Regulatory and molecular properties of citrate synthases from methylotrophs

Abstract Gram-negative methylotrophs contain a high- M _r'large' citrate synthase. Gram-positive methylotrophs, on the other hand, contain a 'small' citrate synthase. These differences in M _r coincided partly with differences in NADH sensitivity. Citrate synthases from obligate Gram-negative and Gram-positive facultative methylotrophs were insensitive to feedback inhibition by NADH; only the enzymes from Gram-negative facultative methylotrophs were inhibited by NADH.     

Gram-positive type citrate synthase in the thermophilic green gliding bacterium Chloroflexus aurantiacus

Abstract Some properties of the citrate synthase from Chloroflexus aurantiacus have been examined in crude cell-free extracts and partially purified preparations. The enzyme had an approximate native molecular size of 140 000, was not inhibited by NADH or 2-oxoglutarate but was inhibited by ATP (about 50% at 5 mM). The K _m for acetyl CoA at pH 8.2 in the presence of 0.5 mM oxaloacetate was determined to be 25 μM.
These properties are characteristic of the 'small' size class of citrate synthases normally associated with gram-positive eubacteria, despite the fact that Chloroflexus stains gram-negatively.     

Molecular size diversity of citrate synthases from Pseudomonas species

Two forms of citrate synthase (EC 4.1.3.7) have been found in several species of Pseudomonas, a 'large' form (Mr congruent to 250,000) which is generally inhibited by NADH and reactivated by AMP, and a 'small' form (Mr congruent to 100,000) which is insensitive to these nucleotide effectors. Other species of Pseudomonas were found to contain either the 'large' or the 'small' form. Gel filtration and ion-exchange with the technique of fast protein liquid chromatography were used to resolve the enzymes. Where both citrate synthases were present, there did not appear to be an equilibrium between the two forms. The results reveal a new and complex diversity of citrate synthase within the genus Pseudomonas.     

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.     

Rhizobium tropici chromosomal citrate synthase gene.

Two genes encoding citrate synthase, a key enzyme in the Krebs cycle, have been found in Rhizobium tropici. One of them is in the bacterial chromosome, while the other is in the symbiotic plasmid. We sequenced the chromosomal gene and found that it is very similar to the previously reported plasmidic gene sequence in its structural region but not in its regulatory region. The chromosomal gene is able to complement an Escherichia coli citrate synthase mutant. In R. tropici, a mutant in the chromosomal citrate synthase gene has a diminished citrate synthase activity (in free-living bacteria), a diminished nodulation capacity, and forms nitrogen-fixing nodules. In contrast, the citrate synthase double mutant forms ineffective nodules devoid of bacteroids and forms less nodules than the single chromosomal mutant. It is inferred that both genes are functional and required during the nodulation process in R. tropici.     

Activities of citrate synthase and other enzymes of Acetobacter xylinum in situ and in vitro.

The activities of a number of enzymes, extracted from Acetobacter xylinum, that are involved in carbohydrate metabolism may be accounted for in situ in permeabilized cells. The kinetic properties of citrate synthase and glycerokinase observed in vitro are also retained in situ. So is the regulatory sensitivity of these enzymes. Both in vitro and in situ, (a) citrate synthase, in contrast with the enzyme for other Gram-negative bacteria, is inhibited by ATP and is insensitive to NADH, and (b) glycerokinase is inhibited by fructose diphosphate and the ratio of its activities towards glycerol and dihydroxyacetone is the same.     

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.     

Probing the roles of key residues in the unique regulatory NADH binding site of type II citrate synthase of Escherichia coli

The citrate synthase of Escherichia coli is an example of a Type II citrate synthase, a hexamer that is subject to allosteric inhibition by NADH. In previous crystallographic work, we defined the NADH binding sites, identifying nine amino acids whose side chains were proposed to make hydrogen bonds with the NADH molecule. Here, we describe the functional properties of nine sequence variants, in which these have been replaced by nonbonding residues. All of the variants show some changes in NADH binding and inhibition and small but significant changes in kinetic parameters for catalysis. In three cases, Y145A, R163L, and K167A, NADH inhibition has become extremely weak. We have used nanospray/time-of-flight mass spectrometry, under non-denaturing conditions, to show that two of these, R163L and K167A, do not form hexamers in response to NADH binding, unlike the wild type enzyme. One variant, R109L, shows tighter NADH binding. We have crystallized this variant and determined its structure, with and without bound NADH. Unexpectedly, the greatest structural changes in the R109L variant are in two regions outside the NADH binding site, both of which, in wild type citrate synthase, have unusually high mobilities as measured by crystallographic thermal factors. In the R109L variant, both regions (residues 260 -311 and 316-342) are much less mobile and have rearranged significantly. We argue that these two regions are elements in the path of communication between the NADH binding sites and the active sites and are centrally involved in the regulatory conformational change in E. coli citrate synthase.     

