Characterization of acetyl-CoA: l-lysine N6-acetyltransferase,which catalyses the first step of carbon catabolism from lysine in Saccharomyces cerevisiae

The carbon catabolism of l-lysine starts in Saccharomyces cerevisiae with acetylation by an acetyl-CoA: l-lysine N⁶-acetyltransferase. The enzyme is strongly induced in cells grown on l-lysine as sole carbon source and has been purified about 530-fold. Its activity was specific for acetyl-CoA and, in addition to l-lysine, 5-hydroxylysine and thialysine act as acetyl acceptor. The following apparent Michaelis constants were determined: acetyl-CoA 0.8 mM, l-lysine 5.8 mM, dl-5-hydroxylysine 2.8 mM, l-thialysine 100 mM. The enzyme had a maximum activity at pH 8.5 and 37°C. Its molecular mass, estimated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, was 52 kDa. Since the native molecular mass, determined by gel filtration, was 48 kDa, the enzyme is a monomer.     

Properties of rat 6-N-trimethyl-l-lysine hydroxylases: Similarities among the kidney,liver, heart,and skeletal muscle activities

Hydroxylation of 6-N-trimethyl-l-lysine(lys(Me₃)) to 3-hydroxy-6-N-trimethyl-l-lysine(3-HO-lys(Me₃)) by several rat tissues has been examined and compared. The kidney enzyme, which previously was shown to require molecular oxygen and α-ketoglutarate as cosubstrates, ferrous iron and ascorbate as cofactors, and to be stimulated by catalase, has a broad pH optimum ranging between 6.5 to 7.5 at 37 °C. As determined with crude tissue extracts from kidney, liver, heart, and skeletal muscle, similar apparent K_m values were obtained for substrate, cosubstrates, and cofactors. In view of similar kinetic parameters among the several lys(Me₃) hydroxylases examined in rat tissues, and the fact that the level of skeletal muscle lys(Me₃) hydroxylase activity is comparable to that of heart, liver, and kidney, because of its large total mass, skeletal muscle may contribute significantly to the biosynthesis of l-carnitine from lys(Me₃). The most effective inhibitors found, competitive with lys(Me₃), were 2-N-acetyl-6-N-trimethyl-l-lysine, 6-N-monomethyl-l-lysine, and 6-N-dimethyl-l-lysine. l-2-Amino-6-N-trimethylammonium-4-hexynoate, d-2-amino-6-N-trimethylammonium-4-hexynoate, and dl2-amino-6-N-trimethylammonium-cis-4-hexenoate, also inhibited hydroxylase activity but by a yet undetermined mechanism. Oxalacetate, succinate, and citrate inhibited the hydroxylation reaction by competing with α-ketoglutarate. The binding of ferrous iron to the enzyme was competitively inhibited by ions of “soft metals” (e.g., Cd²⁺, Zn²⁺) but not by those of “hard metals” (e.g., Ca²⁺, Mg²⁺). Preincubation of the crude kidney enzyme for 15 min at 37 °C with mercuriphenylsulfonate, N-ethylmaleimide, iodoacetate, or iodoacetamide resulted in considerable inhibition of 3-HO-lys(Me₃) formation. The degree of inhibition by N-ethylmaleimide could be reduced by including Zn (II) during preincubation of the enzyme. The effects of “soft” metals and sulfhydryl reagents on the enzyme suggest that sulfhydryl groups are required for ferrous iron binding in the active site.     

Guanosine triphosphate and colchicine affect the activity and the polymeric state of acetyl-CoA carboxylase

Acetyl-CoA carboxylase was purified 300-fold from rat liver, in the absence of added citrate, by precipitation from an 18,000g supernatant in the presence of Triton X-100 at 105,000g and 20 °C, followed by chromatography on phosphocellulose. Acetyl-CoA carboxylase activity in this preparation was activated by preincubation with GTP (0.1–2.0 mm) and with citrate (20 mm). Colchicine (10^?6–10^?3m) inhibited enzyme activity and counteracted the effects of GTP and citrate. Sucrose density gradient centrifugation demonstrated that GTP and citrate preincubation promoted the formation of the polymeric, active enzyme, while colchicine engendered disassembly. Preincubation of the purified acetyl-CoA carboxylase at 4 °C caused inactivation and disassembly, which was countered by preincubation at 37 °C in the presence of GTP or citrate. These results suggest that GTP, like citrate, activates acetyl-CoA carboxylase by enhancing the conversion of the protomeric form of the enzyme to its more active, polymeric state.     

