The molecular basis of pyrazinamide activity on Mycobacterium tuberculosis PanD.

Pyrazinamide has been a mainstay in the multidrug regimens used to treat tuberculosis. It is active against the persistent, non-replicating mycobacteria responsible for the protracted therapy required to cure tuberculosis. Pyrazinamide is a pro-drug that is converted into pyrazinoic acid (POA) by pyrazinamidase, however, the exact target of the drug has been difficult to determine. Here we show the enzyme PanD binds POA in its active site in a manner consistent with competitive inhibition. The active site is not directly accessible to the inhibitor, suggesting the protein must undergo a conformational change to bind the inhibitor. This is consistent with the slow binding kinetics we determined for POA. Drug-resistant mutations cluster near loops that lay on top of the active site. These resistant mutants show reduced affinity and residence time of POA consistent with a model where resistance occurs by destabilizing the closed conformation of the active site.

Effects of adrenergic agents on intracellular Ca2+ homeostasis and metabolism of glucose in astrocytes with an emphasis on pyruvate carboxylation, oxidative decarboxylation and recycling: implications for glutamate neurotransmission and excitotoxicity.

Glucose and glycogen are essential sources of energy for maintaining glutamate homeostasis as well as glutamatergic neurotransmission. The metabolism of glycogen, the location of which is confined to astrocytes, is affected by norepinephrine (NE), and hence, adrenergic signaling in the astrocyte might affect glutamate homeostasis with implications for excitatory neurotransmission and possibly excitotoxic neurodegeneration. In order to study this putative correlation, cultured astrocytes were incubated with 2.5 mM [U-(13)C]glucose in the presence and absence of NE as a time course for 1 h. Employing mass spectrometry, labeling in intracellular metabolites was determined. Moreover, the involvement of Ca(2+) in the noradrenergic response was studied. In unstimulated astrocytes, the labeling pattern of glutamate, aspartate, malate and citrate confirmed important roles for pyruvate carboxylation and oxidative decarboxylation in astrocytic glucose metabolism. Importantly, pyruvate carboxylation was best visualized at 10 min of incubation. The abundance and pattern of labeling in lactate and alanine indicated not only an extensive activity of malic enzyme (initial step for pyruvate recycling) but also a high degree of compartmentalization of the pyruvate pool. Stimulating with 1 µM NE had no effect on labeling patterns and glycogen metabolism, whereas 100 µM NE increased glutamate labeling and decreased labeling in alanine, the latter supposedly due to dilution from degradation of non-labeled glycogen. It is suggested that further experiments uncovering the correlation between adrenergic and glutamatergic pathways should be performed in order to gain further insight into the role of astrocytes in brain function and dysfunction, the latter including excitotoxicity.

Arginine and lysine decarboxylases and the acid tolerance response of Salmonella Typhimurium.

Salmonella Typhimurium CECT 443 inactivation at pH 2.5 in Mineral Medium (MM) and MM supplemented with 0.01% (w/v) arginine, lysine or glutamic acid was studied using stationary-phase cells grown in buffered BHI pH 7.0 (non-acid adapted cells) and acidified BHI up to pH 4.5 with acetic, citric, lactic and hydrochloric acids (acid adapted cells). In all cases, acid adapted cells, with D-values ranging from 23.34 to 86.90 min, showed a significantly higher acid resistance than non-acid adapted cells, with D-values between 8.90 and 10.29 min. Whereas the conditions used for acid adaptation did not exert a significant effect on the acid resistance of the S. Typhimurium CECT 443 resulting cells, the inclusion of lysine and arginine in the challenge medium protected them against acid inactivation, reaching D-values of about 2 and 3 times higher, respectively, than those found in MM or MM supplemented with glutamic acid. None of these three amino acids significantly modified the acid resistance of non-acid adapted cells. The relative expression level of adiA (encoding the arginine decarboxylase), adiY (encoding the transcriptional activator of adiA), cadA (encoding the lysine decarboxylase) and cadB (encoding the lysine/cadaverine transport protein) was examined by quantitative PCR. Acid adapted cells showed higher relative expression levels for both systems, arginine decarboxylase and lysine decarboxylase, which demonstrates that the induction of specialized pH-homeostatic systems plays an important role in S. Typhimurium CECT 443 protection against acid stress. However, the increased acid resistance showed by acid adapted cells challenged in MM arginine or lysine free suggests the existence of other microbial survival strategies.

