The action of trypanocidal arsenical drugs on Trypanosoma brucei and Trypanosoma rhodesiense.
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Animals↗
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Results for “Pyruvate Oxidase”
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Tropical pyomyositis, a thiamine-deficiency disease.
On the basis of clinical observations made in Shanghai which confirmed the clinical and experimental findings of Osawa in Japan, it is suggested that thiamine deficiency is an essential contributary factor in producing tropical pyomyositis (TPM). It was found that the majority of patients affected by TPM suffer or have previously suffered from beriberi. The practical disappearance of TPM in Japan in recent years supports the above view. Thiamine deficiency induces a biochemical lesion (Peters) in the pyruvate oxidase system of the muscle, which breaks down its normal resistance to infection and opens the gate to bacterial agents. It is possible that in different parts of the world other or additional factors may play a similar role to that of thiamine deficiency. It is suggested that a wide survey should be undertaken--perhaps by the World Health Organisation--especially in areas in which TPM is endemic, using all relevant methods, to elucidate the factor or factors responsible for the development of TPM.
Adult↗
Successful dietary control of tyrosinemia II.
A Japanese girl, 2 years, 8 months of age, with palmoplantar keratosis and dendritic corneal opacities, showed increased tyrosine levels in the plasma, urine, and cerebrospinal fluid. The mental and physical growth was not retarded. The hepatorenal functions were within normal limits. Electron microscopically, the epidermal keratinocytes showed increased tonofibrils and no structures suggestive of tyrosine crystals. Cytosol and mitochondrial tyrosine aminotransferase (TAT) activities of the liver were greatly decreased, while p-hydroxyphenyl pyruvate oxidase (p-HPPO) activity was not decreased. The plasma tyrosine levels were controlled for 3 years with low phenylalanine-tyrosine diet.
Amino Acid Metabolism, Inborn Errors↗
The use of choline acetyltransferase for measuring the synthesis of acetyl-coenzyme A and its release from brain mitochondria.
1. A method for measuring small amounts of acetyl-CoA synthesized in subcellular fractions of the brain from pyruvate and released from particles into the incubation medium has been developed by using placental choline acetyltransferase and choline in the incubation medium to transform acetyl-CoA into acetylcholine. Acetylcholine is measured by biological assay. Optimum conditions of incubation are described. 2. With fresh mitochondria, a decrease of acetyl-CoA output into the medium is observed in the presence of ATP or ADP, and an increase in the presence of calcium chloride or 2,4-dinitrophenol. Fluorocitrate and malonate have little or no effect. 3. After the mitochondria had been treated with ether, the release of acetyl-CoA into the medium is much larger; presumably, nearly all acetyl-CoA synthesized is then released and transformed into acetylcholine under the conditions used. The release of acetyl-CoA is diminished in the presence of Krebs-cycle intermediates and ADP. 4. Of all subcellular fractions, the highest acetyl-CoA production from pyruvate is found in the crude mitochondria; rates up to 51 mumoles of acetyl-CoA/g. of original tissue/hr. are observed in ether-treated samples. 5. The activities of acetyl-CoA synthetase and ATP citrate lyase found in homogenates and nerve-ending fractions of brain tissue are considerably lower than those of pyruvate oxidase complex and choline acetyltransferase. 6. The bearing of some of the findings on the question of the source of acetyl radicals for the synthesis of acetylcholine in vivo is discussed.
Acetylcholine↗
Engineering Escherichia coli for efficient conversion of glucose to pyruvate.
Escherichia coli TC44, a derivative of W3110, was engineered for the production of pyruvate from glucose by combining mutations to minimize ATP yield, cell growth, and CO2 production (DeltaatpFH DeltaadhE DeltasucA) with mutations that eliminate acetate production [poxB::FRT (FLP recognition target) DeltaackA] and fermentation products (DeltafocA-pflB DeltafrdBC DeltaldhA DeltaadhE). In mineral salts medium containing glucose as the sole carbon source, strain TC44(DeltafocA-pflB DeltafrdBC DeltaldhA DeltaatpFH DeltaadhE DeltasucA poxB::FRT DeltaackA) converted glucose to pyruvate with a yield of 0.75 g of pyruvate per g of glucose (77.9% of theoretical yield; 1.2 g of pyruvate liters(-1).h(-1)). A maximum of 749 mM pyruvate was produced with excess glucose. Glycolytic flux was >50% faster for TC44 producing pyruvate than for the wild-type W3110 during fully aerobic metabolism. The tolerance of E. coli to such drastic changes in metabolic flow and energy production implies considerable elasticity in permitted pool sizes for key metabolic intermediates such as pyruvate and acetyl-CoA. In strain TC44, pyruvate yield, pyruvate titer, and the rate of pyruvate production in mineral salts medium were equivalent or better than previously reported for other biocatalysts (yeast and bacteria) requiring complex vitamin feeding strategies and complex nutrients. TC44 offers the potential to improve the economics of pyruvate production by reducing the costs of materials, product purification, and waste disposal.
