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1.
The fermentation enzymes, which enable the microaerophilic protist Entamoeba histolytica to parasitize the colonic lumen and tissue abscesses, closely resemble homologues in anaerobic prokaryotes. Here, genes encoding malic enzyme and acetyl-CoA synthetase (nucleoside diphosphate forming) were cloned from E. histolytica, and their evolutionary origins, as well as those encoding two alcohol dehydrogenases (ADHE and ADH1), were inferred by means of phylogenetic reconstruction. The E. histolytica malic enzyme, which decarboxylates malate to pyruvate, closely resembles that of the archaeon Archaeoglobus fulgidus, strongly suggesting a common origin. The E. histolytica acetyl-CoA synthetase, which converts acetyl-CoA to acetate with the production of ATP, appeared to be closely related to the Plasmodium falciparum enzyme, but it was no more closely related to the Giardia lamblia acetyl-CoA synthetase than to those of archaea. Phylogenetic analyses suggested that the adh1 and adhe genes of E. histolytica and Gram-positive eubacteria share a common ancestor. Lateral transfer of genes encoding these fermentation enzymes from archaea or eubacteria to E. histolytica probably occurred early, because the sequences of the amoebic enzymes show considerable divergence from those of prokaryotes, and the amoebic genes encoding these enzymes are in the AT-rich codon usage of the parasite. 相似文献
2.
The gene coding for the acetyl-CoA synthetase (ADP-forming) from the amitochondriate eukaryote Giardia lamblia has been expressed in Escherichia coli. The recombinant enzyme exhibited the same substrate specificity as the native enzyme, utilizing acetyl-CoA and adenine nucleotides as preferred substrates and less efficiently, propionyl- and succinyl-CoA. N- and C-terminal parts of the G. lamblia acetyl-CoA synthetase sequence were found to be homologous to the alpha- and beta-subunits, respectively, of succinyl-CoA synthetase. Sequence analysis of homologous enzymes from various bacteria, archaea, and the eukaryote, Plasmodium falciparum, identified conserved features in their organization, which allowed us to delineate a new superfamily of acyl-CoA synthetases (nucleoside diphosphate-forming) and its signature motifs. The representatives of this new superfamily of thiokinases vary in their domain arrangement, some consisting of separate alpha- and beta-subunits and others comprising fusion proteins in alpha-beta or beta-alpha orientation. The presence of homologs of acetyl-CoA synthetase (ADP-forming) in such human pathogens as G. lamblia, Yersinia pestis, Bordetella pertussis, Pseudomonas aeruginosa, Vibrio cholerae, Salmonella typhi, Porphyromonas gingivalis, and the malaria agent P. falciparum suggests that they might be used as potential drug targets. 相似文献
3.
Simon A. Cobbold Ashley M. Vaughan Ian A. Lewis Heather J. Painter Nelly Camargo David H. Perlman Matthew Fishbaugher Julie Healer Alan F. Cowman Stefan H. I. Kappe Manuel Llinás 《The Journal of biological chemistry》2013,288(51):36338-36350
The malaria parasite Plasmodium falciparum depends on glucose to meet its energy requirements during blood-stage development. Although glycolysis is one of the best understood pathways in the parasite, it is unclear if glucose metabolism appreciably contributes to the acetyl-CoA pools required for tricarboxylic acid metabolism (TCA) cycle and fatty acid biosynthesis. P. falciparum possesses a pyruvate dehydrogenase (PDH) complex that is localized to the apicoplast, a specialized quadruple membrane organelle, suggesting that separate acetyl-CoA pools are likely. Herein, we analyze PDH-deficient parasites using rapid stable-isotope labeling and show that PDH does not appreciably contribute to acetyl-CoA synthesis, tricarboxylic acid metabolism, or fatty acid synthesis in blood stage parasites. Rather, we find that acetyl-CoA demands are supplied through a “PDH-like” enzyme and provide evidence that the branched-chain keto acid dehydrogenase (BCKDH) complex is performing this function. We also show that acetyl-CoA synthetase can be a significant contributor to acetyl-CoA biosynthesis. Interestingly, the PDH-like pathway contributes glucose-derived acetyl-CoA to the TCA cycle in a stage-independent process, whereas anapleurotic carbon enters the TCA cycle via a stage-dependent phosphoenolpyruvate carboxylase/phosphoenolpyruvate carboxykinase process that decreases as the parasite matures. Although PDH-deficient parasites have no blood-stage growth defect, they are unable to progress beyond the oocyst phase of the parasite mosquito stage. 相似文献
4.
