Substrate specificity of citrate lyase deacetylase of Rhodopseudomonas gelatinosa and Rhodopseudomonas palustris.

Citrate lyase (EC 4.1.3.6) isolated from Rhodopseudomonas palustris was investigated with regard to its kinetic properties and its subunit composition. This enzyme was inactivated by citrate lyase deacetylase (EC 3.1.2.-) of Rhodopseudomonas gelatinosa. A corresponding cross-reaction was measured with partially purified deacetylase of R. palustris and citrate lyase of R. gelatinosa. The three different subunit types (alpha, beta, and gamma) of citrate lyase from R. gelatinosa wee purified to homogeneity, and antibodies were prepared against each of the three subunits and against the native enzyme complex. In corresondence with the enzymatic interactions, immunological cross-reactions were found between anti-enzyme and anti-large subunit antibodies and citrate lyase from R. palustris. On the other hand, no immunological cross-reactions were detectable among each of the antibodies and citrate lyases from Enterobacter aerogenes, Streptococcus diacetilactis, and Clostridium sphenoides. Antibodies against the large subunit of citrate lyase inhibited the deacetylase, but antibodies against the middle and small subunits did not, indicating that the large subunits of citrate lyase are involved in binding the deacetylase.     

Citrate lyase from Streptococcus diacetilactis

Citrate lyase (EC 4.1.3.6) was purified 38-fold from cell-free extracts of Streptococcus diacetilactis. The enzyme was homogeneous in analytical ultracentrifugation and polyacrylamide gel electrophoresis The final enzyme preparation contained acetate: HS-citrate lyase ligase—an acetylating enzyme which converts inactive HS-citrate lyase into enzymatically active acetyl-S-citrate lyase. This enzyme activity was purified 25-fold over the crude extract and seemed to be associated with citrate lyase. Partially purified citrate lyase from Leuconostoc citrovorum contained also its acetylating enzyme. Purified citrate lyases from Klebsiella aerogenes and Rhodopseudomonas gelatinosa were devoid of acetylating enzyme activity. The HS-form of citrate lyase from S. diacetilactis was completely acetylated and hence activated by incubation with ATP and acetate for 25 min at 25° C. The enzyme did not acetylate the HS-lyases from R. gelatinosa and K. aerogenes. In contrast to the citrate lyases from R. gelatinosa and K. aerogenes the enzymes from S. diacetilactis and L. citrovorum showed onlya very weak reaction inactivation. It is assumed that this is due to the association of the acetylating enzymes with these lyases.     

The enzyme complex citramalate lyase from Clostridium tetanomorphum.

1. The enzyme citramalate from Clostridium tetanomorphum is not stable in crude extracts. However, the inactive enzyme can be reactivated by incubation with dithioerythritol followed by acetylation with acetic anhydride. Reactivation was also obtained with acetate, ATP, MgCl2 and acetate : SH-enzyme ligases (AMP) from C. tetanomorphum or Klebsiella aerogenes. 2. Incubation of the inactive enzyme with iodoacetate resulted in rapid loss of enzymic activity as determined by reactivation with acetic anhydride whereas the active enzyme was stable in the presence of iodoacetate. Using ido[2-(14)C]acetate the sites of carboxymethylation and acetylation where identified as cysteamine residues of the enzyme. The results demonstrate that the active enzyme contains acetyl thiolester residues which play the central role in the catalytic mechanism. 3. Citramalate lyase was purified by a procedure almost identical to that already described for citrate lyase from K. aerogenes. The molecular weight of citramalate lyase is equal to that of citrate lyase (Mr = 5.2--5.8 X 10(5)) as estimated by gel chromatography and sucrose gradient centrifugation. Polyacrylamide gel elctrophoresis of citramalate lyase in sodium dodecylsulfate yielded three polypeptide chains (Mr: alpha 5.3--5.6 X 10(4); beta 3.3--3.6 X 10(4); gamma 1.0--1.2 X 10(4)) in probably equal molar amounts. These data lead to a hexameric structure (alpha,beta,gamma)6 of the complete enzyme. 4. Pantothenate (5 mol/mol of enzyme) and the essential cysteamine residues were exclusively present in the gamma-chain, the acyl carrier protein of citramalate lyase. The acyl exchange and cleavage functions, probably catalysed by the alpha and beta-subunits, were measured with acyl-CoA derivatives which were able to substitute for the natural acyl carrier. 5. The results demonstrate that citramalate lyase is an enzyme complex with structure and functions closely resembling those of citrate lyase. Although the similarity between citramalate lyase and citrate lyases from various organisms suggests a close evolutionary relationship, these occur in very different, unrelated bacteria. A parallel situation found in the distribution of the nitrogenase system among procaryotes is discussed.     