Escherichia coli↗
Genomic and transcriptomic insights into the virulence and adaptation of shock syndrome-causing Streptococcus anginosus.
Streptococcus anginosus is a common isolate of the oral cavity and an opportunistic pathogen for systemic infections. Although the pyogenic infections caused by S. anginosus are similar to those caused by Streptococcus pyogenes, S. anginosus lacks most of the well-characterized virulence factors of S. pyogenes. To investigate the pathogenicity of S. anginosus, we analysed the genome of a newly identified S. anginosus strain, KH1, which was associated with toxic shock-like syndrome in an immunocompetent adolescent. The genome of KH1 contains nine genomic islands, two Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated systems and many phage-related proteins, indicating that the genome is influenced by prophages and horizontal gene transfer. Comparative genome analysis of 355 S. anginosus strains revealed a significant difference between the sizes of the pan genome and core genome, reflecting notable strain variations. We further analysed the transcriptomes of KH1 under conditions mimicking either the oral cavity or the bloodstream. We found that in an artificial saliva medium, the expression of a putative quorum quenching system and pyruvate oxidase for H2O2 production was upregulated, which could optimize the competitiveness of S. anginosus in the oral ecosystem. Conversely, in a modified serum medium, purine and glucan biosynthesis, competence and bacteriocin production were significantly upregulated, likely facilitating the survival of KH1 in the bloodstream. These findings indicate that S. anginosus can utilize diverse mechanisms to adapt to different environmental niches and establish infection, despite its lack of toxin production.
Streptococcus anginosus↗
Acetate excretion during growth of Salmonella enterica on ethanolamine requires phosphotransacetylase (EutD) activity, and acetate recapture requires acetyl-CoA synthetase (Acs) and phosphotransacetylase (Pta) activities.
This report shows that Salmonella enterica catabolizes ethanolamine to acetyl-CoA (Ac-CoA), which enters the glyoxylate bypass and tricarboxylic acid cycle for the generation of energy and central metabolites. During growth on ethanolamine, S. enterica excreted acetate, whose recapture depended on Ac-CoA synthetase (Acs) and the housekeeping phosphotransacetylase (Pta) enzyme activities. The Pta enzyme did not play a role in acetate excretion during growth of S. enterica on ethanolamine. It is proposed that during growth on ethanolamine, acetate excretion is necessary to maintain a pool of free CoA. Acetate excretion requires the eut operon-encoded phosphotransacetylase (EutD) and acetate kinase (Ack) enzymes. EutD function was not required for growth on ethanolamine, and an eutD strain showed only a slight reduction in growth rate. The existence of an as-yet-unidentified system that releases acetate was revealed during growth of a strain lacking Acs, the housekeeping phosphotransacetylase (Pta), and EutD. The functions of pyruvate oxidase (PoxB), Ack and STM3118 protein [a homologue of the Saccharomyces cerevisiae Ac-CoA hydrolase (Ach1p) enzyme] were not involved in the release of acetate by the acs pta eutD strain.
Acetate-CoA Ligase↗
Changes in the Respiratory, Enzymatic, and Swelling and Contraction Properties of Mitochondria from Colytedons of Phaseolus vulgaris L. during Germination.
Mitochondria isolated from cotyledons of germinating wax beans (Phaseolus vulgaris L.) showed fairly good respiratory control on days 1 and 2 after planting. The respiratory control was completely lost from days 3 to 5. During this period mitochondria were shown to be very leaky, losing about 88% of their total nicotinamide adenine dinucleotide to the suspending medium in a short time. The respiratory control was partially recovered by day 7, after which it completely disappeared again. By the use of differential centrifugation, the mitochondria were divided into subfractions by sequential centrifugation: 10,000g for 5 minutes, 25,000g for 5 minutes, and 40,000g for 5 minutes. The 10,000g subfraction was responsible for the recovery of mitochondrial activity (respiratory control value, adenosine diphosphate to oxygen ratio, and rate of oxygen utilization), on day 7. Activities of succinate dehydrogenase, cytochrome oxidase, pyruvate dehydrogenase, and isocitrate dehydrogenase from different mitochondrial subfractions of aging cotyledons were determined. In general, the enzyme activities, adenosine diphosphate to oxygen ratios, and the ability of mitochondria to swell and contract followed the same pattern as for respiratory control.
