Acetate oxidation to CO2 via a citric acid cycle involving an ATP-citrate lyase: a mechanism for the synthesis of ATP via substrate level phosphorylation in Desulfobacter postgatei growing on acetate and sulfate

Desulfobacter postgatei is an acetate-oxidizing, sulfate-reducing bacterium that metabolizes acetate via the citric acid cycle. The organism has been reported to contain a si-citrate synthase (EC 4.1.3.7) which is activated by AMP and inorganic phosphate. It is show now, that the enzyme mediating citrate formation is an ATP-citrate lyase (EC 4.1.3.8) rather than a citrate synthase. Cell extracts (160,000xg supernatant) catalyzed the conversion of oxaloacetate (apparent K _m=0.2 mM), acetyl-CoA (app. K _m=0.1 mM), ADP (app. K _m=0.06 mM) and phosphate (app. K _m=0.7 mM) to citrate, CoA and ATP with a specific activity of 0.3 mol·min^-1·mg^-1 protein. Per mol citrate formed 1 mol of ATP was generated. Cleavage of citrate (app. K _m=0.05 mM; V _max=1.2 mol · min^-1 · mg^-1 protein) was dependent on ATP (app. K _m=0.4 mM) and CoA (app. K _m=0.05 mM) and yielded oxaloacetate, acetyl-CoA, ADP, and phosphate as products in a stoichiometry of citrate:CoA:oxaloacetate:ADP=1:1:1:1. The use of an ATP-citrate lyase in the citric acid cycle enables D. postgatei to couple the oxidation of acetate to 2 CO₂ with the net synthesis of ATP via substrate level phosphorylation.     

Relationship between fatty acid binding proteins,acetyl-CoA formation and fatty acid synthesis in developing human placenta

The relationship between fatty acid binding proteins, ATP citrate lyase activity and fatty acid synthesis in developing human placenta has been studied. Fatty acid binding proteins reverse the inhibitory efect of palmitoyl-CoA and oleate on ATP citrate lyase and fatty acid synthesis. In the absence of these inhibitors fatty acid binding proteins activate ATP citrate lyase and stimulate [ 1-¹⁴ C] acetate incorporation into placental fatty acids indicating binding of endogenous inhibitors by these proteins. Thus these proteins regulate the supply of acetyl-CoA as well as the synthesis of fatty acids from that substrates. As gestation proceeds and more lipids are required by the developing placenta fatty acid binding protein content, activity of ATP citrate lyase and rate of fatty acid synthesis increase indicating a cause and efect relationship between the demand of lipids and supply of precursor fatty acids during human placental development.     

Synthesis of citrate from phosphoenolpyruvate and acetylcarnitine by mitochondria from rabbit, pigeon and rat liver: Implications for lipogenesis

Rabbit, pigeon and rat liver mitochondria convert exogenous phosphoenolpyruvate and acetylcarnitine to citrate at rates of 14, 74 and 8 nmol/15 min/mg protein. Citrate formation is dependent on exogenous HCO3, is increased consistently by exogenous nucleotides (GDP, IDP, GTP, ADP, ATP) and inhibited strongly by 3-mercaptopicolinate and 1,2,3-benzenetricar☐ylate. Citrate is not made from pyruvate alone or combined with acetylcarnitine. Pigeon and rat liver mitochondria make large amounts of citrate from exogenous succinate, suggesting the presence of an endogenous source of acetyl units or a means of converting oxalacetate to acetyl units. Citrate synthesis from succinate by pigeon and rabbit mitochondria is increased significantly by exogenous acetylcarnitine. Pigeon and rat liver contain 80 and 15 times, respectively, more ATP:citrate lyase activity than does rabbit liver. Data suggest that mitochondrial phosphoenolpyruvate car☐ykinasein vivo could convert glycolysis-derived phosphoenolpyruvate to oxalacetate that, with acetyl CoA, could form citrate for export to support cytosolic lipogenesis as an activator of acetyl CoA car☐ylase, a carbon source via ATP:citrate lyase and NADPH via NADP: malate dehydrogenase or NADP: isocitrate dehydrogenase.     