Modulation of oat arginine decarboxylase gene expression and genome organization in transgenic Trypanosoma cruzi epimastigotes.

We have previously demonstrated that wild-type Trypanosoma cruzi epimastigotes lack arginine decarboxylase (ADC) enzymatic activity as well as its encoding gene. A foreign ADC has recently been expressed in T. cruzi after transformation with a recombinant plasmid containing the complete coding region of the oat ADC gene. In the present study, upon modulation of exogenous ADC expression, we found that ADC activity was detected early after transfection; subsequently it decreased to negligible levels between 2 and 3 weeks after electroporation and was again detected approximately 4 weeks after electroporation. After this period, the ADC activity increased markedly and became expressed permanently. These changes of enzymatic activity showed a close correlation with the corresponding levels of ADC transcripts. To investigate whether the genome organization of the transgenic T. cruzi underwent any modification related to the expression of the heterologous gene, we performed PCR amplification assays, restriction mapping and pulse-field gel electrophoresis with DNA samples or chromosomes obtained from parasites collected at different time-points after transfection. The results indicated that the transforming plasmid remained as free episomes during the transient expression of the foreign gene. Afterwards, the free plasmid disappeared almost completely for several weeks and, finally, when the expression of the ADC gene became stable, two or more copies of the transforming plasmid arranged in tandem were integrated into a parasite chromosome (1.4 Mbp) bearing a ribosomal RNA locus. The sensitivity of transcription to alpha-amanitin strongly suggests involvement of the protozoan RNA polymerase I in the transcription of the exogenous ADC gene.

Biotin regulates the genetic expression of holocarboxylase synthetase and mitochondrial carboxylases in rats.

Biotin is the cofactor of carboxylases [pyruvate (PC), propionyl-CoA (PCC), 3-methyl crotonyl-CoA and acetyl-CoA], to which it is covalently bound by the action of holocarboxylase synthetase (HCS). We have studied whether biotin also regulates their expression, as it does other, nonrelated enzymes (e.g., glucokinase, phosphoenol pyruvate carboxykinase, guanylate cyclase). For this purpose, HCS, PC and PCC mRNAs were studied in biotin-deficient rat liver, kidney, muscle and brain of biotin-deficient rats. PC- and PCC-specific activities and protein masses were also measured. The 24-h time course of HCS mRNA in deficient rats was examined after biotin supplementation. HCS mRNA was significantly reduced during vitamin deficiency. It increased in deficient rats after biotin was injected, reaching control levels 24 h after administration. These changes seem to be the first known instance in mammals of an effect of a water-soluble vitamin on a mRNA functionally related to it. In contrast, the decreased activities of the carboxylases were associated with reductions in the amounts of their enzyme proteins except in brain. However, their mRNA levels were not affected. There are no reports on these types of vitamin affecting the mRNA or protein levels of their apoenzymes or their products. This work provides evidence for biotin being a modulator of the genetic expression of the enzymes involved in its function as a cofactor. As such, it may be a useful model for probing a similar role for other water-soluble vitamins.

Differential effects of biotin deficiency and replenishment on rat liver pyruvate and propionyl-CoA carboxylases and on their mRNAs.

Although the role of vitamins as prosthetic groups of enzymes is well known, their participation in the regulation of their genetic expression has been much less explored. We studied the effect of biotin on the genetic expression of rat liver mitochondrial carboxylases: pyruvate carboxylase (PC), propionyl-CoA carboxylase (PCC), and 3-methylcrotonyl-CoA carboxylase (MCC). Rats were made biotin-deficient and were sacrificed after 8 to 10 weeks, when deficiency manifestations began to appear. At this time, hepatic PCC activity was 20% of the control values or lower, and there was an abnormally high urinary excretion of 3-hydroxyisovaleric acid, a marker of biotin deficiency. Biotin was added to deficient primary cultured hepatocytes. It took at least 24 h after the addition of biotin for PCC to achieve control activity and biotinylation levels, whereas PC became active and fully biotinylated in the first hour. The enzyme's mass was assessed in liver homogenates from biotin-deficient rats and incubated with biotin to convert the apocarboxylases into holocarboylases, which were detected by streptavidin blots. The amount of PC was minimally affected by biotin deficiency, whereas that of the alpha subunits of PCC and of MCC decreased substantially in deficient livers, which likely explains the reactivation and rebiotinylation results. The expression of PC and alphaPCC was studied at the mRNA level by Northern blots and RT/PCR; no significant changes were observed in the deficient livers. These results suggest that biotin regulates the expression of the catabolic carboxylases (PCC and MCC), that this regulation occurs after the posttranscriptional level, and that pyruvate carboxylase, a key enzyme for gluconeogenesis, Krebs cycle anaplerosis, and fatty acid synthesis, is spared of this control.