Citrate Metabolism in Aerobacter cloacae

Growth of Aerobacter cloacae on citrate either anaerobically or aerobically did not require and was not stimulated by the presence of Na(+) in the medium. Citrate was metabolized anaerobically via the fermentation pathway as evidenced by the (i) presence of oxalacetate decarboxylase, (ii) induction of citrate lyase, and (iii) repression of alpha-ketoglutarate dehydrogenase under anaerobic conditions. Thus, although all the other enzymes of the citric acid cycle were present in anaerobic cells, this pathway was not available for the metabolism of citrate. Citrate was metabolized aerobically via the citric acid cycle, since (i) citrate lyase but not oxalacetate decarboxylase was repressed and (ii) alpha-ketoglutarate dehydrogenase was induced under these conditions. The presence of Na(+) in the medium did not lead to a repression of alpha-ketoglutarate dehydrogenase as in the case of Aerobacter aerogenes. The oxalacetate decarboxylase was a soluble, constitutive enzyme, not activated by Na(+) nor inhibited by avidin. It was slightly inhibited by ethylenediaminetetraacetate but was not stimulated by Mg(2+) or Mn(2+). Thus, this enzyme differed markedly in its properties from the same enzyme found in citrate-grown A. aerogenes.     

Effect of growth conditions on the activation and inactivation of citrate lyase of Rhodopseudomonas gelatinosa.

Cells of Rhodopseudomonas gelatinosa growing with citrate anaerobically in the light contained citrate lyase only in the acetylated, enzymatically active form of this enzyme. After exhaustion of citrate in the culture medium citrate lyase was deacetylated to yield the inactive sulfhydryl (HS) enzyme. Acetylation of HS-citrate lyase required light, anaerobic conditions and the availability of citrate as substrate. The acetylation reaction already in progress stopped immediately when the culture was placed in the dark. Deacetylation of citrate lyase occurred anaerobically in the light when citrate was exhausted and under aerobic conditions in the presence or absence of citrate. In cells of R. gelatinosa fermenting citrate in the dark neither the acetylating enzyme nor the deacetylating enzyme was active.     

Purification and properties of citrate lyase from Streptococcus diacetilactis.

Citrate lyase from Streptococcus diacetilactis has been purified to yield a protein that was homogeneous as judged by sedimentation velocity and sedimentation equilibrium experiments. The enzyme's sedimentation coefficient is 16.8 S and its molecular weight is around 585,000. It contains three nonidentical subunits of about 53,000, 34,000, and 10,000 daltons. The enzyme in its active form contains an acetyl group which turns over during the citrate cleavage reaction. Removal of the acetyl group inactivates the enzyme. The deacetyl enzyme can be partially reactivated by acetylation with acetic anhydride. The enzyme undergoes slow "reaction-inactivation." The rate of inactivation is first order and the rate constant of inactivation is much lower than that for a similar inactivation process of the citrate lyase from Klebsiella aerogenes. Like the latter enzyme it contains stoichiometric amounts of phosphopantothenate. The enzyme is inactivated at pH greater than 8.1 and the presence of citrate provides protection against this inactivation. Sedimentation studies of the enzyme at pH 8.7 indicate that the enzyme is dissociated, which may account for the inactivation. The enzyme is immunologically different from citrate lyases of K. aerogenes and Escherichia coli.     

Citrate Metabolism by Pediococcus halophilus

Several strains of non-citrate-metabolizing Pediococcus halophilus have previously been isolated from soy sauce mash or moromi. The factors controlling the metabolism of citrate in soy pediococci were studied. All the soy pediococcal strains tested which failed to decompose citrate did not possess citrate lyase [citrate (pro-3S)-lyase; EC 4.1.3.6] activity. In P. halophilus, citrate lyase was an inducible enzyme, and the optimum pH for activity was 7.0. The metabolism of citrate in P. halophilus was different from that observed in lactic streptococci. The main products from citrate were acetate and formate, and this bacterium produced no acetoin or diacetyl. Formate production from citrate was greatly reduced in the presence of glucose. P. halophilus 7117 (Cit⁺) was proved to contain citrate lyase, pyruvate formate-lyase (EC 2.3.1.54) phosphotransacetylase (phosphate acetyltransferase; EC 2.3.1.8), and acetate kinase (EC 2.7.2.1), i.e., all the enzymes necessary to convert citrate to acetate and formate.     