Stimulation by glutamine of the formation of N6-hydroxylysine in a cell-free extract from aerobacter aerogenes 62-1

Glutamine may serve as an activator and/or regulator of the N⁶-hydroxylase (E.C. 1.14.99) of Aerobacter aerogenes 62-1. Activation and stabilization of N⁶-hydroxylase activity was observed both in vivo and in vitro. Growth in a glutamine-supplemented medium resulted in (1) maximum N⁶-hydroxylase activity at an earlier stage of growth and (2) higher N⁶-hydroxylase activity and continued aerobactin synthesis into stationary phase. Storage of P2 in the presence of L-glutamine (1 mM) significantly increased the lifetime of the labile N⁶-hydroxylase activity. Inclusion of L-glutamine in the incubation mixture typically resulted in a 2-3-fold activation of the hydroxylase activity. The stimulatory effect of glutamine was independent of and additive to the enhancement of N⁶-hydroxylation by the active component(s) in the supernatant, S2 fraction. Glutamic acid-γ-semihydrazide activated slightly in the absence of glutamine but activation of the system by glutamine was decreased by this compound. Azaserine was shown to be an uncompetitive inhibitor with respect to lysine and this inhibition was not reversed by glutamine.     

Structure of mycobactin J

Mycobactin J-1, an iron chelate fromMycobacterium paratuberculosis, was characterized by mass spectrum and by¹H nuclear magnetic resonance (NMR) and¹³C NMR spectra of the parent molecule and of cobactin J-1. The core structure of mycobactin J-1 contained the phenyloxazoline ring system common to the mycobactins. The benzene ring was disubstituted. The two hydroxamate functions were furnished by 1 linear 6-N-hydroxylysine residue and 1 cyclic 6-N-hydroxylysine residue as in other members of this class of compounds. The acyl function at the mycobactic acid hydroxamate center wasn-cis-hexadec-2-enoyl. The hydroxyacid of the cobactin portion of mycobactin J-1 was 2,4-dimethyl-3-hydroxypentanoic acid. This latter residue differs from those of other known mycobactins by the presence of the isopropyl group.     

Identification of the genes and their polypeptide products responsible for aerobactin synthesis by pColV plasmids

Summary Iron acquisition via aerobactin enhances the virulence of Escherichia coli. Genes that specify functions for aerobactin synthesis and iron(III)-aerobactin transport have been identified on several ColV plasmids. Previously, we cloned the locus for aerobactin synthesis from pColV-K311 an dassigned to three loci termed AeA, aerB, and areC the functions for hydroxylation of lysine, acetylation of the 6-amino group of 6-hydroxy-lysine and coupling of N-acetyl-N-hydroxy-lysine with citrate, respectively (Gross et al. 1984). In this paper we show that aerA and aerB determine polypeptides with molecular weights of 50,000 and 35,000, respectively. We identified a fourth gene designated aerD that codes for a polypeptide with a molecular weight of 60,000, and which is required for the linkage of one residue of N-acetyl-N-hydroxy-lysine to citrate. The aerC gene product completes aerobactin synthesis by coupling the secod N-acetyl-N-hydroxy-lysine to the monoacylated derivative citrate. The order of the genes in the operon was found to be aerD-aerB-areC-aerA.     

N6-Trimethyl-lysine metabolism. Structural identification of the metabolite 3-hydroxy-N6-trimethyl-lysine