Effects of sex hormones on the metabolism of tryptophan to niacin and to serotonin in male rats.

It is known that deaths attributable to pellagra, which is considered to be a disease caused by the disturbance of tryptophan metabolism, have been approximately two-fold higher in women than in men. We investigated the effects of the administration of female and male sex hormones on the contents of tryptophan and such metabolites as serotonin, nicotinamide, N1-methylnicotinamide, N1-methyl-2-pyridone-5-carboxamide, and N1-methyl-4-pyridone-3-carboxamide, and on the conversion ratio of tryptophan to niacin in male rats. Feeding a diet containing estrone or testosterone had no effect on the concentrations of tryptophan and serotonin in the blood and brain, or on the concentration of 5-hydroxyindole-3-acetic acid in the brain. On the contrary, feeding a diet containing estrone caused to a decrease in the urinary excretion of nicotinamide, N1-methylnicotinamide, N1-methyl-2-pyridone-5-carboxamide, and N1-methyl-4-pyridone-3-carboxamide, and of the conversion ratio of tryptophan to niacin when compared with the control rats. Feeding a diet containing testosterone had no effect on any parameter. We postulate from these findings that the cause of higher pellagra deaths in women than in men is attributable to the decrease in the formation of niacin from tryptophan, but not in the formation of serotonin by the female hormone. It seems likely that female sex hormones inhibit the synthesis of niacin from tryptophan, and that women, especially during pregnancy, will be more at risk to pellagra than are men.

Inhibition of L-aromatic amino acid decarboxylase by polychlorinated biphenyls.

PC12 cells were used to examine the mechanisms by which polychlorinated biphenyls (PCBs) reduce cellular levels of dopamine (DA). In cells treated 3 days with Aroclor 1254, 2,2',5,5'-tetrachlorobiphenyl (2,2',5,5'-TCB), or 2,2',3,3',4,4'-hexachlorobiphenyl (2,2',3,3',4,4'-HCB), the PCB-mediated reduction in 3H-tyrosine uptake was observed only at high PCB concentrations that produced a reduction in DNA levels. The PCB congener, 2,2',4,4',5,5'-hexachlorobiphenyl (2,2',4,4',5,5'-HCB) did not produce a reduction in 3H-tyrosine uptake at any concentration tested. Thus, there were PCB concentrations at which a reduction in DA levels did not coincide with a decrease in 3H-tyrosine uptake, suggesting that inhibition of tyrosine uptake was not the primary mechanism by which PCBs reduce DA levels. Aroclor 1254-treated cells also exhibited elevated levels of DOPA, further supporting the conclusion that tyrosine levels were not limiting. Incubation of Aroclor 1254-pretreated cells with 3H-tyrosine resulted in a dose-dependent increase in cellular levels of 3H-DOPA and decrease in cellular levels of 3H-DA, suggesting a PCB-mediated inhibition of the conversion of 3H-DOPA to 3H-DA. When the media was supplemented with DOPA, Aroclor 1254-treated cells still exhibited reduced levels of DA, compared to control cells, even though the control and PCB-treated cells had similar cellular levels of DOPA. Thus, one mechanism by which PCBs may reduce cellular levels of DA is by inhibiting L-aromatic amino acid decarboxylase-mediated conversion of DOPA to DA. The PCB congeners, 2,2',4,4'-TCB, 2,2',5,5'-TCB, and 2,2',4,4',5,5'-HCB, also produced dose-dependent increases in DOPA levels. The congener 2,2',3,3',4,4'-HCB did not produce an increase in DOPA levels, although it did mediate reductions in cellular DA levels. However, when PC12 cells were supplemented with DOPA, all four PCB congeners produced a similar reduction in DA levels, suggesting that the conversion of DOPA to DA was inhibited by the PCBs.

Identification of a non-mitochondrial phosphatidylserine decarboxylase activity (PSD2) in the yeast Saccharomyces cerevisiae.