A proposed biochemical mechanism for citric acid accumulation by Aspergillus niger Yang No. 2 growing in solid state fermentation

Aspergillus niger Yang No. 2 and its mutant strain SL1 were grown in solid state fermentation. Samples were taken after 2, 4 and 6 days of incubation and the mycelia were analysed for their intracellular concentrations of some organic acids and adenylates and the activities of selected enzymes. Strain Yang No. 2 contained high concentrations of citrate with very little oxalate, while strain SL1 contained lower concentrations of citrate but considerably higher concentrations of oxalate. As the fermentation proceeded, strain Yang No. 2 showed a much higher ratio of ATP:AMP than did strain SL1. In addition, the enzyme ATP:citrate lyase became undetectable during citrate accumulation in strain Yang No. 2, while its activity remained high during oxalate accumulation in strain SL1. It is proposed that citrate accumulation by strain Yang No. 2 during solid state fermentation is due to blockage of its metabolism in the mitochondrion via inhibition of isocitrate dehydrogenase by the high ATP:AMP ratio, and in the cytosol by repression of ATP:citrate lyase activity.     

Presence and regulation of ATP:citrate lyase from the citric acid producing fungus Aspergillus niger

ATP:citrate lyase (EC 4.1.3.8) has been identified in cell-free extracts from the filamentous fungus Aspergillus niger. The enzyme was located in the cytosol. It exhibits an activity at least ten times that of acetate-CoA-kinase (EC 6.2.1.1) during growth on carbohydrates as carbon sources, and is thus considered responsible for acetyl-CoA formation under these conditions. It is formed constitutively and its biosynthesis does not appear to be controlled by changes in the nitrogen or carbon source or type. ATP:citrate-lyase appears to be very labile during conventional purification procedures; a method involving fast protein liquid anion exchange chromatography was thus developed in order to obtain enzyme preparations sufficiently free of enzymes which could interfere with kinetic investigations. This preparation displays commonly known characteristics of ATP:citrate lyase with respect to substrate affinities and cofactor requirements, with the exception that the affinity for citrate is rather low (2.5 mM). No activator was found. The enzyme is inhibited by nucleoside diphosphates, nucleoside monophosphates and palmitoyl-CoA. Regulation of ATP:citrate lyase be the energy charge of the cytosol in relation to lipid or citric acid accumulation is discussed in view of these findings. Present address: Institut für Allgemeine Biochemie, Universität Wien, Währingerstrasse 38, A-1090 Wien, Austria     

Apparent Inhibition of ATP Citrate Lyase by l-Glutamate In Vitro Is Due to the Presence of Glutamine Synthetase

Preincubation in assay mixture for 30 min at 37 degrees C of ATP citrate lyase from rat brain and liver results in 65-70% inhibition in the presence of 10 mM L-glutamate. This inhibition is specific since none of the known brain metabolites of glutamate shows this effect. ATP and ammonium sulphate-suspended, commercially purified malate dehydrogenase are both important in the generation of inhibition; citrate and NADH are not. The ATP citrate lyase activity in desalted crude extracts and 11% polyethylene glycol-precipitated fractions is inhibited but the enzyme purified by dye affinity chromatography is unaffected. Such purification reveals the presence of a factor responsible for the generation of the inhibition shown to be of Mr 380,000. These lines of evidence implicate endogenous glutamine synthetase, and the involvement of this enzyme is established by the use of its inhibitor L-methionine sulphoximine and by the addition of purified glutamine synthetase to restore the glutamate inhibition of purified ATP citrate lyase. The phenomenon probably arises from the production by glutamine synthetase of ADP, a known product inhibitor of ATP citrate lyase. Therefore contrary to previous reports elsewhere, L-glutamate has no role in the regulation of brain ATP citrate lyase and thus the supply of cytoplasmic acetyl groups for biosynthesis.     