Journal Article↗
Regulatory characteristics and promoter analysis of csiE, a stationary phase-inducible gene under the control of sigma S and the cAMP-CRP complex in Escherichia coli.
Sigma-S and the cAMP-CRP complex are global regulatory factors involved in stationary-phase induction of large groups of genes in Escherichia coli. csiE, a gene located at 57.25 min (co-ordinate 2674) of the physical map of the E. coli chromosome, is under the control of both of these factors. Sigma-S plays a positive, though not absolutely essential, role in the expression of csiE. Regulation by cAMP-CRP has both positive and negative elements, with the latter being dependent on the presence of sigma s, whose expression is negatively influenced by cAMP-CRP. csiE has a single transcriptional start site located 33 bp upstream of the initiation codon. By a 5'-deletion approach, we show that 72 bp upstream of the csiE transcriptional start site are sufficient for regulation by sigma s and cAMP-CRP. A deletion upstream of nucleotide -38 with respect to the start site eliminates positive cAMP-CRP control and makes the remaining expression fully dependent on sigma S. Our results indicate that transcription at the csiE promoter can be initiated in vivo by sigma S-containing RNA polymerase alone as well as by sigma 70-containing RNA polymerase in conjunction with cAMP-CRP or a cAMP-CRP-dependent secondary regulator. The promoter region of poxB, the structural gene for pyruvate oxidase, which is also under the control of sigma S and cAMP-CRP, is very similar to the corresponding region of csiE, suggesting a similar regulatory mechanism also for poxB.
Bacterial Proteins↗
Regulation of pyruvate dehydrogenase complex synthesis in Escherichia coli K 12. Identification of the inducing metabolite.
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Acyltransferases↗
A seven-iron ferredoxin from the thermoacidophilic archaeon Desulfurolobus ambivalens.
A seven-iron ferredoxin was isolated from aerobically grown cells of the hyperthermoacidophilic archaeon Desulfurolobus ambivalens (DSM 3772). The protein is monomeric, with an apparent molecular mass of 15 kDa and contains 7 iron atoms/molecule. The N-terminal sequence shows a large similarity (70% identity) with that of the ferredoxin isolated from the archaeon Sulfolobus acidocaldarius. The EPR characteristics in both the native (oxidized) and dithionite-reduced states of this protein allowed an unequivocal identification of a [3Fe-4S]1+/0 center, with a reduction potential of -270 +/- 20 mV, at pH 7.5. The protein also contains a [4Fe-4S]2+/1+ center with a very low reduction potential (Eo = -540 mV, pH 7.0), which yields a rhombic EPR spectrum upon reduction with sodium dithionite at high pH. The reduction potentials of both centers are slightly pH dependent between pH 6 and 9. The [3Fe-4S] ferredoxin center is able to accept electrons from pyruvate oxidase and NADH oxidase isolated from D. ambivalens. This ferredoxin is present in large amounts (at least 130 mg/kg wet cells), which allowed the unequivocal observation of oxidized [3Fe-4S] clusters in intact D. ambivalens cells.
Amino Acid Sequence↗
Homolactate fermentation by metabolically engineered Escherichia coli strains.
We report the homofermentative production of lactate in Escherichia coli strains containing mutations in the aceEF, pfl, poxB, and pps genes, which encode the pyruvate dehydrogenase complex, pyruvate formate lyase, pyruvate oxidase, and phosphoenolpyruvate synthase, respectively. The process uses a defined medium and two distinct fermentation phases: aerobic growth to an optical density of about 30, followed by nongrowth, anaerobic production. Strain YYC202 (aceEF pfl poxB pps) generated 90 g/liter lactate in 16 h during the anaerobic phase (with a yield of 0.95 g/g and a productivity of 5.6 g/liter . h). Ca(OH)(2) was found to be superior to NaOH for pH control, and interestingly, significant succinate also accumulated (over 7 g/liter) despite the use of N(2) for maintaining anaerobic conditions. Strain ALS961 (YYC202 ppc) prevented succinate accumulation, but growth was very poor. Strain ALS974 (YYC202 frdABCD) reduced succinate formation by 70% to less than 3 g/liter. (13)C nuclear magnetic resonance analysis using uniformly labeled acetate demonstrated that succinate formation by ALS974 was biochemically derived from acetate in the medium. The absence of uniformly labeled succinate, however, demonstrated that glyoxylate did not reenter the tricarboxylic acid cycle via oxaloacetate. By minimizing the residual acetate at the time that the production phase commenced, the process with ALS974 achieved 138 g/liter lactate (1.55 M, 97% of the carbon products), with a yield of 0.99 g/g and a productivity of 6.3 g/liter . h during the anaerobic phase.