《Insect Biochemistry》1984,14(6):639-643
Extracts of tissues of the lower termites, Reticulitermes flavipes and Coptotermes lacteus, and the higher termite, Nasutitermes exitiosus, possess acetyl-CoA synthetase and all the enzymes of the tricarboxylic acid cycle and are thus able to oxidize acetate to CO2. The specific activities of these enzymes in R. flavipes are sufficient to cope with the rate of acetogenesis by the gut microbiota. The presence of the malic enzyme and malate dehydrogenase, but not pyruvate carboxylase or phosphoenolpyruvate carboxykinase, indicates that they may be important as anaplerotic enzymes for the conversion of pyruvate to oxalacetate. An apparent absence of pyruvate dehydrogenase in all termites suggests that they do not convert pyruvate to acetyl-CoA, but rather convert acetate (transported from the hindgut) to this compound. All the enzymes of glycolysis were present in termite extracts. Thus any glucose absorbed from the midgut, and originating from hydrolysis of cellulose by salivary or midgut enzymes, can be metabolized by termites as an energy source. 相似文献
5.
Two routes for synthesis of phosphoenolpyruvate from C4-dicarboxylic acids in Escherichia coli 总被引:6,自引:0,他引:6
Mutants of defective in both phosphoenolpyruvate carboxykinase and phosphoenolpyruvate synthetase are unable to use C4-dicarboxylic acids such as succinate and malate as carbon and energy sources for growth. Revertants that have restored function for either one of these enzymes can grow in a malate-mineral medium, but at a reduced rate compared with the growth of wild-type cells. appears to use two pathways for synthesis of phosphoenolpyruvate from C4-dicarboxylic acids. One of these involves decarboxylation of oxalacetate catalyzed by phosphoenolpyruvate carboxykinase. The second pathway makes use of the combined action of malic enzyme and phosphoenolpyruvate synthetase. 相似文献
6.
Genetic analysis of 119 acetate non-utilizing (acu) mutants in Aspergillus nidulans revealed ten new loci affecting acetate metabolism in addition to the three previously recognized on the basis of resistance to fluoroacetate and acetate non-utilization. The enzyme lesions associated with mutations at seven of the acu loci are described. These are: facA (= acuA), acetyl-CoA synthase; acuD, isocitrate lyase; acuE, malate synthase; acuF, phosphoenolpyruvate carboxykinase; acuG, fructose 1,6-diphosphatase; acuK and acuM, malic enzyme. The acu loci have been mapped and are widely distributed over the genome of A. nidulans. Close linkage has only been found between acuA and acuD (less than 1% recombination). There is no evidence for any pleiotropic mutation in that region affecting the expression of both these genes. Poor induction of the enzymes of the glyoxylate cycle, isocitrate lyase and malate synthase in mutants lacking acetyl-CoA synthase, and also in the other two classes of fluoroacetate-resistant mutants, indicates that the inducer, acetate, may be metabolized to a true metabolic inducer, perhaps acetyl-CoA, to effect formation of the enzymes. There is no evidence of any other class of pleiotropic recessive acu mutations affecting the expression of the acuD and acuE genes, which are therefore thought to be subject to negative rather than positive control. 相似文献
7.
A psychrophilic marine Pseudomonas was found to contain phosphoenolpyruvate (PEP) carboxylase and an adenosine triphosphate-linked PEP carboxykinase. Some properties of these CO(2)-fixing enzymes were compared with those homologous enzymes from the terrestrial mesophile Enterobacter cloacae. The PEP carboxylases from both organisms were activated by acetyl-coenzyme A (CoA) and inhibited by l-aspartate. The enzyme from Pseudomonas was less dependent on the presence of the activator, but maximal activation was attained at acetyl-CoA concentrations much lower (50 mum) than those required to saturate the enzyme from E. cloacae. In both cases the main effect of acetyl-CoA was to decrease the K(m) value for PEP. The activity of PEP carboxylase from Pseudomonas was only slightly inhibited by NaCl, KCl, or NH(4)Cl up to 100 mm, whereas the enzyme from E. cloacae was inhibited by about 70% under similar experimental conditions. Both PEP carboxylase and PEP carboxykinase from Pseudomonas showed considerably lower thermal stability than their counterparts from E. cloacae. Our results suggest that the CO(2)-fixing enzymes from a marine Pseudomonas and E. cloacae are similar in nature and regulation, but they differ in properties related to the peculiar conditions of the marine environment. 相似文献
8.
