Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.3.3.1 (citrate synthase)
 4,488 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The enzymes beta-ketothiolase and citrate synthase from the facultatively methylotrophic Methylobacterium rhodesianum MB 126, which uses the serine pathway, were purified and characterized. The beta-ketothiolase had a relatively high Km for acetyl-CoA (0.5 mM) and was strongly inhibited by CoA (Ki 0.02 mM). The citrate synthase had a much higher affinity for acetyl-CoA (Km 0.07 mM) and was significantly inhibited by NADH (Ki 0.15 mM). The intracellular concentration of CoA metabolites and nucleotides was determined in M. rhodesianum MB 126 during growth on methanol. The level of CoA decreased from about 0.6 nmol (mg dry mass)-1 during growth to the detection limit when poly(beta-hydroxybutyrate) (PHB) accumulated. Nearly unchanged intracellular concentrations of NADH, NADPH, and acetyl-CoA of about 0.5, 0.6-0.7, and 1.0 nmol (mg dry mass)-1, respectively, were determined during growth and PHB synthesis. During growth, the beta-ketothiolase was almost completely inhibited by CoA, and acetyl-CoA was principally consumed by the citrate synthase. During PHB accumulation, the beta-ketothiolase had about 75% of its maximum activity and showed much higher activity than citrate synthase, which at the actual NADH concentration was about 75% inhibited. NADPH concentration was sufficiently high to allow the unlimited activity of acetoacetyl-CoA reductase (Km NADPH 18 microM). PHB synthesis is probably mainly controlled by the CoA concentration in M. rhodesianum MB 126.
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PMID:Competition between beta-ketothiolase and citrate synthase during poly(beta-hydroxybutyrate) synthesis in Methylobacterium rhodesianum. 908 18

2-Methylcitrate synthase (2-MCS1) and citrate synthase (CS) of Ralstonia eutropha strain H16 were separated by affinity chromatography and analyzed for their substrate specificities. 2-MCS1 used not only the primary substrate propionyl-CoA but also acetyl-CoA and, at a low rate, even butyryl-CoA and valeryl-CoA for condensation with oxaloacetate. The KM values for propionyl-CoA and acetyl-CoA were 0.061 or 0.35 mM, respectively. This enzyme is therefore a competitor for acetyl-CoA during biosynthesis of poly(3-hydroxybutyrate) (PHB) and has to be taken into account if metabolic fluxes are calculated for PHB biosynthesis. In contrast, CS could not use propionyl-CoA as a substrate. The gene-encoding CS (cisY) of R. eutropha was cloned and encodes for a protein consisting of 433 amino acids with a calculated molecular weight of 48,600 Da; it is not truncated in the N-terminal region. Furthermore, a gene encoding a second functionally active 2-methylcitrate synthase (2-MCS2, prpC2) was identified in the genome of R. eutropha. The latter was localized in a gene cluster with genes for an NAD(H)-dependent malate dehydrogenase and a putative citrate lyase. RT-PCR analysis of R. eutropha growing on different carbon sources revealed the transcription of prpC2. In addition, cells of recombinant Escherichia coli strains harboring prpC2 of R. eutropha exhibited high 2-MCS activity of 0.544 U mg-1. A prpC2 knockout mutant of R. eutropha exhibited an identical phenotype as the wild type if grown on different media. 2-MCS2 seems to be dispensable, and a function could not be revealed for this enzyme.
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PMID:Occurrence and expression of tricarboxylate synthases in Ralstonia eutropha. 1613 21