Phosphatidylserine decarboxylase (PSD1) plays a central role in the biosynthesis of aminophospholipids in both prokaryotes and eukaryotes by catalyzing the synthesis of phosphatidylethanolamine. Recent reports (Trotter, P. J., Pedretti, J., and Voelker, D. R. (1993) J. Biol. Chem. 268, 21416-21424; Clancey, C. J., Chang, S.-C., and Dowhan, W. (1993) J. Biol. Chem. 268, 24580-24590) described the cloning of a yeast structural gene for this enzyme (PSD1) and the creation of the null allele. Based on the phenotype of strains containing a null allele for PSD1 (psd1-delta 1::TRP1) it was hypothesized that yeast have a second phosphatidylserine decarboxylase. The present studies demonstrate the presence of a second enzyme activity (denoted PSD2), which, depending on the method of evaluation, accounts for 4-12% of the total cellular phosphatidylserine decarboxylase activity found in wild type. Recessive mutations resulting in loss of this enzyme activity (denoted psd2) in cells containing the psd1-delta 1::TRP1 null allele also result in ethanolamine auxotrophy. When incubated with [3H]serine these double mutants accumulate label in phosphatidylserine, while very little (< 5%) is converted to phosphatidylethanolamine. In addition, these mutants have a approximately 70% decrease in the amount of total phosphatidylethanolamine even when grown in the presence of exogenous ethanolamine. Strains containing psd1 or psd2 mutations were utilized for the subcellular localization of the PSD2 enzyme activity. Unlike the PSD1 activity, the PSD2 enzyme activity does not localize to the mitochondria, but to a low density subcellular compartment with fractionation properties similar to both vacuoles and Golgi.

Purification and characterization of a ferulic acid decarboxylase from Pseudomonas fluorescens.

A ferulic acid decarboxylase enzyme which catalyzes the decarboxylation of ferulic acid to 4-hydroxy-3-methoxystyrene was purified from Pseudomonas fluorescens UI 670. The enzyme requires no cofactors and contains no prosthetic groups. Gel filtration estimated an apparent molecular mass of 40.4 (+/- 6%) kDa, whereas sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a molecular mass of 20.4 kDa, indicating that ferulic acid decarboxylase is a homodimer in solution. The purified enzyme displayed an optimum temperature range of 27 to 30 degrees C, exhibited an optimum pH of 7.3 in potassium phosphate buffer, and had a Km of 7.9 mM for ferulic acid. This enzyme also decarboxylated 4-hydroxycinnamic acid but not 2- or 3-hydroxycinnamic acid, indicating that a hydroxy group para to the carboxylic acid-containing side chain is required for the enzymatic reaction. The enzyme was inactivated by Hg2+, Cu2+, p-chloromercuribenzoic acid, and N-ethylmaleimide, suggesting that sulfhydryl groups are necessary for enzyme activity. Diethyl pyrocarbonate, a histidine-specific inhibitor, did not affect enzyme activity.

Carboxylases in de novo purine biosynthesis. Characterization of the Gallus gallus bifunctional enzyme.

Two successive steps in de novo purine biosynthesis are catalyzed by the enzymes 5-aminoimidazole ribonucleotide (AIR) carboxylase and 4-[(N-succinylamino)carbonyl]-5-aminoimidazole ribonucleotide (SAICAR) synthetase. Amino acid sequence alignments of the proteins from various sources suggested that several unusual differences exist within the structure and function of these enzymes. In vertebrates, a bifunctional enzyme (PurCE) catalyzes successive carboxylation and aspartylation steps of AIR to form SAICAR. This is in contrast to the three proteins, PurK, PurE, and PurC, from Escherichia coli which have recently been shown to require 2 equiv of ATP for the AIR to SAICAR conversion in the presence of physiological HCO3- concentrations (Meyer et al., 1992). A comparative study of these proteins has been initiated using a high-production, heterologous expression system for the Gallus gallus AIR carboxylase-SAICAR synthetase and yields purified enzyme following a two-step procedure. Selective assays have been developed for all the enzymatic activities of the bifunctional protein. The G. gallus AIR carboxylase has no ATP dependence and displays a Km for HCO3- that is 10-fold lower than that for the related PurE protein from E. coli, supporting the hypothesis that the two enzymes require different substrates. No common cofactors or metals are required for catalysis. Each catalytic activity has been shown to be independent by selective inactivation of SAICAR synthetase with the affinity agent 5'-[4-(fluorosulfonyl)benzoyl]-adenosine (FSBA) and inhibition of AIR carboxylase with a tight-binding inhibitor 4-nitro-5-aminoimidazole ribonucleotide (NAIR). The native protein aggregates, and limited proteolysis indicates that the global structure of the protein involves two independent folding domains, each containing a different catalytic site.