Role of acid metabolism in Streptomyces coelicolor morphological differentiation and antibiotic biosynthesis

Studies of citrate synthase (CitA) were carried out to investigate its role in morphological development and biosynthesis of antibiotics in Streptomyces coelicolor. Purification of CitA, the major vegetative enzyme activity, allowed characterization of its kinetic properties. The apparent K(m) values of CitA for acetyl coenzyme A (acetyl-CoA) (32 microM) and oxaloacetate (17 microM) were similar to those of citrate synthases from other gram-positive bacteria and eukaryotes. CitA was not strongly inhibited by various allosteric feedback inhibitors (NAD(+), NADH, ATP, ADP, isocitrate, or alpha-ketoglutarate). The corresponding gene (citA) was cloned and sequenced, allowing construction of a citA mutant (BZ2). BZ2 was a glutamate auxotroph, indicating that citA encoded the major citrate synthase allowing flow of acetyl-CoA into the tricarboxylic acid (TCA) cycle. Interruption of aerobic TCA cycle-based metabolism resulted in acidification of the medium and defects in morphological differentiation and antibiotic biosynthesis. These developmental defects of the citA mutant were in part due to a glucose-dependent medium acidification that was also exhibited by some other bald mutants. Unlike other acidogenic bald strains, citA and bldJ mutants were able to produce aerial mycelia and pigments when the medium was buffered sufficiently to maintain neutrality. Extracellular complementation studies suggested that citA defines a new stage of the Streptomyces developmental cascade.     

Site-directed mutagenesis of citrate synthase; the role of the active-site aspartate in the binding of acetyl-CoA but not oxaloacetate

Asp-362, a potential key catalytic residue of Escherichia coli citrate synthase (citrate oxaloacetate-lyase [pro-3S)-CH2COO- ----acetyl-CoA), EC 4.1.3.7) has been converted to Gly-362 by oligonucleotide-directed mutagenesis. The mutant gene was completely sequenced, using a series of synthetic oligodeoxynucleotides spanning the structural gene to confirm that no additional mutations had occurred during genetic manipulation. The mutant gene was expressed in M13 bacteriophage and produced a protein which migrated in an identical manner to wild-type E. coli citrate synthase on SDS-polyacrylamide gels and which cross-reacted with E. coli citrate synthase antiserum. The mutant gene was subsequently recloned into pBR322 for large scale purification of the protein, and the resulting plasmid, pCS31, used to transform the citrate synthase deletion strain, W620. The mutant enzyme purified in an analogous manner to wild-type E. coli citrate synthase and expressed less than 2% of wild-type enzyme activity. The activity of the partial reactions catalysed by citrate synthase was similarly affected suggesting that this residual activity may be due to contaminating wild-type enzyme activity. The mutant citrate synthase retains a high-affinity NADH-binding site consistent with the protein preserving its overall structural integrity. Oxaloacetate binding to the protein is unaffected by the Asp-362 to Gly-362 mutation. Binding of the acetyl-CoA analogue, carboxymethyl-CoA, could not be detected in the mutant protein indicating that the lack of catalytic competence is due primarily to the inability of the protein to bind the second substrate, acetyl-CoA.     

The interactions of adenylates with allosteric citrate synthase.

Evidence is presented that a number of derivatives of adenylic acid may bind to the allosteric NADH binding site of Escherichia coli citrate synthase. This evidence includes the facts that all the adenylates inhibit NADH binding in a competitive manner and that those which have been tested protect an enzyme sulfhydryl group from reaction with 5,5'-dithiobis-(2-nitrobenzoic acid) in the same way that NADH does. However, whereas NADH is a potent inhibitor of citrate synthase, most of the adenylates are activators. The best activator, ADP-ribose, increases the affinity of the enzyme for the substrate, acetyl-CoA, and saturates the enzyme in a sigmoid manner. A fluorescence technique, involving the displacement of 8-anilino-1-naphthalenesulfonate from its complex with citrate synthase, is used to obtain saturation curves for several nucleotides under nonassay conditions. It is found that acetyl-coenzyme A, coenzyme A, and ADP-ribose all bind to the enzyme cooperatively, and that the binding of each becomes tighter in the presence of KCl, the activator, and oxaloacetic acid (OAA), the second substrate. Another inhibitor, alpha-ketoglutarate, can complete with OAA in the absence of KCl but not in its presence. The nature of the allosteric site of citrate synthase, and the modes of action of several activators and inhibitors, are discussed in the light of this evidence.     

The binding of reduced nicotinamide adenine dinucleotide to citrate synthase of Escherichia coli K12.