Aerobiosis↗
Expression of the xylulose 5-phosphate phosphoketolase gene, xpkA, from Lactobacillus pentosus MD363 is induced by sugars that are fermented via the phosphoketolase pathway and is repressed by glucose mediated by CcpA and the mannose phosphoenolpyruvate phosphotransferase system.
Purification of xylulose 5-phosphate phosphoketolase (XpkA), the central enzyme of the phosphoketolase pathway (PKP) in lactic acid bacteria, and cloning and sequence analysis of the encoding gene, xpkA, from Lactobacillus pentosus MD363 are described. xpkA encodes a 788-amino-acid protein with a calculated mass of 88,705 Da. Expression of xpkA in Escherichia coli led to an increase in XpkA activity, while an xpkA knockout mutant of L. pentosus lost XpkA activity and was not able to grow on energy sources that are fermented via the PKP, indicating that xpkA encodes an enzyme with phosphoketolase activity. A database search revealed that there are high levels of similarity between XpkA and a phosphoketolase from Bifidobacterium lactis and between XpkA and a (putative) protein present in a number of evolutionarily distantly related organisms (up to 54% identical residues). Expression of xpkA in L. pentosus was induced by sugars that are fermented via the PKP and was repressed by glucose mediated by carbon catabolite protein A (CcpA) and by the mannose phosphoenolpyruvate phosphotransferase system. Most of the residues involved in correct binding of the cofactor thiamine pyrophosphate (TPP) that are conserved in transketolase, pyruvate decarboxylase, and pyruvate oxidase were also conserved at a similar position in XpkA, implying that there is a similar TPP-binding fold in XpkA.
Aldehyde-Lyases↗
Effect of acidic pH on expression of surface-associated proteins of Streptococcus oralis.
Streptococcus oralis, a member of the mitis group of oral streptococci, is implicated in the pathogenesis of infective endocarditis and is the predominant aciduric non-mutans-group streptococcus in dental plaque. We undertook to identify the most abundant surface-associated proteins of S. oralis and to investigate changes in protein expression when the organism was grown under acidic culture conditions. Surface-associated proteins were extracted from cells grown in batch culture, separated by two-dimensional gel electrophoresis, excised, digested with trypsin, and analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry and liquid chromatography-tandem mass spectrometry. Putative functions were assigned by homology to a translated genomic database of Streptococcus pneumoniae. A total of 27 proteins were identified; these included a lipoprotein, a ribosome recycling factor, and the glycolytic enzymes phosphoglycerate kinase, fructose bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase, and enolase. The most abundant protein, phosphocarrier protein HPr, was present as three isoforms. Neither lactate dehydrogenase nor pyruvate oxidase, dominant intracellular proteins, were present among the proteins on the gels, demonstrating that proteins in the surface-associated pool did not arise as a result of cell lysis. Eleven of the proteins identified were differentially expressed when cells were grown at pH 5.2 versus pH 7.0, and these included superoxide dismutase, a homologue of dipeptidase V from Lactococcus lactis, and the protein translation elongation factors G, Tu, and Ts. This study has extended the range of streptococcal proteins known to be expressed at the cell surface. Further investigations are required to ascertain their functions at this extracellular location and determine how their expression is influenced by other environmental conditions.
Bacterial Proteins↗
Molecular and functional characterization of Salmonella enterica serovar typhimurium poxA gene: effect on attenuation of virulence and protection.
