Models of proteolysis of oligomeric enzymes and their applications to the trypsinolysis of citrate synthases.

A simple statistical approach was used to generate predictive models of the proteolysis of multisubunit enzymes in order to correlate the loss of enzyme activity with the loss of native subunit. The models were applied to the trypsinolysis of the citrate synthases of pig heart, Bacillus megaterium and Escherichia coli. With the dimeric citrate synthases (pig heart and B. megaterium) trypsinolysis of one of the subunits appears to destroy the activity of the whole enzymic molecule. The hexameric E. coli citrate synthase behaves like a trimer of dimeric units, each of the dimers behaving similarly to the B. megaterium and pig heart enzymes. Palmitoyl-CoA is required for the trypsinolysis of pig heart citrate synthase, and at relatively high concentrations of this compound trypsinolysis of one subunit leaves the other subunit fully active. Palmitoyl-CoA is not required for the trypsinolysis of the other citrate synthases, and high concentrations of this metabolite do not affect the correlation of proteolysis with inactivation of these enzymes.     

Citrate synthase: an immunochemical investigation of interspecies diversity

Rabbit antibodies have been raised to pig heart citrate synthase. Using purified IgG, competitive enzyme-linked immunoassays and assays of citrate synthase activity indicate the presence of antibodies to a number of antigenic sites on the enzyme, only some of which are essential for catalytic activity. From a comparison of citrate synthases from prokaryotic and eukaryotic organisms, the degree of interaction between antibody and enzyme was in the order: pig heart greater than pigeon breast greater than Bacillus megaterium greater than Escherichia coli. These findings are discussed in terms of the known interspecies diversity of the enzyme.     

In vitro mutagenesis of Escherichia coli citrate synthase to clarify the locations of ligand binding sites

In vitro mutagenesis techniques have been used to investigate two structure-function questions relating to the allosteric citrate synthase of Escherichia coli. The first question concerns the binding site of alpha-keto-glutarate, which is a structural analogue of the substrate oxaloacetate and yet has been suggested to be an allosteric inhibitor of the enzyme. Using oligonucleotide-directed mutagenesis of the cloned E. coli citrate synthase gene, we prepared missense mutants, designated CS226H----Q and CS229H----Q, in which histidine residues at positions 226 and 229, respectively, were replaced by glutamine. In the homologous pig heart citrate synthase it is known (Wiegand, G., and Remington, S. J. (1986) Annu. Rev. Biophys. Biophys. Chem. 15, 97-117) that the equivalent of His-229 helps to bind oxaloacetate, while the equivalent of His-226 is nearby. Kinetic and ligand binding measurements showed that CS226H----Q had a reduced affinity for oxaloacetate and alpha-ketoglutarate, while CS229H----Q bound oxaloacetate even less effectively, and was not inhibited by alpha-ketoglutarate at all under our conditions. This parallel loss of binding affinities for oxaloacetate and alpha-ketoglutarate, in two mutants altered in residues at the active site of E. coli citrate synthase, strongly suggests that inhibition of this enzyme by alpha-ketoglutarate is not allosteric but occurs by competitive inhibition at the active site. The second question investigated was whether the known inhibition by acetyl-CoA of binding of NADH, an allosteric inhibitor of E. coli citrate synthase, occurs heterotropically, as an indirect result of acetyl-CoA binding at the active site, or directly, by competition at the allosteric NADH binding site. Using existing restriction sites in the cloned E. coli citrate synthase gene, we prepared a deletion mutant which lacked 24 amino acids near what is predicted to the acetyl-CoA-binding portion of the active site. The mutant protein was inactive, and acetyl-CoA did not bind to the active site but still inhibited NADH binding. Thus acetyl-CoA can interact with both the allosteric and the active sites of this enzyme.     