Carboxylation of phenylphosphate by phenol carboxylase, an enzyme system of anaerobic phenol metabolism.

Several lines of evidence indicate that the first step in the anaerobic metabolism of phenol is phenol carboxylation to 4-hydroxybenzoate; this reaction is considered a biological Kolbe-Schmitt carboxylation. A phenol carboxylase system was characterized by using a denitrifying Pseudomonas strain, K 172, which catalyzes an isotope exchange between 14CO2 and the carboxyl group of 4-hydroxybenzoate. The enzymatic isotope exchange activity (100 nmol min-1 mg-1 of protein) requires Mn2+ and K+. We show that this system also catalyzes the carboxylation of phenylphosphate (the phosphoric acid monophenyl ester) to 4-hydroxybenzoate and phosphate. The specific activity of phenylphosphate carboxylation at the optimal pH of 6.5 is 12 nmol of CO2 fixed min-1 mg-1 of protein. Phenylphosphate cannot be replaced by Mg(2+)-ATP and phenol. The carboxylase activity requires Mn2+ but, in contrast to the isotope exchange activity, does not require K+. The apparent Km values are 1.5 mM dissolved CO2 and 0.2 mM phenylphosphate. Several convenient assays for phenylophosphate carboxylation are described. The isotope exchange reaction and the net carboxylation reaction are catalyzed by the same oxygen-sensitive enzyme, which has a half-life in an air-saturated solution of less than 1 min. Both activities cochromatographed with a protein with a Mr of 280,000, and both activities were induced only after anaerobic growth on phenol. The carboxylation of phenylphosphate suggests that phenylphosphate itself is the physiological CO2 acceptor molecular of this novel CO2 fixation reaction. Alternatively, phenylphosphate could simulate the unknown natural precursor. It is suggested that the formation of an enzyme-bound phenolate anion from the activated phenolic compound is the rate-determining step in the carboxylation reaction.

The sodium ion pumping oxaloacetate decarboxylase of Klebsiella pneumoniae. Metal ion content, inhibitors and proteolytic degradation studies.

Oxaloacetate decarboxylase of Klebsiella pneumoniae was shown to contain between 0.6 and 1.0 mol zinc per mol enzyme in different preparations. The decarboxylase activity was completely abolished after 15 min incubation with 1 mM Hg(NO3)2 in phosphate buffer, while the activity decreased only 20% if the incubation was performed in MES/Tris buffer. Treatment of the isolated subunits with Hg(NO3)2 indicated that the binding site for Hg2+ ions is on the alpha subunit. Other inhibitors of the decarboxylase are KSCN and diethylstilbestrol. Inactivation of the enzyme with 2% 1-butanol was significantly reduced by 100 mM NaCl. Sodium ions also protected the isolated beta + gamma subunits from a digestion with trypsin.

Protection of wheat against leaf and stem rust and powdery mildew diseases by inhibition of polyamine metabolism.

In higher plants, polyamines arise from arginine by one of two pathways: via ornithine and ornithine decarboxylase or via agmatine and arginine decarboxylase but in fungi, only the ornithine decarboxylase pathway is present. Since polyamines are required for normal growth of microorganisms and plants and since the ornithine pathway can be irreversibly blocked by alpha-difluoromethylornithine (DFMO) which has no effect on arginine decarboxylase, fungal infection of green plants might be controlled by the site-directed use of such a specific metabolic inhibitor. DFMO at relatively low concentrations provided effective control of the three biotrophic fungal pathogens studied, Puccinia recondita (leaf rust), P. graminis f. sp. tritici (stem rust), and Erysiphe graminis (powdery mildew) on wheat (Triticum aestivum L.) Effective control of infection by leaf or stem rust fungi was obtained with sprays of DFMO that ranged from about 0.01 to 0.20 mM in experiments where the inhibitor was applied after spore inoculation. The powdery mildew fungus was somewhat more tolerant of DFMO, but good control of the pathogen was obtained at less than 1.0 mM. In general, application of DFMO after spore inoculation was more effective than application before inoculation. Less control was obtained following treatment with alpha-difluoromethylarginine (DFMA) but the relatively high degree of control obtained raises the possibility of a DFMA to DFMO conversion by arginase.