NADPH, a major reducing power in microorganisms, is mostly generated from the pentose phosphate (PP) pathway by glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) expressed by the zwf and gnd genes, respectively. The characteristics of these two genes in Escherichia coli were compared after their re-introduction into the parent strain for over-expression. zwf encoding G6PDH increased the level of NADPH 3 folds compared to gnd encoding 6PGDH. An excess of NADPH depressed cell growth mainly due to the inhibition of citrate synthase in the TCA cycle. Recombinant plasmids containing zwf or gnd co-integrated with the phbCAB operon from Ralstonia eutropha were constructed, and introduced into E. coli for the oddball biosynthesis of PHB. The amount of PHB increased after enforcing the genes; especially the zwf gene an increase of around 41%, due to the rise in NADPH and the depressed TCA cycle, leading to the metabolic flux of intermediates to the pathway for the biosynthesis of PHB.
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PMID:Amplification of the NADPH-related genes zwf and gnd for the oddball biosynthesis of PHB in an E. coli transformant harboring a cloned phbCAB operon. 1623 47

The enzymatic capacity for metabolism of poly-(beta)-hydroxybutyrate (PHB) has been examined in nitrogen-fixing symbioses of soybean (Glycine max L.) plants, which may accumulate substantial amounts of PHB, and chickpea (Cicer arietinum L.) plants, which contain little or no PHB. In the free-living state, both Bradyrhizobium japonicum CB 1809 and Rhizobium sp. (Cicer) CC 1192, which form nodules on soybean and chickpea plants, respectively, produced substantial amounts of PHB. To obtain information on why chickpea bacteroids do not accumulate PHB, the specific activities of enzymes of PHB metabolism (3-ketothiolase, acetoacetyl-coenzyme A reductase, PHB depolymerase, and 3-hydroxybutyrate dehydrogenase), the tricarboxylic acid cycle (malate dehydrogenase, citrate synthase, and isocitrate dehydrogenase), and related reactions (malic enzyme, pyruvate dehydrogenase, and glutamate:2-oxoglutarate transaminase) were compared in extracts from chickpea and soybean bacteroids and the respective free-living bacteria. Significant differences were noted between soybean and chickpea bacteroids and between the bacteroid and free-living forms of Rhizobium sp. (Cicer) CC 1192, with respect to the capacity for some of these reactions. It is suggested that a greater potential for oxidizing malate to oxaloacetate in chickpea bacteroids may be a factor that favors the utilization of acetyl-coenzyme A in the tricarboxylic acid cycle over PHB synthesis.
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PMID:Enzymes of Poly-(beta)-Hydroxybutyrate Metabolism in Soybean and Chickpea Bacteroids. 1653 45

Regulatory roles of nicotinamide nucleotides and three key enzymes, beta-ketothiolase (KT), NADPH-dependent acetoacetyl-CoA reductase (AAR), and citrate synthase (CS), on poly(3-hydroxybutyrate) (PHB) synthesis in recombinant Escherichia coli harboring a plasmid containing the Alcaligenes eutrophus polyhydroxyalkanoate (PHA) biosynthesis genes were examined. Cells were grown in various media and were subsequently compared for PHB concentration, PHB content, the activities of the key enzymes, and the levels of nicotinamide nucleotides. Cells of recombinant E. coli accumulated the largest amount of PHB in LB+glucose medium among those tested. PHB synthesis was not enhanced by limiting inorganic ions. The activity of CS, which competes with KT for acetyl-CoA, was lower when cells were grown in LB+glucose compared with other media. The NADPH level and the NADPH/NADP ratio were high in LB+glucose. Examination of the time profiles of the specific PHB synthesis rate, key enzyme activities, and the levels of nicotinamide nucleotides showed that PHB synthesis is most significantly affected by the NADPH level. Even though the NADH level and the NADH/NAD ratio were also high during the synthesis of PHB, no direct evidence of their positive effect on PHB synthesis was found. Low activity of CS was beneficial for PHB synthesis due to the availability of more acetyl-CoA to PHB biosynthetic pathway. In recombinant E. coli, the level of NADPH and/or the NADPH/NADP ratio seem to be the most critical factor regulating the activity of AAR and, subsequently, PHB synthesis. (c) 1996 John Wiley & Sons, Inc.
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PMID:Regulatory effects of cellular nicotinamide nucleotides and enzyme activities on poly(3-hydroxybutyrate) synthesis in recombinant Escherichia coli. 1862 49