Amino acid sequence of Escherichia coli citrate synthase

Detailed evidence for the amino acid sequence of allosteric citrate synthase from Escherichia coli is presented. The evidence confirms all but 11 of the residues inferred from the sequence of the gene as reported previously [Ner, S. S., Bhayana, V., Bell, A. W., Giles, I. G., Duckworth, H. W., & Bloxham, D. P. (1983) Biochemistry 22, 5243]; no information has been obtained about 10 of these (residues 101-108 and 217-218), and we find aspartic acid rather than asparagine at position 10. Substantial regions of sequence homology are noted between the E. coli enzyme and citrate synthase from pig heart, especially near residues thought to be involved in the active site. Deletions or insertions must be assumed in a number of places in order to maximize homology. Either of two lysines, at positions 355 and 356, could be formally homologous to the trimethyllysine of pig heart enzyme, but neither of these is methylated. It appears that E. coli and pig heart citrate synthases are formed of basically similar subunits but that considerable differences exist, which must explain why the E. coli enzyme is hexameric and allosterically inhibited by NADH, while the pig heart enzyme is dimeric and insensitive to that nucleotide.     

Expression and base sequence of the citrate synthase gene of Acinetobacter anitratum

The sequence of 1895 base pairs of Acinetobacter anitratum genomic DNA, containing the structural gene for the allosteric citrate synthase of that Gram-negative bacterium, is presented. The sequence contains an open reading frame of 424 codons, the 5' end of which is the same as the N-terminal sequence of A. anitratum citrate synthase, less the initiator methionine. The inferred amino acid sequence of the enzyme is about 70% identical with that of citrate synthase from Escherichia coli, which like the A. anitratum enzyme is sensitive to allosteric inhibition by NADH. There is also a more distant homology with the nonallosteric citrate synthases of pig heart and yeast. The gene contains sequences that strongly resemble those found in E. coli promoters, an E. coli type of ribosomal binding site, and a hyphenated dyad sequence at the 3' end of the gene which resembles the rho-independent terminators found in some E. coli genes. The plasmid clone containing the A. anitratum citrate synthase gene pLJD1, originally isolated because it hybridized with the cloned E. coli citrate synthase gene under conditions of reduced stringency, produces large amounts of A. anitratum citrate synthase in an E. coli host which lacks citrate synthase. This work completes proof of the hypothesis that the three major kinds of citrate synthases are formed of similar subunits, although their functional properties are different.     

Studies on the citryl-CoA-dependent inhibition of citrate-synthase with source variants from baker's yeast, Escherichia coli and Sulfolobus solfataricus

1) Citrate synthase from pig heart has previously been shown to display complex kinetic characteristics in the reactions with citryl-CoA, resulting in inhibition. The synthase from another eukaryotic source, baker's yeast, yields the same complex kinetics. 2) Synthases from a Gram-negative prokaryote, E. coli, and from an archaebacterium, S. solfataricus, catalyse the reactions of citryl-CoA in kinetics of the Michaelis-Menten type. A comparison of the rates of citryl-CoA hydrolysis (V') and physiological reaction (V), determined with these enzymes, corresponds to ratios of V'/V approximately 1 and approximately 2, respectively. Thus, and for the first time, there is no reason left to doubt the intermediate formation of citryl-CoA in the physiological reaction. 3) The complex kinetics indicated under 1) are related to efficient formation of citrate from citryl-CoA-derived acetyl-CoA and oxaloacetate in the presence of NADH and malate dehydrogenase. These conditions are not met by the enzymes from E. coli, S. solfataricus and by proteolytically nicked synthase species from pig heart. All these enzyme variants have low affinities to either one or both of the physiological substrates. Consistent with earlier ideas, the results indicate that the inhibition mechanism is related to high affinities of the enzyme for both acetyl-CoA and oxaloacetate.     

Biosynthesis in Escherichia coli of sn-glycerol 3-phosphate, a precursor of phospholipid. Palmitoyl-CoA inhibition of the biosynthetic sn-glycerol-3-phosphate dehydrogenase.