¹H and ¹³C nuclear-magnetic-resonance spectroscopy and functional-group analysis were used to determine the molecular structure of an isolated metabolite (II_b) of trimethyl-lysine as 3-hydroxy-N⁶-trimethyl-lysine, an important intermediate in the conversion of trimethyl-lysine into trimethylammoniobutyrate and carnitine [Hoppel, Cox & Novak (1980) Biochem. J. 188, 509–519]. Functional-group analysis revealed the presence of a primary amine and reaction of metabolite (II_b) with periodate yielded 4-N-trimethylammoniobutyrate as a product, showing 2,3-substitution on the molecule and suggesting that the 3-substitution on the molecule may be an alcohol ([unk]CH–OH), amine ([unk]CH[unk]–NH₂) or carbonyl ([unk]C=O) functional group. ¹H integration ratios, ¹H and ¹³C chemical-shift data and ¹H and ¹³C signal multiplicities from the sample (II_b) were used to complete the identification of metabolite (II_b) as 3-hydroxy-N⁶-trimethyl-lysine. For example, the proton multiplet at δ 4.2p.p.m. and doublet at δ 4.1p.p.m., positions representative of amine or alcohol substitution on methylene carbon atoms, integration ratios of 1:1:2:9:4 and a positive ninhydrin test suggest 3-hydroxy-N⁶-trimethyl-lysine as the molecular structure for metabolite (II_b). ¹³C chemical-shift data obtained from the sample (II_b) and compared with several model compounds (trimethylammoniohexanoate, trimethyl-lysine and 3-hydroxylysine) resulted in generation of the spectrum of the metabolite and allowed independent identification of metabolite (II_b) as 3-hydroxy-N⁶-trimethyl-lysine. The ¹H spectrum of erythro- and threo-3-hydroxylysine are presented for comparison, and the ¹H and ¹³C n.m.r. spectra of the erythro-isomer support this analysis.     

Metabolism of cytokinin: ribosylation of cytokinin bases by adenosine phosphorylase from wheat germ

As part of the study of cytokinin metabolic pathways, an enzyme, adenosine phosphorylase (EC 2.4.2.-), which catalyzed the ribosylation of N⁶-(Δ²-isopentenyl)adenine, N⁶-furfuryladenine, and adenine to form the corresponding nucleosides, was partially purified from wheat (Triticum aestivum) germ. The pH optimum for the ribosylation of the cytokinins and adenine was from 6.5 to 7.8; for guanine and hypoxanthine it was from 7.0 to 8.5 At pH 7.2 (63 millimolar N-2-hydroxyethyl piperazine-N′-ethanesulfonic acid) and 37 C the K_m for N⁶-(Δ²-isopentenyl)adenine was 57.1 micromolar; N⁶-furfuryladenine, 46.5 micromolar; adenine, 32.2 micromolar; and the V_max for N⁶-(Δ²-isopentenyl)adenine, N⁶-furfuryladenine, and adenine were 134.7, 137.1, and 193.1 nanomoles per milligram protein per minute, respectively. The equilibrium constants of the phosphorolysis of N⁶-(Δ²-isopentenyl)adenosine and adenosine by this enzyme indicated that the reaction strongly favored nucleoside formation. This enzyme was shown to be distinct from inosine-guanosine phosphorylase based on the differences in the Sephadex G-100 gel filtration behaviors, pH optima, and the product and p-hydroxymercuribenzoate inhibitor studies. These results suggest that adenosine phosphorylase may play a significant role in the regulation of cytokinin metabolism.     

Formation of coA esters of cinnamic acid derivatives by extracts of Brassica napo-brassica root tissue

An enzyme fraction from aged swede root disks catalyses the formation of CoA thioesters of cinnamic acids in the presence of CoA, ATP and Mg²⁺. The enzyme shows activity only to those cinnamic acid derivatives bearing a phenolic OH group, p-coumaric and ferulic acids being the most active substrates. The requirement for Mg²⁺ can be replaced by Mn²⁺, Co²⁺ or Ni²⁺. The requirement for ATP could not be replaced by GTP, CTP, UTP, ADP or AMP. ADP and AMP, but not pyrophosphate, inhibited the ATP dependent activation of p-coumarate. The activity was inhibited by N-ethylmaleimide and p-chloro-mercuribenzoate which suggests a requirement for -SH groups for activation. The activity of the enzyme is low in freshly prepared disks but rises during ageing, particularly if the ageing is carried out in the presence of low concentrations of ethylene.     

