Characterization of the acyl substrate binding pocket of acetyl-CoA synthetase

AMP-forming acetyl-CoA synthetase [ACS; acetate:CoA ligase (AMP-forming), EC 6.2.1.1] catalyzes the activation of acetate to acetyl-CoA in a two-step reaction. This enzyme is a member of the adenylate-forming enzyme superfamily that includes firefly luciferase, nonribosomal peptide synthetases, and acyl- and aryl-CoA synthetases/ligases. Although the structures of several superfamily members demonstrate that these enzymes have a similar fold and domain structure, the low sequence conservation and diversity of the substrates utilized have limited the utility of these structures in understanding substrate binding in more distantly related enzymes in this superfamily. The crystal structures of the Salmonella enterica ACS and Saccharomyces cerevisiae ACS1 have allowed a directed approach to investigating substrate binding and catalysis in ACS. In the S. enterica ACS structure, the propyl group of adenosine 5'-propylphosphate, which mimics the acyl-adenylate intermediate, lies in a hydrophobic pocket. Modeling of the Methanothermobacter thermautotrophicus Z245 ACS (MT-ACS1) on the S. cerevisiae ACS structure showed similar active site architecture, and alignment of the amino acid sequences of proven ACSs indicates that the four residues that compose the putative acetate binding pocket are well conserved. These four residues, Ile312, Thr313, Val388, and Trp416 of MT-ACS1, were targeted for alteration, and our results support that they do indeed form the acetate binding pocket and that alterations at these positions significantly alter the enzyme's affinity for acetate as well as the range of acyl substrates that can be utilized. In particular, Trp416 appears to be the primary determinant for acyl chain length that can be accommodated in the binding site.     

The 1.75 A crystal structure of acetyl-CoA synthetase bound to adenosine-5'-propylphosphate and coenzyme A

Acetyl-coenzyme A synthetase catalyzes the two-step synthesis of acetyl-CoA from acetate, ATP, and CoA and belongs to a family of adenylate-forming enzymes that generate an acyl-AMP intermediate. This family includes other acyl- and aryl-CoA synthetases, firefly luciferase, and the adenylation domains of the modular nonribosomal peptide synthetases. We have determined the X-ray crystal structure of acetyl-CoA synthetase complexed with adenosine-5'-propylphosphate and CoA. The structure identifies the CoA binding pocket as well as a new conformation for members of this enzyme family in which the approximately 110-residue C-terminal domain exhibits a large rotation compared to structures of peptide synthetase adenylation domains. This domain movement presents a new set of residues to the active site and removes a conserved lysine residue that was previously shown to be important for catalysis of the adenylation half-reaction. Comparison of our structure with kinetic and structural data of closely related enzymes suggests that the members of the adenylate-forming family of enzymes may adopt two different orientations to catalyze the two half-reactions. Additionally, we provide a structural explanation for the recently shown control of enzyme activity by acetylation of an active site lysine.     

AMP-forming acetyl-CoA synthetases in Archaea show unexpected diversity in substrate utilization

Adenosine monophosphate (AMP)-forming acetyl-CoA synthetase (ACS; acetate:CoA ligase (AMP-forming), EC 6.2.1.1) is a key enzyme for conversion of acetate to acetyl-CoA, an essential intermediate at the junction of anabolic and catabolic pathways. Phylogenetic analysis of putative short and medium chain acyl-CoA synthetase sequences indicates that the ACSs form a distinct clade from other acyl-CoA synthetases. Within this clade, the archaeal ACSs are not monophyletic and fall into three groups composed of both bacterial and archaeal sequences. Kinetic analysis of two archaeal enzymes, an ACS from Methanothermobacter thermautotrophicus (designated as MT-ACS1) and an ACS from Archaeoglobus fulgidus (designated as AF-ACS2), revealed that these enzymes have very different properties. MT-ACS1 has nearly 11-fold higher affinity and 14-fold higher catalytic efficiency with acetate than with propionate, a property shared by most ACSs. However, AF-ACS2 has only 2.3-fold higher affinity and catalytic efficiency with acetate than with propionate. This enzyme has an affinity for propionate that is almost identical to that of MT-ACS1 for acetate and nearly tenfold higher than the affinity of MT-ACS1 for propionate. Furthermore, MT-ACS1 is limited to acetate and propionate as acyl substrates, whereas AF-ACS2 can also utilize longer straight and branched chain acyl substrates. Phylogenetic analysis, sequence alignment and structural modeling suggest a molecular basis for the altered substrate preference and expanded substrate range of AF-ACS2 versus MT-ACS1.     