Homogeneous biosynthetic sn-glycerol-3-phosphate dehydrogenase (EC 1.1.1.8) of Escherichia coli was potently inhibited by palmitoyl-CoA and other long chain acyl-CoA thioesters. The concentration dependence of this inhibition was not cooperative. Enzyme activity was inhibited 50% at 1 microM palmitoyl-CoA; thus, this inhibition occurred at concentrations below the critical micellar concentration of palmitoyl-CoA. Palmitoyl-CoA was a reversible, noncompetitive inhibitor with respect to both NADPH and dihydroxyacetone phosphate. Palmitoyl-CoA did not affect the quaternary structure of the enzyme. This inhibition could be prevented or reversed by the addition of phospholipid vesicles prepared from E. coli phospholipids. Palmitoyl-CoA did not alter the kinetics of inhibition by sn-glycerol 3-phosphate, which is a proven physiological regulator of this enzyme. Decanoyl-CoA, dodecanoyl-CoA, myristoyl-CoA, palmitoyl-(1,N6-etheno)CoA, stearoyl-CoA, and oleoyl-CoA inhibited sn-glycerol-3-phosphate dehydrogenase at concentrations below their critical micellar concentrations. Palmitate inhibited sn-glycerol-3-phosphate dehydrogenase activity 50% at 200 microM. Palmitoyl-carnitine, deoxycholate, taurocholate, and dodecyl sulfate were more potent inhibitors than Triton X-100, Tween-20, or Tween-80. Palmitoyl-acyl carrier protein at concentrations up to 50 microM had no effect on sn-glycerol-3-phosphate dehydrogenase activity. The possible physiological role of long chain fatty acyl-CoA thioesters in the regulation of sn-glycerol 3-phosphate and phospholipid biosynthesis in E. coli is discussed.     

Characterization of mutant TMK368K pig citrate synthase expressed in and isolated from Escherichia coli

A cDNA that encodes pig citrate synthase (PCS) was inserted into a plasmid T7 vector and was expressed in an E. coli gltA mutant. Up to 10 mg of purified PCS was obtained from 2 liters of E. coli. The mammalian protein produced in E. coli comigrated with the enzyme purified from pig heart on a SDS-polyacrylamide gel (SDS-PAGE) with an Mr of 50,000, and reacted with a polyclonal antibody directed against pig heart citrate synthase. The Vmax and Km of the expressed PCS were indistinguishable from those of the pig heart enzyme. The PCS produced in E. coli did not contain the trimethylation modification of Lys 368, characteristic of the pig heart enzyme. These data suggest that the PCS protein produced in E. coli is catalytically similar to the enzyme purified from pig heart and methylation of Lys 368 is not essential for catalysis.     

A comparison of the citrate synthases of Escherichia coli and Acinetobacter anitratum

Citrate synthase has been purified to homogeneity from a strain of the Gram-negative aerobic bacterium Acinetobacter anitratum in a form which retains its sensitivity to the allosteric inhibitor NADH. In subunit size, amino acid composition, and antigenic reactivity the enzyme shows a marked structural resemblance to the citrate synthase of the Gram-negative facultative anaerobe Escherichia coli. Whereas the E. coli enzyme is subject to a strong, hyperbolic inhibition by NADH (Hill's number n = 1.0, Ki = 2 microM), the A. anitratum enzyme shows a weak, sigmoid response (n = 1.6, I0.5 = 140 microM) to this nucleotide. With E. coli, NADH inhibition is competitive with acetyl-CoA, and noncompetitive with oxaloacetate; with A. anitratum, NADH is noncompetitive with both substrates. Acinetobacter anitratum citrate synthase shows hyperbolic saturation with acetyl-CoA (n = 1.8). The finding of Weitzman and Jones (Nature (London) 219, 270 (1968) that NADH inhibition of the enzyme from Acinetobacter spp. is reversible by AMP, while that from E. coli is not, is explained by the much greater affinity of the E. coli enzyme for NADH. Unlike E. coli citrate synthase, the A. anitratum enzyme does not react with the sulfhydryl reagent 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) in the absence of denaturation. With a second sulfhydryl reagent, 4,4'-dithiodipyridine (4,4'-PDS), the A. anitratum enzyme reacts with 1 equiv. of subunit; this modification induces a partial activity loss (attributable to a arise in the Km for acetyl-CoA) and an increase in the sensitivity to NADH. With the E. coli enzyme, 4,4'-PDS causes complete inactivation. Acinetobacter anitratum citrate synthase is much more resistant to urea denaturation than the E. coli enzyme is; the resistance of both enzymes to urea is greatly improved in the presence of 1 M KCl. It is suggested that the amino acid sequences of the subunits of the citrate synthases of these two bacteria are about 90% homologous, and that the 10% differences are in key residues, perhaps largely in the subunit contact regions, which account for the differences in allosteric properties.     