Citrate synthase from Escherichia coli enhances the fluorescence of its allosteric inhibitor, NADH, and shifts the peak of emission of the coenzyme from 457 to 428 nm. These effects have been used to measure the binding of NADH to this enzyme under various conditions. The dissociation constant for the NADH-citrate synthase complex is about 0.28 muM at pH 6.2, but increases toward alkaline pH as if binding depends on protonation of a group with a pKa of about 7.05. Over the pH range 6.2-8.7, the number of binding sites decreases from about 0.65 to about 0.25 per citrate synthase subunit. The midpoint of this transition is at about pH 7.7, and it may be one reflection of the partial depolymerization of the enzyme which is known to occur in this pH range. A gel filtration method has been used to verify that the fluorescence enhancement technique accurately reveals all of the NADH molecules bound to the enzyme in the concentration range of interest. NAD+ and NADP+ were weak competitive inhibitors of NADH binding at pH 7.8 (Ki values greater than 1 mM), but stronger inhibition was shown by 5'-AMP and 3'-AMP, with Ki values of 83 +/- 5 and 65 +/- 4 muM, respectively. Acetyl-CoA, one of the substrates, and KCl, an activator, also inhibit the binding in a weakly cooperative manner. All of these effects are consistent with kinetic observations on this system. We interpret our results in terms of two types of binding site for nucleotides on citrate synthase: an active site which binds acetyl-CoA, the substrate, or its analogue 3'-AMP; and an allosteric site which binds NADH or its analogue 5'-AMP and has a lesser affinity for other nicotinamide adenine dinucloetides. When the active site is occupied, we propose that NADH cannot bind to the allosteric site, but 5'-AMP can; conversely, when NADH is the in the allosteric site, the active site cannot be occupied. In addition to these two classes of sites, there must be points for interaction with KCl and other salts. Oxaloacetate, the second substrate, and alpha-ketoglutarate, an inhibitor whose mode of action is believed to be allosteric, have no effect on NADH binding to citrate synthase at pH 7.8. When NADH is bound to citrate synthase, it quenches the intrinsic tryptophan fluorescence of the enzyme. The amount of quenching is proportional to the amount of NADH bound, at least up to a binding ratio of 0.50 NADH per enzyme subunit. This amount of binding leads to the quenching of 53 +/- 5% of the enzyme fluorescence, which means that one NADH molecule can quench all the intrinsic fluorescence of the subunit to which it binds.     

The role of cysteine 206 in allosteric inhibition of Escherichia coli citrate synthase. Studies by chemical modification, site-directed mutagenesis, and 19F NMR

Escherichia coli citrate synthase is strongly and specifically inhibited by NADH, but this inhibition can be prevented by reacting the enzyme with Ellman's reagent. We have now labeled the single reactive cysteine covalently with monobromobimane and isolated and sequenced the bimane-containing cyanogen bromide peptide and identified the cysteine as Cys-206. Modeling studies suggest that this residue is on the subunit surface, 25-30 A from the active site. Mutation of Cys-206 to serine (C206S), or of Gly-207 to alanine (E207A), weakened NADH binding and inhibition; when these mutations were present together, NADH binding was weaker by 18-fold and inhibition by 250-fold. The mutations also had small effects on substrate binding at the active site. Cys-206 of wild type enzyme and of the mutant E207A was alkylated with 1,1,1-trifluorobromoacetone and the environment of the fluorine nuclei studied by 19F NMR. With wild type enzyme, the NMR spectrum consisted of two peaks of about equal intensity but different line widths, at -8.65 ppm (line width 11.2 +/- 0.5 Hz) and -7.6 ppm (line width 57 +/- 4 Hz). As the labeled wild type citrate synthase was titrated with KCl, the narrow peak converted to the broad one. The same range of KCl concentrations was needed for this conversion as for the allosteric activation of E. coli citrate synthase. The E207A mutant gave the broader NMR peak almost exclusively. We propose that the fluorine label in wild type citrate synthase exists in two conformational states with different mobilities, exchanging slowly on the NMR time scale, and that treatment with KCl, or truncation of the Glu-207 side chain by mutagenesis, stabilizes one of these states. Consistent with this explanation is the finding that Cys-206 reacts more quickly with Ellman's reagent in the presence of KCl, and that this rate is faster yet in the E207A mutant.     

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.     

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.     