ATP citrate lyase activity is post‐translationally regulated by sink strength and impacts the wax,cutin and rubber biosynthetic pathways

Cytosolic acetyl‐CoA is involved in the synthesis of a variety of compounds, including waxes, sterols and rubber, and is generated by the ATP citrate lyase (ACL). Plants over‐expressing ACL were generated in an effort to understand the contribution of ACL activity to the carbon flux of acetyl‐CoA to metabolic pathways occurring in the cytosol. Transgenic Arabidopsis plants synthesizing the polyester polyhydroxybutyrate (PHB) from cytosolic acetyl‐CoA have reduced growth and wax content, consistent with a reduction in the availability of cytosolic acetyl‐CoA to endogenous pathways. Increasing the ACL activity via the over‐expression of the ACLA and ACLB subunits reversed the phenotypes associated with PHB synthesis while maintaining polymer synthesis. PHB production by itself was associated with an increase in ACL activity that occurred in the absence of changes in steady‐state mRNA or protein level, indicating a post‐translational regulation of ACL activity in response to sink strength. Over‐expression of ACL in Arabidopsis was associated with a 30% increase in wax on stems, while over‐expression of a chimeric homomeric ACL in the laticifer of roots of dandelion led to a four‐ and two‐fold increase in rubber and triterpene content, respectively. Synthesis of PHB and over‐expression of ACL also changed the amount of the cutin monomer octadecadien‐1,18‐dioic acid, revealing an unsuspected link between cytosolic acetyl‐CoA and cutin biosynthesis. Together, these results reveal the complexity of ACL regulation and its central role in influencing the carbon flux to metabolic pathways using cytosolic acetyl‐CoA, including wax and polyisoprenoids.     

Plastid targeting and transient expression of rat liver ATP: citrate lyase in pea protoplasts

Protoplasts isolated from pea leaves (Pisum sativum L. cv. Hurst Greenshaft) were electroporated in the presence of plasmid pDR#1, which contains the rat liver ATP:citrate lyase gene fused to a duplex 35S cauliflower mosaic virus promoter with a transit peptide sequence of the Rubisco small subunit. The level of enzyme expression and viability of protoplasts were both influenced by polyethylene glycol treatment before electroporation. Under the optimised electroporation conditions, an average increase of ATP:citrate lyase activity of 14% was observed in the transfected cells after 24 h, with a similar magnitude of change in the abundance of the corresponding mRNA. Immunoblot analysis confirmed the correct expression and targeting of ATP:citrate lyase protein in the chloroplasts of pea protoplasts. These results provide a basis for the establishment of a procedure for targeting heterologous protein into pea plastids in the presence of a transit peptide. Received: 14 June 1996 / Revision received: 24 November 1996 / Accepted: 4 January 1997     

Widespread occurrence of ATP:citrate lyase and carnitine acetyltransferase in filamentous fungi

Ten filamentous fungi, belonging to five different genera of both higher and lower fungi, including oleaginous fungi and fungi known to produce secondary metabolites, all possessed both ATP:citrate lyase (17–84nmol min–1 (mg protein)–1) and carnitine acetyltransferase activity (9–62nmol min–1 (mg protein)–1). The possession of these two enzymes appears to be a common feature in filamentous fungi.     

Cytosolic 3-hydroxy-3-methyl glutaryl coenzyme a synthase in rat brain: Properties and developmental change

The properties and developmental change in the activity of cytosolic 3-hydroxy-3-methyl glutaryl coenzyme A (HMG-CoA) synthase in brain was examined and whether or not HMG-CoA lyase is present in cytosol and mitochondria from brain was determined. Although mitochondrial fractions contained significant HMG-CoA lyase activity, the enzyme activity was not detected in brain cytosol. The synthase activity was present in both mitochondrial and cytosolic fraction. The HMG-CoA synthesis by brain cytosol was optimal at pH 8.0 and did not require Mg²⁺ or exogenous acetoacetyl CoA. This indicates that brain cytosol can synthesize sufficient quantity of acetoacetyl CoA from acetyl CoA to be utilized for HMG-CoA synthesis. Our results also showed that the specific activity (nmol acetyl CoA incorporated/mg protein) of HMG-CoA synthase in brain cytosol was high (between 2–11 days of postnatal age) when the cholesterol content of brain is increasing rapidly, and the activity declined slowly thereafter. This suggests that in brain, cytosolic enzyme HMG-CoA synthase plays a role in the regulation of cholesterol synthesis.     