Chimeric allosteric citrate synthases: construction and properties of citrate synthases containing domains from two different enzymes.

The citrate synthases of the gram-negative bacteria, Escherichia coli and Acinetobacter anitratum, are allosterically inhibited by NADH. The kinetic properties, however, suggest that the equilibrium between active (R) and inactive (T) conformational states is shifted toward the T state in the E. coli enzyme. We have now manipulated the cloned genes for the two bacterial enzymes to produce two chimeric proteins, in which one folding domain of each subunit is derived from each enzyme. One chimera (the large domain from A. anitratum and the small domain from the E. coli enzyme) is designated CS ACI::eco; the other is called CS ECO::aci. Both chimeras are roughly as active as the wild type parents, but their Km values for both substrates are lower than those for the E. coli enzyme, and NADH inhibition is markedly sigmoid, while that for E. coli citrate synthases is hyperbolic. Curve-fitting to the allosteric equation suggests that these differences are the result of the destabilization of the T state in the chimeras. The ACI::eco chimera exists almost entirely as a hexamer, like the A. anitratum enzyme, while the ECO::aci chimera, like the E. coli synthase, forms three major bands on nondenaturing polyacrylamide gels, two of them hexamers of different net charge, and one a dimer. These findings indicate that subunit interactions leading to hexamer formation in allosteric citrate synthases of gram-negative bacteria involve mainly the large domains. The chimeras are also used to show that the NADH binding site of E. coli citrate synthase is located entirely in the large domain. Sensitivity of the chimeras to denaturation by urea, to which the A. anitratum enzyme is much more resistant than the E. coli enzyme, is determined by the large domains. Sensitivity to inactivation by subtilisin is intermediate between those shown by the E. coli (very sensitive) and A. anitratum (quite resistant) synthases. This result suggests that digestibility by subtilisin is determined by conformational factors as well as the amino acid sequences of the target regions.     

Immunological mapping of fine molecular surface structures of citrate synthase enzymes from different cell types.

Citrate synthase (EC 4.1.3.7), which is present in all living organisms as a key enzyme in aerobic energy metabolism, is one of the most highly phylogenetically conserved enzymes known in terms of its primary and active site structure. However, in terms of other parameters such as in vitro stability, tolerance to changes in pH, degree of self-polymerization, etc., citrate synthases from different sources are markedly different. These divergences can be observed even between isoforms of the enzyme within the same species. Data documenting these diversities suggest that a high degree of difference in tertiary structures may occur. Therefore, the surface profiles of citrate synthase enzymes from yeast, pig, rat, tomato and Escherichia coli were investigated with immunological methods using monoclonal antibody families generated against either pig citrate synthase (alpha-PCS) or yeast citrate synthase-2 (alpha-YCS-2). A high degree of homology of enzyme epitopes was detected on the mitochondrial citrate synthases originating from yeast, tomato, pig and rat cells. Major differences were found between the hexameric citrate synthase originating from E. coli compared with those dimeric forms prepared from eukaryotic cells. Only modest similarities were detected between the highly homologous peroxisomal and mitochondrial yeast citrate synthases. Furthermore, a point mutation of one of the catalytic residues (H274R on recombinant pig and H313R on yeast enzyme) of mitochondrial citrate synthase (CS-1) resulted in a significant increase in immunological similarity with the peroxisomal isoenzyme (CS-2). These findings are discussed in terms of the possible mechanism of evolution of CS-2 in yeast.     