《Process Biochemistry》2010,45(9):1459-1467
The aim of this work was to understand the relevance of central carbon metabolism in salt stress adaptation of Escherichia coli. The cells were grown anaerobically in batch and chemostat reactors at different NaCl concentrations using glycerol as a carbon source. Enzyme activities of the main metabolic pathways, external metabolites, ATP level, NADH/NAD+ ratio, l-carnitine production and the expression level of the main genes related to stress response were used to characterize the metabolic state under the osmotic stress. The results provided the first experimental evidence of the important role played by central metabolism adaptation and cell survival after long-term exposure to salt stress. Increased glycolytic fluxes and higher production of fermentation products indicated the importance of energy metabolism. Carbon fluxes under stress conditions were controlled by the decrease in the isocitrate dehydrogenase/isocitrate lyase ratio and the phosphoenolpyruvate carboxykinase/phosphoenolpyruvate carboxylase ratio, and the increase in the phosphotransferase/acetyl-CoA synthetase ratio. Altogether, the results demonstrate that, under salt stress, E. coli enhances energy production by substrate-level phosphorylation (Pta–Ack pathway) and the anaplerotic function of the TCA cycle, in order to provide precursors for biosynthesis. The results are discussed in relation with the general stress response and metabolic adaptation of E. coli. 相似文献
9.
Yoko Chiba ) Ryoma Kamikawa ) Kumiko Nakada-Tsukui ) Yumiko Saito-Nakano ) Tomoyoshi Nozaki ) 《The Journal of biological chemistry》2015,290(39):23960-23970
Phosphoenolpyruvate carboxykinase (PEPCK) is one of the pivotal enzymes that regulates the carbon flow of the central metabolism by fixing CO2 to phosphoenolpyruvate (PEP) to produce oxaloacetate or vice versa. Whereas ATP- and GTP-type PEPCKs have been well studied, and their protein identities are established, inorganic pyrophosphate (PPi)-type PEPCK (PPi-PEPCK) is poorly characterized. Despite extensive enzymological studies, its protein identity and encoding gene remain unknown. In this study, PPi-PEPCK has been identified for the first time from a eukaryotic human parasite, Entamoeba histolytica, by conventional purification and mass spectrometric identification of the native enzyme, followed by demonstration of its enzymatic activity. A homolog of the amebic PPi-PEPCK from an anaerobic bacterium Propionibacterium freudenreichii subsp. shermanii also exhibited PPi-PEPCK activity. The primary structure of PPi-PEPCK has no similarity to the functional homologs ATP/GTP-PEPCKs and PEP carboxylase, strongly suggesting that PPi-PEPCK arose independently from the other functional homologues and very likely has unique catalytic sites. PPi-PEPCK homologs were found in a variety of bacteria and some eukaryotes but not in archaea. The molecular identification of this long forgotten enzyme shows us the diversity and functional redundancy of enzymes involved in the central metabolism and can help us to understand the central metabolism more deeply. 相似文献
10.
Thyroxine elicits divergent changes in mRNA levels for two urea cycle enzymes and one gluconeogenic enzyme in tadpole liver 总被引:1,自引:0,他引:1
S M Morris 《Archives of biochemistry and biophysics》1987,259(1):144-148
Thyroxine-induced metamorphosis of the tadpole to the frog (Rana catesbeiana) is marked by increased activities of the urea cycle enzymes in liver. Cloned cDNAs for two mammalian urea cycle enzymes--carbamyl-phosphate synthetase I and argininosuccinate synthetase--were shown to cross-hybridize with the corresponding mRNAs in tadpole liver. Thyroxine treatment produced nearly 10-fold, coordinate increases in hybridizable mRNA levels for these two enzymes in tadpole liver. This increase is sufficient to account for reported increases in enzyme levels and synthesis rates, demonstrating that thyroxine largely regulates concentrations of these enzymes at a pretranslational step(s). In contrast, levels of phosphoenolpyruvate carboxykinase mRNA in tadpole liver decreased by more than 90% following thyroxine treatment. This differs from the thyroxine-induced increases in synthesis rates of enzyme and mRNA reported for phosphoenolpyruvate carboxykinase in rat liver. However, the decreased levels of this mRNA in tadpole liver may represent a secondary response due to thyroxine-stimulated release of insulin. 相似文献
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12.