Fermentation of pyruvate by 7 species of phototrophic purple bacteria]

The dark, anaerobic fermentation of pyruvate under growth conditions was examined with the following species of phototrophic purple bacteria: Rhodospirillum rubrum strains Ha and S1, Rhodopseudomonas gelatinosa strain 2150, Rhodopseudomonas acidophila strain 7050, Rhodopseudomonas palustris strain ATCC 17001, Rhodopseudomonas capsulata strains Kb1 and 6950, Rhodopseudomonas sphaeroides strain ATCC 17023, and Chromatium vinosum strain D. Fermentation balances were established for all experiments. Under fermentative conditions cell protein and dry weight increased only slightly, if at all. The species differed considerably in their fermentative activity; R. rubrum and R. gelatinosa exhibited the highest rates (2-8 mumoles pyruvate/mg protein-h). R. acidophila and R. capsulata showed an intermediate fermentation rate (0.4--2.0 mumoles pyruvate/mg protein-h), while the other strains tested fermented at quite low rates (0.2-0.4 mumoles pyruvate/mg protein-h). The extremes of fermentation times were from 30-380 hours. Based on the products of fermentation which were formed in addition to acetate, formate, and CO2, the species can be grouped as follows: a) R. rubrum, R. gelatinosa, and R. sphaeroides additionally form propionate. b) R. gelatinosa, R. palustris, R. capsulata, R. sphaeroides, and C. vinosum additionally form lactate. R. palustris also produces butyrate. c) R. acidophila and R. capsulata additionally form much 2,3-butanediol, acetoin, and diacetyl. Small amounts of acetoin were formed by the rest of the strains. A comparison of the fermentation of pyruvate by normal and starved cells (4 days in the light without a carbon source) of R. rubrum and R. gelatinosa shows that the latter ferment more slowly and produce less acetate and formate, but more propionate or lactate. The fermentation of pyruvate by R. rubrum was also studied in cultures in which the pH fell (7.2--6.6). Compared with the fermentation at neutral pH (7.3, 7.4), the following differences were found: a slower fermentation rate, an increased production of dry weight, an increased formation of propionate, but a reduced formation of acetate and a very low production of formate.     

Nonenzymatic reactivation of des-acetyl citrate lyase by acetyl adenylate. First example of enzyme activation by chemotrophic modification.

The experimental conditions of nonenzymatic reactivation of des-acetyl citrate lyase from K. aerogenes were studied. It was found that at pH 8.5 0.2 MM acetyl AMP causes a fast reactivation of the enzyme. The pH dependence of activity and the Km for citrate are very similar for both native and reactivated enzyme.     

Photoaffinity labeling of Klebsiella aerogenes citrate lyase by p-azidobenzoyl coenzyme A

p-Azidobenzoyl coenzyme A functions as a linear competitive inhibitor for (3S)-citryl-CoA in the citryl-CoA oxaloacetate-lyase reaction catalyzed by the Klebsiella aerogenes deacetylcitrate lyase complex (Ki = 80 microM; (3S)-citryl-CoA Km = 67 microM). Inactivation is irreversible on photolysis of p-azidobenzoyl-CoA in the presence of the deacetylcitrate lyase complex. Mg2+ is not required for the inactivation. Inactivation is blocked by (3S)-citryl-CoA in the presence of ethylenediaminetetraacetic acid. p-Azidobenzoyl-CoA has no effect on the acetyl-CoA:citrate CoA transferase activity of both the deacetylcitrate lyase complex and its isolated transferase subunit. The stoichiometry of the CoA ester binding has been investigated by the use of p-azido[14C]benzoyl-CoA as a photoaffinity reagent. The labeling is exclusively on the lyase beta subunit of the citrate lyase complex.     

Energy-dependent inactivation of citrate lyase in Enterobacter aerogenes.

Enterobacter aerogenes was grown in continous culture with ammonia as the growth-limiting substrate, and changes in citrate lyase and citrate synthase activities were monitored after growth shifts from anaerobic growth on citrate to aerobic growth on citrate, aerobic growth on glucose, anaerobic growth on glucose, and anaerobic growth on glucose plus nitrate. Citrate lyase was inactivated during aerobic growth on glucose and during anaerobic growth with glucose plus nitrate. Inactivation did not occur during anaerobic growth on glucose, and as a result of the simultaneous presence of citrate lyase and citrate synthase, growth difficulties were observed. Citrate lyase inactivation consisted of deacetylation of the enzyme. The corresponding deacetylase could not be demonstrated in cell extracts, and it is concluded that, as in a number of other inactivations, electron transport to oxygen or nitrate was required for inactivation.     