Properties of pyruvate kinase from soybean nodule cytosol

The properties of pyruvate kinase from soybean (Glycine max L.) nodule cytosol were examined to determine what influence the N₂ fixation process might have on this supposed key control enzyme. A crude enzyme preparation was prepared by chromatography of cytosol extract on a diethylaminoethyl-cellulose column. ATP and citrate at 5 mm concentrations inhibited pyruvate kinase 27 and 34%, respectively. Enzyme activation was hyperbolic with respect to both K⁺ and NH₄⁺ concentrations. In the presence of physiological concentrations of K⁺ and high phosphoenolpyruvate (PEP) concentrations, NH₄⁺ inhibited enzyme activity. Comparisons of kinetic parameters (V_max and apparent K_a) for NH₄⁺ and K⁺ with inhibition curves indicated that inhibition was very likely a result of competition of the ions for activation site(s) on the pyruvate kinase. In addition, apparent K_a (monovalent cation) and K_m (PEP) were influenced by PEP and monovalent cation concentrations, respectively. This effect may reflect a fundamental difference between plant and animal pyruvate kinases. It is concluded that control of cytosol pyruvate kinase may be closely related to reactions involved in the assimilation of NH₄⁺.     

Regulation of rat liver phosphofructokinase by glucagon-induced phosphorylation

In perfused rat liver, the effects of various hormones on the stimulation of phosphorylation and allosteric properties of purified phosphorfructokinase were investigated. Rat livers were perfused with [³²P]phosphate followed with various hormones or cyclicAMP, and ³²P-labeled phosphofructokinase was isolated. ³²P incorporation into the enzyme and enzyme inhibition by ATP or citrate were determined. Only glucagon increased the ³²P incorporation into phosphofructokinase and this increase was approximately threefold. The cyclicAMP level was increased simultaneously approximately four- to fivefold compared to the control perfused liver. Similar results were obtained by perfusing the liver with cyclicAMP (0.1 mm). The phosphorylated phosphofructokinase showed a decrease in the K_i values for ATP (from 0.4 to 0.2 mm) and citrate (from 2 to 0.6 mm). Neither epinephrine nor insulin affected the extent of phosphorylation or the allosteric properties of the enzyme. The half-maximal concentration of glucagon required for phosphorylation of phosphofructokinase and modification of its allosteric properties was approximately 6 × 10^?11m. It is concluded that glucagon increases the inhibition of liver phosphofructokinase by ATP and citrate through phosphorylation of the enzyme involving a β-receptor-mediated cyclicAMP-dependent mechanism.     

Affinity chromatography of phosphofructokinase.

The behavior of mammalian phosphofructokinase on immobilized adenine nucleotides was investigated. Three different insolubilized ligands were compared using a pure rabbit muscle phosphofructokinase. N⁶-[(6-aminohexyl)-carbamoyl-methyl]-ATP-Sepharose bound at least 90 times more enzyme than either N⁶-(6-aminohexyl)-AMP-agarose or ATP-adipic acid hydrazide-Sepharose. The elution of phosphofructokinase from the ATP-Sepharose with various metabolites and combinations of metabolites was investigated. The enzyme is eluted specifically from N⁶-[(6-aminohexyl)-carbamoyl]-ATP-Sepharose with a mixture of 25 μm each of fructose 6-phosphate and ADP (±Mg²⁺). The enzyme is not eluted either with ATP (25 μm), fructose 1,6-diphosphate (1 mm), ADP (25 μm), fructose 6-phosphate (1 mm) alone, or with a mixture of fructose 1,6-diphosphate (25 μm) and ATP (25 μm). The recovery of bound enzyme was usually greater than 90%. A mixture of glucose 6-phosphate and ADP or a mixture of IDP and fructose 6-phosphate also elutes the enzyme, but the recovery with these eluants was only about 40%. It was concluded that the “dead-end” complex is the most effective in the elution. Using this method, phosphofructokinase has been prepared in an essentially homogeneous form from muscle and brain of rabbit and rat. The overall isolation procedure involves a high speed centrifugation of crude extracts which sediments phosphofructokinase as a pellet, followed with adsorption on N⁶-[(6-aminohexyl)-carbamoyl-methyl]-ATP-Sepharose and specific elution with the mixture of fructose 6-phosphate and ADP.     