The concentrations of cyclic AMP and cyclic GMP in brown fat and liver of both suckling and adult rats at fixed times after injection of insulin (2.5 U/100 g body weight) or prednisolone (2.5 mg/100 g body weight) were compared with the activity of phosphoenolpyruvate carboxykinase assayed 24 h after the injections. A stimulus that produced an increase in cyclic AMP content also produced an increase in the enzyme activity. If the content of cyclic GMP was also increased there was no rise in phosphoenolpyruvate carboxykinase activity. A rise in the content of cyclic GMP alone was associated with a reduction in the activity of the enzyme. These preliminary results indicate that cyclic AMP could be involved in the induction of phosphoenolpyruvate carboxykinase and that cyclic GMP may somehow be related to its repression. The known differences in the response of phosphoenolpyruvate carboxykinase activity to insulin and prednisolone in different tissues and at different stages of ontogenic development may thus be linked to differences in the responsiveness of enzymes concerned with the metabolism of cyclic nucleotides. 相似文献
13.
C A Casey P M Anderson 《Comparative biochemistry and physiology. B, Comparative biochemistry》1985,82(2):307-315
The submitochondrial localization of the four mitochondrial enzymes associated with urea synthesis in liver of Squalus acanthias (spiny dogfish), a representative elasmobranch, was determined. Glutamine- and acetylglutamate-dependent carbamoyl-phosphate synthetase, ornithine carbamoyltransferase, glutamine synthetase, and arginase were all localized within the matrix of liver mitochondria. The subcellular and submitochondrial localization and activities of several related enzymes involved in nitrogen metabolism and gluconeogenesis in liver and dogfish are also reported. Pyruvate carboxylase and phosphoenolpyruvate carboxykinase were localized in the mitochondrial matrix. Synthesis of citrulline by isolated mitochondria from ornithine proceeds at a near optimal rate at ornithine concentrations as low as 0.08 mM. The same stoichiometry and rates of citrulline synthesis are observed when ornithine is replaced by arginine. The mitochondrial location of arginase does not appear to reflect a mechanism for regulating ornithine availability. 相似文献
14.
Phosphoenolpyruvate carboxykinase in plants exhibiting crassulacean Acid metabolism 总被引:8,自引:14,他引:8
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Phosphoenolpyruvate carboxykinase has been found in significant activities in a number of plants exhibiting Crassulacean acid metabolism. Thirty-five species were surveyed for phosphoenolpyruvate carboxykinase, phosphoenolpyruvate carboxylase, ribulose diphosphate carboxylase, malic enzyme, and malate dehydrogenase (NAD). Plants which showed high activities of malic enzyme contained no detectable phosphoenolpyruvate carboxykinase, while plants with high activities of the latter enzyme contained little malic enzyme. It is proposed that phosphoenolpyruvate carboxykinase acts as a decarboxylase during the light period, furnishing CO2 for the pentose cycle and phosphoenolpyruvate for gluconeogenesis. 相似文献
15.