Discovery of furan carboxylate derivatives as novel inhibitors of ATP-citrate lyase via virtual high-throughput screening

The enzyme ATP citrate lyase (ACL) catalyzes the formation of cytosolic acetyl CoA, the starting material for de novo lipid and cholesterol biosynthesis. The dysfunction and upregulation of ACL in numerous cancers makes it an attractive target for developing anticancer therapies. ACL inhibition by shRNA knockdown limits cancer cell proliferation and reduces cancer stemness. We designed and implemented a dual docking protocol to select virtual ACL inhibitors that were scored among the top 10 percentiles by both the Autodock Vina and the Glamdock algorithms. Via this in silico screens of a focused furoic acid library, we discovered four subtypes of furans and benzofurans as novel ACL inhibitors. The hit rate of our in silico protocol was 45.8% with 11 of 24 virtual hits confirmed as active in an in vitro ACL enzymatic assay. The IC₅₀ of the most potent ACL inhibitor A1 is 4.1 μM. Our results demonstrated remarkable hit rate by the dual docking approach and provided novel chemical scaffolds for the development of ACL inhibitors for the treatment of cancer.     

Inhibition of the synthesis of acetylcholine in rat brain slices by (-)-hydroxycitrate and citrate

Abstract: Slices of rat caudate nucleus were incubated in a solution of 123 mM-NaCl, 5 mM-KCl, 1.2 mM-MgCl₂, 1.2 mM-NaH₂PO₄, 25 mM-NaHCO₃, 0.2 mM-choline chloride, 0.058 mM-paraoxon, 1 mM-EGTA, and oxidizable substrates. (−)-Hydroxycitrate, a specific inhibitor of ATP-citrate lyase (EC 4.1.3.8), used at a concentration of 2.5 mM, inhibited the synthesis of acetylcholine (ACh) from [1,5-¹⁴C]citrate by 82–86%, but that from [U-¹⁴C]glucose by only 33%, from [2-¹⁴C]pyruvate by 24% and from [1-¹⁴C-acetyl]carnitine by 8%; the production of ¹⁴CO₂ from these substrates was not substantially changed. The synthesis of ACh from glucose and pyruvate was in hibited also by citrate; 2.5 mM- and 5 mM-citrate diminished it by 43% and 66%, respectively; the production of from [U-¹⁴C]glucose and from [1-¹⁴C]pyruvate was not affected. The mechanism of the inhibitory effect of citrate on the synthesis of ACh is not clear; the possibility is discussed that citrate alters the intracellular milieu in cholinergic neurons by chelating the intracellular Ca²⁺ and decreases the supply of mitochondrial acetyl-CoA to the cytosol. The results with (−)-hydroxycitrate indicate that the cleavage of citrate by ATP-citrate lyase is not responsible for the supply of more than about one-third of the acetyl-CoA which is used for the synthesis of ACh when glucose or pyruvate are the main oxidizable substrates. This proportion may be even smaller, since (−)-hydroxycitrate possibly affects the synthesis of ACh from glucose and pyruvate by a mechanism (unknown) similar to that of citrate, rather than by the inhibition of ATP-citrate lyase.     

ATP Citrate lyase from Ipomoea batatas root tissue infected with Ceratocystis fimbriata

ATP citrate lyase was detected in sweet potato (Ipomoea batatas) root tissue infected with Ceratocystis fimbriata. The activity increased strik     

Carnitine acetyltransferase activity in oleaginous yeasts

Abstract The highest activities of carnitine acetyltransferase (CAT) were found in non-oleaginous yeasts ( Candida utilis and Saccharomyces cerevisiae ); lower activities, ranging from 50% down to 3% of the highest values, were found in various strains of oleaginous yeasts ( Candida curvata, Lipomyces starkeyi, Rhodosporidium toruloides and Trichosporon cutaneum ). Supply of acetyl units into the cytosol of the latter, but not of the former yeasts, was therefore necessarily reliant on the action of ATP: citrate lyase (ACL), which was present in all oleaginous yeasts. There was no correlation, however, between the amount of lipid in the oleaginous yeasts and the specific activities of either CAT or ACL. Activity of CAT was increased up to 30-fold by growing yeasts on a triacylglycerol.     