Characterization of citrate synthase from Geobacter sulfurreducens and evidence for a family of citrate synthases similar to those of eukaryotes throughout the Geobacteraceae

Members of the family Geobacteraceae are commonly the predominant Fe(III)-reducing microorganisms in sedimentary environments, as well as on the surface of energy-harvesting electrodes, and are able to effectively couple the oxidation of acetate to the reduction of external electron acceptors. Citrate synthase activity of these organisms is of interest due to its key role in acetate metabolism. Prior sequencing of the genome of Geobacter sulfurreducens revealed a putative citrate synthase sequence related to the citrate synthases of eukaryotes. All citrate synthase activity in G. sulfurreducens could be resolved to a single 49-kDa protein via affinity chromatography. The enzyme was successfully expressed at high levels in Escherichia coli with similar properties as the native enzyme, and kinetic parameters were comparable to related citrate synthases (kcat= 8.3 s(-1); Km= 14.1 and 4.3 microM for acetyl coenzyme A and oxaloacetate, respectively). The enzyme was dimeric and was slightly inhibited by ATP (Ki= 1.9 mM for acetyl coenzyme A), which is a known inhibitor for many eukaryotic, dimeric citrate synthases. NADH, an allosteric inhibitor of prokaryotic hexameric citrate synthases, did not affect enzyme activity. Unlike most prokaryotic dimeric citrate synthases, the enzyme did not have any methylcitrate synthase activity. A unique feature of the enzyme, in contrast to citrate synthases from both eukaryotes and prokaryotes, was a lack of stimulation by K+ ions. Similar citrate synthase sequences were detected in a diversity of other Geobacteraceae members. This first characterization of a eukaryotic-like citrate synthase from a prokaryote provides new insight into acetate metabolism in Geobacteraceae members and suggests a molecular target for tracking the presence and activity of these organisms in the environment.     

Citrate synthase from a Gram-positive bacterium. Purification and characterization of the Bacillus megaterium enzyme.

Citrate synthase was purified to homogeneity from a Gram-positive bacterium (Bacillus megaterium) for the first time. The Mr of the native enzyme was determined to be 84 000 (S.E.M. +/- 5000). Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and gel filtration in guanidinium chloride revealed a single protein species of Mr 40 300 (S.E.M. +/- 4400), indicating a dimeric enzyme. This dimeric structure was confirmed by cross-linking the native enzyme with dimethyl suberimidate and with glutaraldehyde, followed by electrophoretic analysis. The enzyme follows Michaelis-Menten kinetics with respect to both substrates, acetyl-CoA and oxaloacetate, and is sensitive to non-specific inhibition by a range of adenine nucleotides. In both molecular and catalytic properties the citrate synthase closely resembles the enzyme from eukaryotic sources and contrasts markedly with the larger, hexameric, enzyme from Gram-negative bacteria.     