Acetate-nonutilizing Mutants of Neurospora crassa II. Biochemical Deficiencies and the Roles of Certain Enzymes
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The levels of Krebs cycle, glyoxylate cycle, and certain other enzymes were measured in a wild-type strain and in seven groups of acetate-nonutilizing (acu) mutants of Neurospora crassa, both after growth on a medium containing sucrose and after a subsequent 6-hr incubation in a similar medium, containing acetate as the sole source of carbon. In the wild strain, incubation in acetate medium caused a rise in the levels of isocitrate lyase, malate synthase, phosphoenolpyruvate carboxykinase, acetyl-coenzyme A synthetase, nicotinamide adenine dinucleotide phosphate-linked isocitrate dehydrogenase, citrate synthase, and fumarate hydratase. Isocitrate lyase activity was absent in acu-3 mutants; acu-5 mutants lacked acetyl-coenzyme A synthetase activity; and no oxoglutarate dehydrogenase activity (or only low levels) could be detected in acu-2 and acu-7 mutants. In acu-6 mutants, phosphoenolpyruvate carboxykinase activity was either very low or absent. No specific biochemical deficiencies could be attributed to the acu-1 and acu-4 mutations. The role of several of these enzymes during growth on acetate is discussed. 相似文献
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18.
Johannes Lehmann 《Planta》1973,114(1):51-61
Summary Tests for enzymes of gluconeogenesis and of the synthesis and degradation of sucrose and polysaccharides have been carried out in the phloem exudate of Cucurbita pepo. All the enzymes which are necessary for the synthesis of sucrose and polysaccharides from metabolites of the citric acid cycle were found to be present in the exudate, except phosphoenolpyruvate carboxykinase. The polysaccharide synthetase was found to exhibit higher activity with glycogen (which is an unnatural polysaccharide in higher plants) than with starch. In addition, polysaccharide synthetase activity could be increased remarkably with 2 mM glucose-6-phosphate and glycogen as primer. Among the enzymes which catabolize sucrose and polysaccharides (phosphorylase, invertase, sucrose phosphorylase), only sucrose phosphorylase showed activity. 相似文献
19.
Glycogen phosphorylase genes or messages from four amitochondriate eukaryotes, Trichomonas vaginalis, Mastigamoeba balamuthi, Entamoeba histolytica (two genes) and Giardia intestinalis, have been isolated and sequenced. The sequences of the amitochondriate protist enzymes appear to share a most recent common ancestor. The clade containing these sequences is closest to that of another protist, the slime mold (Dictyostelium discoideum), and is more closely related to fungal and plant phosphorylases than to mammalian and eubacterial homologs. Structure-based amino acid alignment shows conservation of the residues and domains involved in catalysis and allosteric regulation by glucose 6-phosphate but high divergence at domains involved in phosphorylation-dependent regulation and AMP binding in fungi and animals. Protist phosphorylases, as their prokaryotic and plant counterparts, are probably not regulated by phosphorylation. 相似文献
20.
Stereoselectivity of interaction of phosphoenolpyruvate analogues with various phosphoenolpyruvate-utilizing enzymes 总被引:1,自引:0,他引:1
The halogenated phosphoenolpyruvate analogues (Z)-phosphoenol-3-fluoropyruvate, (E)-phosphoenol-3-fluoropyruvate, and (Z)-phosphoenol-3-bromopyruvate were synthesized and purified. The analogues were characterized by 1H and by 19F NMR where applicable. Absolute stereoselectivity of the fluorophosphoenolpyruvate isomers as substrates with the enzymes phosphoenolpyruvate carboxykinase, enolase, and pyruvate phosphate dikinase was observed. The Z isomer exhibited substrate activity with these enzymes while no substrate activity was measured with the E isomer. Both isomers exhibited substrate activity with the enzyme pyruvate kinase, however, with a substantial decrease in the Vmax/Km ratio compared to phosphoenolpyruvate as the substrate. A metal ion dependent stereoselectivity of inhibition was measured for these analogues with the enzymes phosphoenolpyruvate carboxykinase, enolase, and pyruvate kinase. The cation activator appears to affect the specificity and thus the catalytic site of these enzymes. Proton longitudinal relaxation rate titrations demonstrate that the dissociation constants, K3, of the fluorophosphoenolpyruvate isomers from the enzyme-Mn complex agree, in most cases, with the measured KI values and analogue binding resembles phosphoenolpyruvate binding. With the enzyme phosphoenolpyruvate carboxykinase, the KI not equal to K3 for (E)-fluorophosphoenolpyruvate which suggests that the binding of the E isomer is affected by the presence of the other substrates. The halogenated derivatives apparently undergo an enzyme-Mn catalyzed Michael-type addition reaction with the bromo-substituted analogue decomposing much faster than the fluoro analogues. 相似文献