were investigated. Pseudomonas putida IFO 12996, which is a d-stereospecific hydantoinase producer, has dihydropyrimidinase activity, and Comamonas sp. E222c and Blastobacter sp. A17p-4, which are N-carbamoyl-D-amino acid amidohydrolase producers, have β-ureidopropionase activity. Blastobacter sp. also possesses both d-stereospecific hydantoinase and dihydropyrimidinase activities. Thus, two amide ring-opening activities and/or two N -carbamoyl amino acid-hydrolyzing activities coexist in these bacteria. However, the differences of the induction levels of each enzyme activities for the several pyrimidine- and hydantoin-related compounds suggest that these corresponding amide ring-opening or N -carbamoyl amino acid-hydrolyzing activities are not always catalyzed by the same enzymes.