Citrate synthases from germinating castor bean seeds – II. Time course of synthesis and immunochemical comparison

The time course of total citrate synthase activity in castor bean ( Ricinus communis L., type Sanzibariensis) endosperm showed a 7-fold increase during the initial 5 days of germination and a decrease thereafter. All citrate synthase activity in the ungerminated seeds was due to the mitochondrial isoenzyme. After two days of germination the glyoxysomal isoenzyme began to appear. After 5 days the glyoxysomal citrate synthase represented 50 to 55% of the total activity and the mitochondrial enzyme the remainder. This was estimated from (a) inactivation of the glyoxysomal citrate synthase by 5,5'-dithiobis(2-nitrobenzoic acid); (b) solid phase adsorption of the glyoxysomal synthase by a specific antiserum; (c) separation of isoenzymes by (NH₄)₂SO₄ gradient solubilization.
The increase of both citrate synthases during the initial 4 days of germination could be prevented by 10 μg cycloheximide ml⁻¹, but not by 40 or 400 μg chloramphenicol ml⁻¹, indicating a synthesis on 80 S ribosomes. Actinomycin D completely inhibited the appearance of the glyoxysomal enzyme while the mitochondrial enzyme was not affected. Antisera against the two isoenzymes revealed major structural differences between two citrate synthases, however, also some common determinants. No cross-reaction was observed with the citrate synthase from pig heart or E. coli.     

Inhibition of glutamate dehydrogenase and malate dehydrogenases by palmitoyl coenzyme A.

In extension of a previous study with yeast glucose-6-P dehydrogenase (Kawaguchi, A., and Bloch, K. (1974) J. Biol. Chem. 249, 5793-5800), the structural changes accompanying the inhibition of glutamate dehydrogenase and several malate dehydrogenases by palmitoyl-CoA and by sodium dodecyl sulfate have been investigated. Palmitoyl-CoA converts liver glutamate dehydrogenase to enzymatically inactive dimeric subunits (Mr = 1.2 X 10(5)) and tightly binds to the dissociated enzyme. Removal of the inhibitor from the palmitoyl-CoA-dimer complex fails to regenerate enzyme activity. The Ki values for palmitoyl-CoA inhibition of malate dehydrogenases (oxalacetate reduction) are, for the enzyme from pig heart mitochondria, 1.8 muM, 500 muM from pig heart supernatant, and 10 muM from chicken heart supernatant. These inhibitions are readily reversible. Palmitoyl-CoA does not alter the quaternary structure of any of the malate dehydrogenases and binds only weakly to these enzymes. Mitochondrial malate dehydrogenase assayed in the direction malate to oxalacetate is much less sensitive to palmitoyl-CoA, with Ki values of 50 muM at pH 10 and greater than 50 muM at pH 7.4. While the differences in palmitoyl-CoA sensitivity in the forward and backward reactions catalyzed by mitochondrial dehydrogenase are unexplained, a physiological rationale for these differential effects is offered. Sodium dodecyl sulfate dissociates the various dehydrogenases to monomeric subunits in contrast to the more selective effects of palmitoyl-CoA.     

Proton uptake accompanies formation of the ternary complex of citrate synthase, oxaloacetate, and the transition-state analog inhibitor, carboxymethyl-CoA. Evidence that a neutral enol is the activated form of acetyl-CoA in the citrate synthase reaction.

Citrate synthase complexes with the transition-state analog inhibitor, carboxymethyl-CoA (CM-CoA), are believed to mimic those with the activated form of acetyl-CoA. The X-ray structure [Karpusas, M., Branchaud, B., & Remington, S.J. (1990) Biochemistry 29, 2213] of the ternary complex of the enzyme, oxaloacetate, and CMCoA has been used as the basis for a proposal that a neutral enol of acetyl-CoA is that activated form. Since the inhibitor carboxyl has a pKa of 3.90, analogy with an enolic acetyl-CoA intermediate leads to the prediction that a proton should be taken up from solution upon formation of the analog complex so that the transition-state analog carboxyl is protonated when bound. We have obtained evidence in solution for this proposal by comparing the isoelectric points and the pH dependence of the dissociation constants of the ternary complexes of the pig heart enzyme with the neutral ground-state analog inhibitor, acetonyl-CoA (KCoA), and the anionic transition-state analog inhibitor (CMCoA) and by studying the NMR spectra of the transition-state analog complexes of allosteric (Escherichia coli) and nonallosteric (pig heart) enzymes. The pH dependence of the dissociation constant of the ground-state analog indicates no proton uptake, while that for the transition-state analog indicates that 0.55 +/- 0.04 proton is taken up when the analog binds to the citrate synthase-oxaloacetate binary complex. The overall charges of ternary complexes of the pig heart enzyme with the transition-state and ground-state analog inhibitors are the same, as monitored by their isoelectric points.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sequence alignment of citrate synthase proteins using a multiple sequence alignment algorithm and multiple scoring matrices