Cloning, sequencing, and expression of the gene for NADH-sensitive citrate synthase of Pseudomonas aeruginosa

The structural gene for the allosteric citrate synthase of Pseudomonas aeruginosa has been cloned from a genomic library by using the Escherichia coli citrate synthase gene as a hybridization probe under conditions of reduced stringency. Subcloning of portions of the original 10-kilobase-pair (kbp) clone led to isolation of the structural gene, with its promoter, within a 2,083-bp length of DNA flanked by sites for KpnI and BamHI. The nucleotide sequence of this fragment is presented; the inferred amino acid sequence was 70 and 76% identical, respectively, with the citrate synthase sequences from E. coli and Acinetobacter anitratum, two other gram-negative bacteria. DEAE-cellulose chromatography of P. aeruginosa citrate synthase from an E. coli host harboring the cloned P. aeruginosa gene gave three peaks of activity. All three enzyme peaks had subunit molecular weights of 48,000; the proteins were identical by immunological criteria and very similar in kinetics of substrate saturation and NADH inhibition. Because the cloned gene contained only one open reading frame large enough to encode a polypeptide of such a size, the three peaks must represent different forms of the same protein. A portion of the cloned P. aeruginosa gene was used as a hybridization probe under stringent conditions to identify highly homologous sequences in genomic DNA of a second strain classified as P. aeruginosa and isolates of P. putida, P. stutzeri, and P. alcaligenes. When crude extracts of each of these four isolates were mixed with antiserum raised against purified P. aeruginosa citrate synthase, however, only the P. alcaligenes extract cross-reacted.     

Isolation, nucleotide sequence, and expression of a cDNA encoding pig citrate synthase

Citrate synthase is a key enzyme of the Krebs tricarboxylic acid cycle and catalyzes the stereospecific synthesis of citrate from acetyl coenzyme A and oxalacetate. The amino acid sequence and three-dimensional structure of pig citrate synthase dimers are known, and regions of the enzyme involved in substrate binding and catalysis have been identified. A cloned complementary DNA sequence encoding pig citrate synthase has been isolated from a pig kidney lambda gt11 cDNA library after screening with a synthetic oligonucleotide probe. The complete nucleotide sequence of the 1.5-kilobase cDNA was determined. The coding region consists of 1395 base pairs and confirms the amino acid sequence of purified pig citrate synthase. The derived amino acid sequence of pig citrate synthase predicts the presence of a 27 amino acid N-terminal leader peptide whose sequence is consistent with the sequences of other mitochondrial signal peptides. A conserved amino acid sequence in the mitochondrial leader peptides of pig citrate synthase and yeast mitochondrial citrate synthase was identified. To express the pig citrate synthase cDNA in Escherichia coli, we employed the inducible T7 RNA polymerase/promoter double plasmid expression vectors pGP1-2 and pT7-7 [Tabor, S., & Richardson, C. C. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 1074-1078]. The pig citrate synthase cDNA was modified to delete the N-terminal leader sequence; then by use of a synthetic oligonucleotide linker, the modified cDNA was cloned into pT7-7 immediately following the initiator Met. A glutamate-requiring (citrate synthase deficient), recA- E. coli mutant, DEK15, was transformed with pGP1-2 and then pT7-7PCS. pT7-7PCS complemented the E. coli gltA mutation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Separation of two forms of glutamate synthase in leaves of tomato (Lycopersicon esculentum)

Two forms of glutamate synthase, one dependent on NAD(P)H, and the other on ferredoxin, have been completely separated by ionic exchange chromatography on DEAE cellulose. The NAD(P)H dependent enzyme was further purified by affinity chromatography with Blue Sepharose, showing Km values of 0.5 mM, 0.3 mM and 1.7 μM for glutamine, 2-oxoglutarate and NADH, respectively. Ferredoxin dependent enzyme was also purified to electrophoretic homogeneity; the Km values were 0.5 mM, 0.2 mM and 0.2 μM for glutamine, 2-oxoglutarate and ferredoxin, respectively. These results support the glutamine synthetase-glutamate synthase pathway for nitrogen assimilation.     

Cloning and nucleotide sequence of the gene coding for citrate synthase from a thermotolerant Bacillus sp.

The structural gene coding for citrate synthase from the gram-positive soil isolate Bacillus sp. strain C4 (ATCC 55182) capable of secreting acetic acid at pH 5.0 to 7.0 in the presence of dolime has been cloned from a genomic library by complementation of an Escherichia coli auxotrophic mutant lacking citrate synthase. The nucleotide sequence of the entire 3.1-kb HindIII fragment has been determined, and one major open reading frame was found coding for citrate synthase (ctsA). Citrate synthase from Bacillus sp. strain C4 was found to be a dimer (Mr, 84,500) with a subunit with an Mr of 42,000. The N-terminal sequence was found to be identical with that predicted from the gene sequence. The kinetics were best fit to a bisubstrate enzyme with an ordered mechanism. Bacillus sp. strain C4 citrate synthase was not activated by potassium chloride and was not inhibited by NADH, ATP, ADP, or AMP at levels up to 1 mM. The predicted amino acid sequence was compared with that of the E. coli, Acinetobacter anitratum, Pseudomonas aeruginosa, Rickettsia prowazekii, porcine heart, and Saccharomyces cerevisiae cytoplasmic and mitochondrial enzymes.