JNK and Ceramide Kinase Govern the Biogenesis of Lipid Droplets through Activation of Group IVA Phospholipase A2

The biogenesis of lipid droplets (LD) induced by serum depends on group IVA phospholipase A₂ (cPLA₂α). This work dissects the pathway leading to cPLA₂α activation and LD biogenesis. Both processes were Ca²⁺-independent, as they took place after pharmacological blockade of Ca²⁺ transients elicited by serum or chelation with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester). The single mutation D43N in cPLA₂α, which abrogates its Ca²⁺ binding capacity and translocation to membranes, did not affect enzyme activation and formation of LD. In contrast, the mutation S505A did not affect membrane relocation of the enzyme in response to Ca²⁺ but prevented its phosphorylation, activation, and the appearance of LD. Expression of specific activators of different mitogen-activated protein kinases showed that phosphorylation of cPLA₂α at Ser-505 is due to JNK. This was confirmed by pharmacological inhibition and expression of a dominant-negative form of the upstream activator MEKK1. LD biogenesis was accompanied by increased synthesis of ceramide 1-phosphate. Overexpression of its synthesizing enzyme ceramide kinase increased phosphorylation of cPLA₂α at Ser-505 and formation of LD, and its down-regulation blocked the phosphorylation of cPLA₂α and LD biogenesis. These results demonstrate that LD biogenesis induced by serum is regulated by JNK and ceramide kinase.     

Direct evidence for the participation of pyruvate in N-hydroxylation of lysine

The contribution of pyruvate to the formation of N6-acetyl-N6-hydroxylysine by a cell-free system of Aerobacter aerogenes 62-1 involved in the production of the dihydroxamate siderophore, aerobactin, has been assessed by a study of the influence of its analogs as well as of inhibitors of thiamine pyrophosphate-dependent decarboxylation reactions. These studies have provided unequivocal evidence for pyruvate functioning not only as a source of reducing equivalents in the initial step of N-hydroxylation of lysine but also as a precursor of the acetyl moiety in the subsequent conversion of the N-hydroxy amino to its N6-acetyl derivative.     

Partial Purification and Characterization of a Ca2+-Dependent Protein Kinase from the Green Alga, Dunaliella salina

A calcium-dependent protein kinase was partially purified and characterized from the green alga Dunaliella salina. The enzyme was activated at free Ca²⁺ concentrations above 10⁻⁷ molar. and half-maximal activation was at about 3 × 10⁻⁷ molar. The optimum pH for its Ca²⁺-dependent activity was 7.5. The addition of various phospholipids and diolein had no effects on enzyme activity and did not alter the sensitivity of the enzyme toward Ca²⁺. The enzyme was inhibited by calmodulin antagonists, N-(6-aminohexyl)-1-naphthalene sulfonamide and N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide in a dose-dependent manner while the protein kinase C inhibitor, sphingosine, had little effect on enzyme activity up to 800 micromolar. Immunoassay showed some calmodulin was present in the kinase preparations. However, it is unlikely the kinase was calmodulin regulated, since it still showed stimulation by Ca²⁺ in gel assays after being electrophoretically separted from calmodulin by two different methods. This gel method of detection of the enzyme indicated that a protein band with an apparent molecular weight of 40,000 showed protein kinase activity at each one of the several steps in the purification procedure. Gel assay analysis also showed that after native gel isoelectric focusing the partially purified kinase preparations had two bands with calcium-dependent activity, at isoelectric points 6.7 and 7.1. By molecular weight, by isoelectric point, and by a comparative immunoassay, the Dunaliella kinase appears to differ from at least some of the calcium-dependent, but calmodulin and phospholipid independent kinases described from higher plants.     

Synthesis and characterization of oligodeoxyribonucleotides containing a site-specifically incorporated N6-carboxymethyl-2′-deoxyadenosine or N4-carboxymethyl-2′-deoxycytidine