Salmonella enterica poxA mutants exhibit a pleiotropic phenotype, including reduced pyruvate oxidase activity; reduced growth rate; and hypersensitivity to the herbicide sulfometuron methyl, alpha-ketobutyrate, and amino acid analogs. These mutants also failed to grow in the presence of the host antimicrobial peptide, protamine. In this study, PoxA- mutants of S. enterica serovar Typhimurium (S. typhimurium) were found to be 10,000-fold attenuated in orally inoculated BALB/c mice and 1,000-fold attenuated in intraperitoneally inoculated BALB/c mice, compared to wild-type S. typhimurium UK-1. In addition, poxA mutants were found to be capable of colonizing the spleen, mesenteric lymph nodes, and Peyer's patches; to induce strong humoral immune responses; and to protect mice against a lethal wild-type Salmonella challenge. A 2-kb DNA fragment was isolated from wild-type S. typhimurium UK-1 based on its ability to complement an isogenic poxA mutant. The nucleotide sequence of this DNA fragment revealed an open reading frame of 325 amino acids capable of encoding a polypeptide of 36.8 kDa that was confirmed in the bacteriophage T7 expression system. Comparison of the translated sequence to the available databases indicated high homology to a family of lysyl-tRNA synthetases. Our results indicate that a mutation of poxA has an attenuating effect on Salmonella virulence. Further, poxA mutants are immunogenic and could be useful in designing live vaccines with a variety of bacterial species. To our knowledge, this is the first report on the effect of poxA mutation on bacterial virulence.
Animals↗
Effect of halofenate and clofibrate on growth and lipid synthesis in Saccharomyces cerevisiae.
Halofenate-free acid (HFA) inhibited the growth of Saccharomyces cerevisiae by 50% at a concentration of 0.34 mm. This inhibitory effect was prevented by addition of either oleate or acetate, but not by pyruvate. When cell growth was supported by oleate, HFA inhibited the incorporation of radioactive carbon from glucose-U-(14)C or pyruvate-2-(14)C into fatty acids and sterols. The incorporation of radioactive carbon into fatty acids and sterols from acetate-2-(14)C was unaffected by the compound. When cell growth was supported by either oleate or acetate, HFA inhibited the conversion of pyruvate-1-(14)C to (14)CO(2). These results suggest that HFA inhibits the conversion of pyruvate to acetate in yeast. Partially purified yeast pyruvate dehydrogenase was inhibited 50% by 5.5 mm HFA; however, the concentration required for 50% inhibition was considerably reduced when the enzyme was preincubated with the compound at room temperature. In a similar manner, the hypolipidemic agent clofibrate-free acid inhibited the growth of yeast by 50% at 3.0 mm. This inhibition was also prevented by acetate and not by pyruvate. In addition, clofibrate-free acid inhibited partially purified pyruvate dehydrogenase by 50% at a concentration of 37.0 mm.
Acetates↗
Role of pyruvate and S-adenosylmethioine in activating the pyruvate formate-lyase of Escherichia coli.
The pyruvate formate-lyase activity of extracts of Escherichia coli is stimulated and the dilution effect is abolished by the addition of pyruvate to the extract. The activity can be purified fourfold from pyruvate-supplemented extracts by isoelectric precipitation under anaerobic conditions. The activity of extracts not supplemented with pyruvate has been separated into two fractions by treatment with protamine sulfate-fraction PS, the soluble portion, and fraction N, an extract of the precipitate formed upon the addition of protamine sulfate. After treatment of these fractions with charcoal, pyruvate formate-lyase activity is stimulated by the addition of S-adenosylmethionine. When sodium pyruvate is added to the crude extract before the fractionation, fraction PS has full enzymatic activity and is not stimulated by fraction N or by S-adenosylmethionine. Incubation of the inactive fractions with pyruvate and S-adenosylmethionine in the absence of other substrates similarly results in a highly active preparation, not subject to the "dilution effect" obtained when the fractions are added separately to the assay. These observations suggest that the component in the protamine supernatant fraction is activated by the other fraction and that S-adenosylmethionine and pyruvate are required for the activation reaction. The activating factor present in the protamine precipitate fraction may be further purified by heating for 10 min at 100 C under H(2) atmosphere. The yield of this factor from crude extract is not affected by activation of the pyruvate formate-lyase of the extract, indicating that the factor acts catalytically. The requirement for pyruvate is only partially satisfied by alpha-ketobutyrate and not at all by other alpha-keto acids, acetyl phosphate, or adenosine triphosphate. The rate of activation is maximal at 0.01 m sodium pyruvate and 3 x 10(-4)mS-adenosylmethionine; it is linearly dependent on the amount of activating factor added. The rate of activation is the same when the activation reaction is initiated by addition of any of the four required components, indicating that no slow step of activation can be carried out by any three of the components. A similar pyruvate formate-lyase system was found in extracts of the methionine/B(12) autotroph 113-3, grown with methionine supplement, indicating that vitamin B(12) derivatives do not participate in the system.
Acyltransferases↗
Catalytic functions of thiamin diphosphate.
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Aldehyde-Lyases↗