Electron microscopical investigation of citrate lyase single molecules

Electron micrographs of citrate lyase from Rhodopseudomonas gelatinosa and Klebsiella aerogenes reveal two characteristic molecular forms. The "basket" form and the "star" form were subjected to two-dimensional image reconstruction using a technique involving averaging of superposed single molecular images after rotational correlation. A three-dimensional image reconstruction shows that the images of these forms can be interconverted by rotation and that they therefore represent different views of the same structure.     

On the significance of the prosthetic group composition of citrate lyase.

1. Klebsiella aerogenes contains two different acyl carrier proteins, one specific for citrate lyase, the other for fatty acid synthetase. 2. The acyl carrier protein of fatty acid synthetase from K. aerogenes was isolated and compared with the corresponding protein from Escherichia coli and with the acyl carrier protein of citrate lyase from K. aerogenes. 3. As judged from prosthetic group compositions as well as amino acid and fingerprint analyses, the acyl carrier proteins of the two fatty acid synthetases are nearly identical but different from that of citrate lyase from K. aerogenes. 4. Therefore, the different prosthetic groups alone cannot be responsible for the different specificities of the acyl carrier proteins of fatty acid synthetase and citrate lyase in K. aerogenes. 5. The prosthetic group of citrate lyase, phosphoribosyl dephospho-CoA, apparently represents no incidental, phosphopantetheine-replacing aberration. The requirement of citrate lyase for the CoA-like prosthetic group may arise from the substrate requirement of both subunit enzymes of the enzyme complex.     

Purification and characterization of ATP:citrate lyase from Hydrogenobacter thermophilus TK-6.

ATP:citrate lyase [ATP citrate (pro-3S)-lyase; EC 4.1.3.8] was purified and characterized from the cells of Hydrogenobacter thermophilus, an aerobic, thermophilic, hydrogen-oxidizing bacterium which fixes carbon dioxide by a reductive carboxylic acid cycle. The enzyme was quite stable, even in the absence of sulfhydryl reagents. Optimum pH for reaction was 6.7 to 6.9, and optimum temperature was around 80 degrees C. The molecular weight of native enzyme was estimated to be 260,000 by gel filtration analysis, and that of a subunit was estimated to be 43,000 by sodium dodecyl sulfate-polyacrylamide gel analysis. Km values for reaction components were as follows: citrate, 6.25 mM; ATP, 650 microM; coenzyme A, 40.8 microM; and Mg2+, 8 mM. The enzyme showed citrate synthase activity in the presence of Mg2+, but the reaction rate was very low (less than 1/200 of the lyase activity).     

End Products of Glucose Fermentation by Brochothrix thermosphacta

Anaerobically, Brochothrix thermosphacta fermented glucose primarily to l-lactate, acetate, formate, and ethanol. The ratio of these end products varied with growth conditions. Both the presence of acetate and formate and a pH below about 6 increased l-lactate production from glucose. Small amounts of butane-2,3-diol were also produced when the pH of the culture was low (相似文献   

ATP Citrate Lyase from Germinating Castor Bean Endosperm: Localization and some Properties

ATP citrate lyase (EC 4.1.3.8) has been found in crude extracts from endosperm tissue of germinating castor bean and shows its maximum activity in 4- to 5-day-old seedlings. A strict requirement for coenzyme A and adenosine 5′-triphosphate was demonstrated. The pH optimum for the reaction is around 7.5. The unstable enzyme can be stabilized by freezing and addition of citrate and glycerol. (−)-Hydroxycitrate is a potent inhibitor. The molecular weight is about 400,000. The adenosine 5′-triphosphate citrate lyase is localized in the plastids, where it possibly plays a role in providing acetyl coenzyme A for lipid biosynthesis.     