Humans are exposed to both endogenous and exogenous N-nitroso compounds (NOCs), and many NOCs can be metabolically activated to generate a highly reactive species, diazoacetate, which is capable of inducing carboxymethylation of nucleobases in DNA. Here we report, for the first time, the chemical syntheses of authentic N⁶-carboxymethyl-2′-deoxyadenosine (N⁶-CMdA) and N⁴-carboxymethyl-2′-deoxycytidine (N⁴-CMdC), liquid chromatography–ESI tandem MS confirmation of their formation in calf thymus DNA upon diazoacetate exposure, and the preparation of oligodeoxyribonucleotides containing a site-specifically incorporated N⁶-CMdA or N⁴-CMdC. Additionally, thermodynamic studies showed that the substitutions of a dA with N⁶-CMdA and dC with N⁴-CMdC in a 12-mer duplex increased Gibbs free energy for duplex formation at 25°C by 5.3 and 6.8 kcal/mol, respectively. Moreover, primer extension assay revealed that N⁴-CMdC was a stronger blockade to Klenow fragment-mediated primer extension than N⁶-CMdA. The polymerase displayed substantial frequency of misincorporation of dAMP opposite N⁶-CMdA and, to a lesser extent, misinsertion of dAMP and dTMP opposite N⁴-CMdC. The formation and the mutagenic potential of N⁶-CMdA and N⁴-CMdC suggest that these lesions may bear important implications in the etiology of NOC-induced tumor development.     

Construction and biochemical characterization of recombinant cytoplasmic forms of the IucD protein (lysine:N6-hydroxylase) encoded by the pColV-K30 aerobactin gene cluster.

The aerobactin gene cluster in pColV-K30 consists of five genes (iucABCD iutA); four of these (iucABCD) are involved in aerobactin biosynthesis, whereas the fifth one (iutA) encodes the ferriaerobactin outer membrane receptor. iucD encodes lysine:N6-hydroxylase, which catalyzes the first step in aerobactin biosynthesis. Regardless of the method used for cell rupture, we have consistently found that IucD remains membrane bound, and repeated efforts to achieve a purified and active soluble form of the enzyme have been unsuccessful. To circumvent this problem, we have constructed recombinant IucD proteins with modified amino termini by creating three in-frame gene fusions of IucD to the amino-terminal amino acids of the cytoplasmic enzyme beta-galactosidase. Two of these constructs resulted in the addition to the iucD coding region of a hydrophilic leader sequence of 13 and 30 amino acids. The other construct involved the deletion of the first 47 amino acids of the IucD amino terminus and the addition of 19 amino acids of the amino terminus of beta-galactosidase. Cells expressing any of the three recombinant IucD forms were found to produce soluble N6-hydroxylysine. One of these proteins, IucD439, was purified to homogeneity from the soluble fraction of the cell lysates, and it was capable of participating in the biosynthesis of aerobactin, as determined in vitro by a cell-free system and in vivo by a cross-feeding bioassay. A medium ionic strength of 0.25 (250 mM NaCl) or higher was required to maintain the protein in a catalytically functional, tetrameric state.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of S-Adenosylmethionine: Ribosomal Ribonucleic Acid-Adenine (N6-) Methyltransferase of Escherichia coli Strain B

This study is concerned with the isolation and characterization of the enzyme, S-adenosylmethionine:ribosomal ribonucleic acid-adenine (N⁶⁻) methyl-transferase [rRNA-adenine (N⁶-) methylase] of Escherichia coli strain B, which is responsible for the formation of N⁶-methyladenine moieties in ribosomal ribonucleic acids (rRNA). A 1,500-fold purified preparation of the species-specific methyltransferase methylates a limited number of adenine moieties in heterologous rRNA (Micrococcus lysodeikticus and Bacillus subtilis) and methyl-deficient homologous rRNA. The site recognition mechanism does not require intact 16 or 23S rRNA. The enzyme does not utilize transfer ribonucleic acid as a methyl acceptor nor does it synthesize 2-methyladenine or N⁶-dimethyladenine moieties. Mg²⁺, spermine, K⁺, and Na⁺ increase the reaction rate but not the extent of methylation; elevated concentrations of the cations inhibit markedly. The purified preparations utilize 9-β-ribosyl-2,6-diaminopurine (DAPR) as a methyl acceptor with the synthesis of 9-β-ribosyl-6-amino-2-methylaminopurine. A comparison of the two activities demonstrated that one methyltransferase is responsible for the methylation of both DAPR and rRNA. This property provides a sensitive assay procedure unaffected by ribonucleases and independent of any specificity exhibited by rRNA methyl acceptors.     

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 (k_cat = 8.3 s⁻¹; K_m = 14.1 and 4.3 μM for acetyl coenzyme A and oxaloacetate, respectively). The enzyme was dimeric and was slightly inhibited by ATP (K_i = 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.