Purification and characterization of two reversible and ADP-dependent acetyl coenzyme A synthetases from the hyperthermophilic archaeon Pyrococcus furiosus.

Pyrococcus furiosus is a strictly anaerobic archaeon (archaebacterium) that grows at temperatures up to 105 degrees C by fermenting carbohydrates and peptides. Cell extracts have been previously shown to contain an unusual acetyl coenzyme A (acetyl-CoA) synthetase (ACS) which catalyzes the formation of acetate and ATP from acetyl-CoA by using ADP and phosphate rather than AMP and PPi. We show here that P. furiosus contains two distinct isoenzymes of ACS, and both have been purified. One, termed ACS I, uses acetyl-CoA and isobutyryl-CoA but not indoleacetyl-CoA or phenylacetyl-CoA as substrates, while the other, ACS II, utilizes all four CoA derivatives. Succinyl-CoA did not serve as a substrate for either enzyme. ACS I and ACS II have similar molecular masses (approximately 140 kDa), and both appear to be heterotetramers (alpha2beta2) of two different subunits of 45 (alpha) and 23 (beta) kDa. They lack metal ions such as Fe2+, Cu2+, Zn2+, and Mg2+ and are stable to oxygen. At 25 degrees C, both enzymes were virtually inactive and exhibited optimal activities above 90 degrees C (at pH 8.0) and at pH 9.0 (at 80 degrees C). The times required to lose 50% of their activity at 80 degrees C were about 18 h for ACS I and 8 h for ACS II. With both enzymes in the acid formation reactions, ADP and phosphate could be replaced by GDP and phosphate but not by CDP and phosphate or by AMP and PPi. The apparent Km values for ADP, GDP, and phosphate were approximately 150, 132, and 396 microM, respectively, for ACS I (using acetyl-CoA) and 61, 236, and 580 microM, respectively, for ACS II (using indoleacetyl-CoA). With ADP and phosphate as substrates, the apparent Km values for acetyl-CoA and isobutyryl-CoA were 25 and 29 microM, respectively, for ACS I and 26 and 12 microM, respectively, for ACS II. With ACS II, the apparent Km value for phenylacetyl-CoA was 4 microM. Both enzymes also catalyzed the reverse reaction, the ATP-dependent formation of the CoA derivatives of acetate (I and II), isobutyrate (I and II), phenylacetate (II only), and indoleacetate (II only). The N-terminal amino acid sequences of the two subunits of ACS I were similar to those of ACS II and to that of a hypothetical 67-kDa protein from Escherichia coli but showed no similarity to mesophilic ACS-type enzymes. To our knowledge, ACS I and II are the first ATP-utilizing enzymes to be purified from a hyperthermophile, and ACS II is the first enzyme of the ACS type to utilize aromatic CoA derivatives.     

Studies on Acetyl-Coenzyme A Synthetase of Yeast: Inhibition by Long-Chain Acyl-Coenzyme A Esters

Long-chain acyl-coenzyme A (CoA) compounds (palmityl, stearyl, and oleyl) were found to be potent inhibitors of acetyl-CoA synthetase (ACS) of Saccharomyces cerevisiae strain LK2G12 from aerobic, but not from nonaerobic, cells. The effectiveness of the inhibitors of the aerobic enzyme was in the following order: palmityl-CoA < stearyl-CoA < oleyl-CoA. Short-chain acyl-CoA compounds (propionyl, butyryl, and valeryl) and long-chain fatty acids had no effect on ACS from either source. The inhibition by oleyl-CoA was found to be dependent on enzyme concentration, whereas the inhibition by palmityl- and stearyl-CoA was independent of ACS concentration. Inhibition by palmityl-CoA was noncompetitive with respect to both acetate and CoA, and with increasing concentration of inhibitor the pattern was sigmoidal, with a Hill value of 3.24. At maximally inhibitory concentrations of palmityl-CoA, a small amount of enzyme activity remained. This noninhibitable enzyme in aerobic cells was shown not to be of nonaerobic origin.     