Purification of L-glutamate-dependent citrate lyase from Clostridium sphenoides and electron microscopic analysis of citrate lyase isolated from Rhodopseudomonas gelatinosa, Streptococcus diacetilactis and C. sphenoides

Citrate lyase from Clostridium sphenoides was purified 72-fold with a yield of 11%. In contrast to citrate lyase from other sources the activity of this enzyme was strictly dependent on the presence of L-glutamate. The purified enzyme was only stable in the presence of 150 mM L-glutamate or 7 mM L-glutamate plus glycerol, sucrose or bovine serum albumin. Changes of the L-glutamate pool and of enzyme activity in growing cells of C. sphenoides indicated that citrate lyase activity in this organism was regulated by the intracellular L-glutamate concentration. Citrate lyase isolated from C. sphenoides, Rhodopseudomonas gelatinosa and Streptococcus diacetilactis was investigated by electron microscopy using the negative staining technique. Three different projections of enzyme molecules were observed: 'star' form, 'ring' form and 'triangle' form. In samples from R. gelatinosa and S. diacetilactis, star and ring forms occurred in a ratio of about 1:9. Using the enzyme from S. diacetilactis it was demonstrated that this ratio could be altered in favour of the star form by the addition of citrate or tricarballylate. The triangle form was observed in less than 1% of all evaluated molecules and may represent a transition form. In lyase samples from C. sphenoides there existed a correlation between enzyme activity and the proportion of stars and rings at varying concentrations of L-glutamate.     

Purification and properties of citrate lyase from Escherichia coli

Citrate lyase (EC 4.1.3.6) has been purified from Escherichia coli and the homogeneity of the preparation established from the three-component subunits obtained on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The purified enzyme has a specific activity of 120 mumol min-1 mg-1 and requires optimally 10 mM Mg2+ and a pH of 8.0 for the cleavage reaction. The native enzyme is polydispersed in the ultracentrifuge and in polyacrylamide gel electrophoresis. The enzyme complex is composed of three different polypeptide chains of 85 000, 54 000, 32 000 daltons. An estimate of subunit stoichiometry indicates that 1 mol of the largest polypeptide chain is associated with 6 mol each of the smaller ones. The polypeptide subunits have been isolated in pure state and their biological functions characterize. The 54 000-dalton subunit functions as the acyltransferase alpha subunit catalyzing the formation of citryl coenzyme A from citrate in the presence of acetyl coenzyme A and ethylenediaminetetraacetic acid. The 32 000-dalton subunit functions as the acyllyase beta subunit catalyzing the cleavage of (3S)-citryl coenzyme A to oxal-acetate and acetyl coenzyme A. The 85 000-dalton subunit, which carries exclusively the prosthetic group components, functions as the acyl-carrier protein gamma subunit in the cleavage of citrate in the presence of mg2+ and the alpha and beta subunits. The presence of a large ACP subunit and the unusual stoichiometry of the different subunits distinguish the complex from other citrate lyases. A ligase which acetylates the deacetyl[citrate lyase] in the presence of acetate and ATP has ben shown to be present in the organism.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acetate metabolism in Escherichia coli.

Levels of several intermediary metabolites were measured in cells grown in acetate medium in order to test the hypothesis that the glyoxylate cycle is repressed by phosphoenolpyruvate (PEP). Wild-type cells had less PEP than either isocitrate dehydrogenase - deficient cells (which had greater isocitrate lyase activity than the wild type) or isocitrate dehydrogenase - deficient, citrate synthase-deficient cells (which are poorly inducible). Thus induction of the glyoxylate cycle is more complicated than a simple function of PEP concentration. No correlation between enzyme activity and the level of oxaloacetate, pyruvate, or citrate was found either. Citrate was synthesized in citrate synthase-deficient mutants, possibly via citrate lyase.     

Citrate metabolism in anaerobic bacteria

Abstract The regulation of anaerobic citrate metabolism is very diverse among different groups of bacteria. In organisms like Streptococcus lactis and Clostridium sporosphaeroides which lack citrate synthase, the activity of its antagonistic enzyme, citrate lyase, need not be regulated. Many anaerobes like Rhodocyclus gelatinosus and Clostridium sphenoides are able to synthesize their own l -glutamate and contain citrate synthase. In these bacteria the activity of citrate metabolizing enzymes which are involved in a cascade system are under strict control. In Rc. gelatinosus activation/inactivation of citrate lyase is controlled by acetylation/deacetylation which is catalyzed by its corresponding regulatory enzymes, citrate lyase ligase and citrate lyase deacetylase. In C. sphenoides inactivation of citrate lyase is accomplished by deacetylation as well as by changing in the enzyme conformation. Activation of citrate lyase is catalyzed by citrate lyase ligase whose activity in addition is modulated by phosphorylation/dephosphorylation. Further, electron transport process also seems to play a role in the inactivation of citrate metabolizing enzymes in enteric bacteria.