Carbon assimilation pathways in sulfate-reducing bacteria II. Enzymes of a reductive citric acid cycle in the autotrophic Desulfobacter hydrogenophilus

The strict anaerobe Desulfobacter hydrogenophilus is able to grow autotrophically with CO₂, H₂, and sulfate as sole carbon and energy sources. The generation time at 30°C under autotrophic conditions in a pure mineral medium was 15 h, the growth yield was 8 g cell dry mass per mol sulfate reduced to H₂S. Enzymes of the autotrophic CO₂ assimilation pathway were investigated. Key enzymes of the Calvin cycle and of the acetyl CoA pathway could not be found. All enzymes of a reductive citric acid cycle were present at specific activities sufficient to account for the observed growth rate. Notably, an ATP-citrate lyase (1.3 mol · min^-1 · mg cell protein^-1) was present both in autotrophically and in heterotrophically grown cells, which was rapidly inactivated in the absence of ATP. The data indicate that in D. hydrogenophilus a reductive citric acid cycle is operating in autotrophic CO₂ fixation. Since other autotrophic sulfate reducers possess an acetyl CoA pathway for CO₂ fixation, two different autotrophic pathways occur in the same physiological group.Dedicated to Prof. H. G. Wood on the occasion of his 80th birthday     

The Interaction of Dithiothreitol and Acetyl Coenzyme A in a Radiochemical Assay for Rat Brain ATP:Citrate Oxaloacetate Lyase

Abstract: [¹⁴C]Acetyl-CoA was found to react spontaneously with dithiothreitol to give a relatively apolar product which was readily extractable into a butanol-toluene scintillant. This technique was used in a rapid, reproducible assay for rat brain ATP:citrate lyase using [1,5-¹⁴C]citrate as substrate. The tissue extract, a 14,000 g supernatant, exhibited a lyase activity of approximately 7 nmol acetyl-CoA produced/min per mg supernatant protein, and was inhibited ≥79% by α-ketoglutaric acid (10 m m ), Cu²⁺ (1 m m )and Zn²⁺(1 m m ). [¹⁴C]Oxaloacetate, [¹⁴C]malate and endogenous citrate synthase were found not to interfere significantly with lyase estimations, but NADH was required in the reaction mixture to inhibit acetyl-CoA hydrolase activity.     

Acetyl-CoA-carboxylase activity in normally developing wheat leaves

In order to investigate the role of acetyl CoA carboxylase (ACC) in the regulation of fatty-acid biosynthesis in chloroplasts, the activities and relative amounts of the enzyme have been measured in the tissue of wheat (Triticum aestivum L.) leaves undergoing development and cellular differentiation. The total activity in the first leaves of 5- to 7-d-old plants was similar but decreased to less than half in 9-d-old plants. The activity of ACC in the cells of the first leaf of 7-d-old plants doubled when cell age increased from 24 to 48 h, remained relatively constant for a further 24 h and then declined. The amount of ACC in cells increased 15-fold during the first 36 h of cell enlargement. Cells more than 36 h old contained about two-thirds the maximum amount of ACC found in younger cells. The most rapid phase of fatty-acyl accumulation in lipids was in cells aged between 60 and 84 h. Tenfold changes in the activity of ACC were observed when the assay conditions with respect to ATP, ADP, Mg²⁺ and pH were changed to correspond to the physiological conditions in chloroplasts during light/dark transitions. This observation and the magnitude of the changes in the optimum activity and amount of ACC in leaf cells undergoing development are consistent with a role for ACC in the regulation of the flow of carbon from acetyl CoA to fatty acids in chloroplasts.Abbreviation ACC acetyl CoA carboxylase     

The Contribution of Citrate to the Synthesis of Acetyl Units in Synaptosomes of Developing Rat Brain