The alignment of Escherichia coli citrate synthase to pig heart citrate synthase and the multiple alignment of the known sequences of the citrate synthase family of enzymes have been performed using six different amino acid similarity scoring matrices and a large range of gap penalty ratios for insertions and deletions of amino acids. The alignment studies have been performed as the first step in a project aimed at homology modelling E. coli citrate synthase (a hexamer) from pig heart citrate synthase (a dimer) in a molecular modelling approach to the study of multi-subunit enzymes. The effects of several important variables in producing realistic alignments have been investigated. The difference between multiple alignment of the family of enzymes versus simple pairwise alignment of the pig heart and E. coli proteins was explored. The effects of initial separate multiple alignments of the most highly related or most homologous species of the family of enzymes upon a subsequent pairwise alignment between species was evaluated. The value of 'fingerprinting' certain residues to bias the alignment in favour of matching those residues, as well as the worth of the computerized approach compared to an intuitive alignment technique, were assessed.     

Inhibitory effect of long chain fatty acyl CoAs on RNA polymerase from Escherichia coli

RNA polymerase from Escherichia coli was inhibited by long chain fatty acyl CoAs, such as myristoyl CoA (Ki = 17.2 microM), palmitoyl CoA (Ki = 8.9 microM), oleoyl CoA (Ki = 5.5 microM), and stearoyl CoA (Ki = 0.94 microM). The inhibition by these CoA thioesters was non-competitive against nucleoside triphosphates. Short chain fatty acyl CoAs, such as acetyl CoA, propionyl CoA, acetoacetyl CoA, butyryl CoA, and decanoyl CoA, failed to inhibit RNA polymerase. CoA, Na-myristate, Na-palmitate, Na-oleate, Na-stearate, palmitoyl carnitine, and carnitine did not inhibit the enzyme. The inhibition of RNA polymerase by long chain fatty acyl CoAs was competitive against template DNA.     

Identification and characterization of a re-citrate synthase in Dehalococcoides strain CBDB1

The genome annotations of all sequenced Dehalococcoides strains lack a citrate synthase, although physiological experiments have indicated that such an activity should be encoded. We here report that a Re face-specific citrate synthase is synthesized by Dehalococcoides strain CBDB1 and that this function is encoded by the gene cbdbA1708 (NCBI accession number CAI83711), previously annotated as encoding homocitrate synthase. Gene cbdbA1708 was heterologously expressed in Escherichia coli, and the recombinant enzyme was purified. The enzyme catalyzed the condensation of oxaloacetate and acetyl coenzyme A (acetyl-CoA) to citrate. The protein did not have homocitrate synthase activity and was inhibited by citrate, and Mn2+ was needed for full activity. The stereospecificity of the heterologously expressed citrate synthase was determined by electrospray ionization liquid chromatography-mass spectrometry (ESI LC/MS). Citrate was synthesized from [2-(13)C]acetyl-CoA and oxaloacetate by the Dehalococcoides recombinant citrate synthase and then converted to acetate and malate by commercial citrate lyase plus malate dehydrogenase. The formation of unlabeled acetate and 13C-labeled malate proved the Re face-specific activity of the enzyme. Shotgun proteome analyses of cell extracts of strain CBDB1 demonstrated that cbdbA1708 is expressed in strain CBDB1.