Characterization of an Archaeal Medium-Chain Acyl Coenzyme A Synthetase from Methanosarcina acetivorans

Short- and medium-chain acyl coenzyme A (acyl-CoA) synthetases catalyze the formation of acyl-CoA from an acyl substrate, ATP, and CoA. These enzymes catalyze mechanistically similar two-step reactions that proceed through an enzyme-bound acyl-AMP intermediate. Here we describe the characterization of a member of this enzyme family from the methane-producing archaeon Methanosarcina acetivorans. This enzyme, a medium-chain acyl-CoA synthetase designated Macs_Ma, utilizes 2-methylbutyrate as its preferred substrate for acyl-CoA synthesis but cannot utilize acetate and thus cannot catalyze the first step of acetoclastic methanogenesis in M. acetivorans. When propionate or other less favorable acyl substrates, such as butyrate, 2-methylpropionate, or 2-methylvalerate, were utilized, the acyl-CoA was not produced or was produced at reduced levels. Instead, acyl-AMP and PP_i were released in the absence of CoA, whereas in the presence of CoA, the intermediate was broken down into AMP and the acyl substrate, which were released along with PP_i. These results suggest that although acyl-CoA synthetases may have the ability to utilize a broad range of substrates for the acyl-adenylate-forming first step of the reaction, the intermediate may not be suitable for the thioester-forming second step. The Macs_Ma structure has revealed the putative acyl substrate- and CoA-binding pockets. Six residues proposed to form the acyl substrate-binding pocket, Lys²⁵⁶, Cys²⁹⁸, Gly³⁵¹, Trp²⁵⁹, Trp²³⁷, and Trp²⁵⁴, were targeted for alteration. Characterization of the enzyme variants indicates that these six residues are critical in acyl substrate binding and catalysis, and even conservative alterations significantly reduced the catalytic ability of the enzyme.AMP-forming acetyl coenzyme A (acetyl-CoA) synthetase (Acs; acetate:CoA ligase [AMP forming], EC 6.2.1.1), which catalyzes the activation of acetate to acetyl-CoA, is a member of the acyl-adenylate-forming enzyme superfamily (8), which consists of acyl- and aryl-CoA ligases, nonribosomal peptide synthetases that mediate the synthesis of peptide and polyketide secondary metabolites, such as gramicidin and tyrocidine, and the enzymes firefly luciferase and α-aminoadipate reductase. Although these enzymes share the property of forming an acyl-adenylate intermediate and are structurally related, they share limited sequence homology and catalyze unrelated reactions in which the intermediate serves different functions for different members of this enzyme family.A two-step mechanism for Acs (equations 1 and 2) in which the reaction proceeds through an acetyl-AMP intermediate has been proposed based on evidence including detection of an enzyme-bound acetyl-AMP (2-4, 38): (1) (2)In the CoA-dependent first step of the reaction, an enzyme-bound acetyl-AMP intermediate is formed from acetate and ATP, and inorganic pyrophosphate (PP_i) is released. In the second step, the acetyl group is transferred to the sulfhydryl group of CoA and AMP is released. Other short- (Sacs) and medium-chain acyl-CoA synthetases (Macs) follow a similar reaction mechanism using acyl substrates other than acetate (8, 15).In the 2.3-Å structure of trimeric Saccharomyces cerevisiae Acs1 in a binary complex with AMP (19), the C-terminal domain is positioned away from the N-terminal domain in a conformation for catalysis of the first step of the reaction (equation 1). The 1.75-Å structure of the monomeric Salmonella enterica Acs (Acs_Se) (13) in complex with both CoA and adenosine-5′-propylphosphate, an inhibitor of the related propionyl-CoA synthetase (12, 15), which mimics the acetyl-adenylate intermediate, reveals that the C-terminal domain of Acs is rotated approximately 140° toward the N-terminal domain to form the complete active site for catalysis of the second half-reaction (equation 2). In this orientation, the CoA thiol is properly positioned for nucleophilic attack on the acetyl group. In structure/function studies of 4-chlorobenzoate:CoA ligase (CBAL), a distant member of the acyl- and aryl-CoA synthetase subfamily of the acyl-adenylate-forming enzyme superfamily, Wu et al. (39) and Reger et al. (28) provide evidence that PP_i produced in the first step of the reaction dissociates from the enzyme before the switch from the first conformation to the second conformation required for CoA binding and catalysis of the second step of the reaction.Acs and Sacs/Macs are widespread in all three domains of life and play a key role in archaea, as suggested by the finding that several thermophilic archaea have multiple open reading frames (ORFs) (up to seven) that encode putative Sacs or Macs (33). The chemolithoautotrophic methanoarchaeon Methanothermobacter thermautotrophicus has two ORFs with high identity to Acs and a third ORF that is likely to encode a Macs. M. thermautotrophicus Acs1 (Acs1_Mt) has been biochemically and kinetically characterized, has been shown to have a strong preference for acetate as the acyl substrate, and can also utilize propionate but not butyrate (16, 17).Methanosarcina and Methanosaeta are the only two methanoarchaea isolated that are able to utilize acetate as substrate for methane production. Unlike Methanosaeta species, which utilize Acs for catalyzing the first step of methanogenesis (18, 34), Methanosarcina species employ the acetate kinase-phosphotransacetylase pathway for activation of acetate to acetyl-CoA, and an Acs activity has not been observed in Methanosarcina (1, 23, 30, 32). Surprisingly, an Acs-related sequence was identified in the Methanosarcina acetivorans genome. Here we describe the kinetic characterization this enzyme, designated Macs_Ma, and show that it utilizes longer acyl substrates than Acs. The preferred acyl substrate was shown to be 2-methylbutyrate, and 2-methylbutyryl-CoA, AMP, and PP_i were the products of the reaction, as expected. However, when propionate was used as the acyl substrate, propionyl-CoA was not produced. Instead, in the absence of CoA, propionyl-AMP and PP_i were released, whereas in the presence of CoA, the propionyl-AMP intermediate was broken down into AMP and propionate and released along with PP_i. Intermediate results were obtained with other acyl substrates, with both acyl-CoA and acyl-AMP production observed.The 2.1-Å crystal structure of Macs_Ma (31), determined in the absence of any substrate, revealed the enzyme to be in a conformation similar to that of the S. enterica Acs (13) with respect to the position of the C-terminal domain. Through inspection of the Macs_Ma structure and alignment of Acs, Sacs, and Macs sequences, we identified six residues that form the putative acyl substrate-binding pocket. Individual alterations at these residues dramatically diminished enzyme activity and indicate that the acyl substrate-binding pocket of Macs_Ma has a very precise architecture that cannot be perturbed.     