Abstract: The activities of pyruvate dehydrogenase, citrate synthase, and choline acetyltransferase in rat brain synaptosomes increased during on-togenesis by 3 and 14 times, respectively. Activity of ATP-citrate lyase decreased by 26% during the same period. Pyruvate consumption by synapto-somes from 1-day-old animals was 40% lower than that found in older rats; however, citrate efflux from intrasynaptosomal mitochondria in immature synaptosomes was over twice as high as that in mature ones. The rates of production of synaptoplasmic acetyl-CoA, ATP-citrate lyase were 1.03, 1.40, and 0.49 nmol/min/mg protein in 1-, 10-day-old, and adult rats, respectively. 3-Bromopyruvate (0.5 m M ) inhibited pyruvate consumption by 70% and caused a complete block of citrate utilization by citrate lyase in every age group. Parameters of citrate metabolism in cerebellar synaptosomes were the same as those in cerebral ones. These data indicate that production of acetyl-CoA. from citrate in synaptoplasm may be regulated either by adaptative, age-dependent changes in permeability and carrier capacity of the mitochondrial membrane or by the inhibition of synthesis of intramitochondrial acetyl-CoA. ATP-citrate lyase activity is not a rate-limiting factor in this process. Metabolic fluxes of pyruvate to cytoplasmic citrate and acetyl-CoA. are presumably the same in both cholinergic and noncholinergic nerve endings. The significance of citrate release from intrasynaptosomal mitochondria as a regulatory step in acetylcholine synthesis is discussed.     

Energy transformation coupled to formate oxidation during anaerobic fermentation

A correlation between the rate of ATP synthesis by F₀F₁ ATP synthase and formate oxidation by formate hydrogen lyase (FHL) has been found in inside-out membrane vesicles of the Escherichia coli mutant JW 136 (Δhya/Δhyb) with double deletions of hydrogenases 1 and 2, grown anaerobically on glucose in the absence of external electron acceptors at pH 6.5. ATP synthesis was suppressed by the H⁺-ATPase inhibitors N,N′-dicyclohexylcarbodiimide, sodium azide, and the uncoupler carbonyl cyanide m-chlorophenylhydrazone. Copper ions inhibited formate-dependent hydrogenase and ATP-synthase activities but did not affect the ATPase activity of the vesicles. The maximal rate of ATP synthesis (0.83 μmol/min per mg protein) was determined at simultaneous application of sodium formate, ADP, and inorganic phosphate, and was stimulated by K⁺ ions. The results confirm the assumption of a dual role of hydrogenase 3, the formate hydrogen lyase subunit that can couple the reduction of protons to H₂ and their translocation through membrane with chemiosmotic synthesis of ATP.     

A novel direct homogeneous assay for ATP citrate lyase

ATP citrate lyase (ACL) is a cytosolic enzyme that catalyzes the synthesis of acetyl-CoA and oxaloacetate using citrate, CoA, and ATP as substrates and Mg²⁺ as a necessary cofactor. The ACL-dependent synthesis of acetyl-CoA is thought to be an essential step for the de novo synthesis of fatty acids and cholesterol. For this reason, inhibition of ACL has been pursued as a strategy to treat dyslipidemia and obesity. Traditionally, ACL enzyme activity is measured indirectly by coupling to enzymes such as malate dehydrogenase or chloramphenicol acetyl transferase. In this report, however, we describe a novel procedure to directly measure ACL enzyme activity. We first identified a convenient method to specifically detect [¹⁴C]acetyl-CoA without detecting [¹⁴C]citrate by MicroScint-O. Using this detection system, we devised a simple, direct, and homogeneous ACL assay in 384-well plate format that is suitable for high-throughput screening. The current assay consists of 1) incubation of ACL enzyme with [¹⁴C]citrate and other substrates/cofactors CoA, ATP, and Mg²⁺, 2) EDTA quench, 3) addition of MicroScint-O, the agent that specifically detects product [¹⁴C]acetyl-CoA, and 4) detection of signal by TopCount. This unique ACL assay may provide more efficient identification of new ACL inhibitors and allow detailed mechanistic characterization of ACL/inhibitor interactions.