Reaction mechanism and structural model of ADP-forming Acetyl-CoA synthetase from the hyperthermophilic archaeon Pyrococcus furiosus: evidence for a second active site histidine residue

In Archaea, acetate formation and ATP synthesis from acetyl-CoA is catalyzed by an unusual ADP-forming acetyl-CoA synthetase (ACD) (acetyl-CoA + ADP + P(i) acetate + ATP + HS-CoA) catalyzing the formation of acetate from acetyl-CoA and concomitant ATP synthesis by the mechanism of substrate level phosphorylation. ACD belongs to the protein superfamily of nucleoside diphosphate-forming acyl-CoA synthetases, which also include succinyl-CoA synthetases (SCSs). ACD differs from SCS in domain organization of subunits and in the presence of a second highly conserved histidine residue in the beta-subunit, which is absent in SCS. The influence of these differences on structure and reaction mechanism of ACD was studied with heterotetrameric ACD (alpha(2)beta(2)) from the hyperthermophilic archaeon Pyrococcus furiosus in comparison with heterotetrameric SCS. A structural model of P. furiosus ACD was constructed suggesting a novel spatial arrangement of the subunits different from SCS, however, maintaining a similar catalytic site. Furthermore, kinetic and molecular properties and enzyme phosphorylation as well as the ability to catalyze arsenolysis of acetyl-CoA were studied in wild type ACD and several mutant enzymes. The data indicate that the formation of enzyme-bound acetyl phosphate and enzyme phosphorylation at His-257alpha, respectively, proceed in analogy to SCS. In contrast to SCS, in ACD the phosphoryl group is transferred from the His-257alpha to ADP via transient phosphorylation of a second conserved histidine residue in the beta-subunit, His-71beta. It is proposed that ACD reaction follows a novel four-step mechanism including transient phosphorylation of two active site histidine residues:     

Functional analyses of two acetyl coenzyme A synthetases in the ascomycete Gibberella zeae

Acetyl coenzyme A (acetyl-CoA) is a crucial metabolite for energy metabolism and biosynthetic pathways and is produced in various cellular compartments with spatial and temporal precision. Our previous study on ATP citrate lyase (ACL) in Gibberella zeae revealed that ACL-dependent acetyl-CoA production is important for histone acetylation, especially in sexual development, but is not involved in lipid synthesis. In this study, we deleted additional acetyl-CoA synthetic genes, the acetyl-CoA synthetases (ACS genes ACS1 and ACS2), to identify alternative acetyl-CoA production mechanisms for ACL. The ACS1 deletion resulted in a defect in sexual development that was mainly due to a reduction in 1-palmitoyl-2-oleoyl-3-linoleoyl-rac-glycerol production, which is required for perithecium development and maturation. Another ACS coding gene, ACS2, has accessorial functions for ACS1 and has compensatory functions for ACL as a nuclear acetyl-CoA producer. This study showed that acetate is readily generated during the entire life cycle of G. zeae and has a pivotal role in fungal metabolism. Because ACSs are components of the pyruvate-acetaldehyde-acetate pathway, this fermentation process might have crucial roles in various physiological processes for filamentous fungi.     

Mammalian long-chain acyl-CoA synthetases

Acyl-CoA synthetase enzymes are essential for de novo lipid synthesis, fatty acid catabolism, and remodeling of membranes. Activation of fatty acids requires a two-step reaction catalyzed by these enzymes. In the first step, an acyl-AMP intermediate is formed from ATP. AMP is then exchanged with CoA to produce the activated acyl-CoA. The release of AMP in this reaction defines the superfamily of AMP-forming enzymes. The length of the carbon chain of the fatty acid species defines the substrate specificity for the different acyl-CoA synthetases (ACS). On this basis, five sub-families of ACS have been characterized. The purpose of this review is to report on the large family of mammalian long-chain acyl-CoA synthetases (ACSL), which activate fatty acids with chain lengths of 12 to 20 carbon atoms. Five genes and several isoforms generated by alternative splicing have been identified and limited information is available on their localization. The structure of these membrane proteins has not been solved for the mammalian ACSLs but homology to a bacterial form, whose structure has been determined, points at specific structural features that are important for these enzymes across species. The bacterial form acts as a dimer and has a conserved short motif, called the fatty acid Gate domain, that seems to determine substrate specificity. We will discuss the characterization and identification of the different spliced isoforms, draw attention to the inconsistencies and errors in their annotations, and their cellular localizations. These membrane proteins act on membrane-bound substrates probably as homo- and as heterodimer complexes but have often been expressed as single recombinant isoforms, apparently purified as monomers and tested in Triton X-100 micelles. We will argue that such studies have failed to provide an accurate assessment of the activity and of the distinct function of these enzymes in mammalian cells.     

Enhanced isoamyl acetate production upon manipulation of the acetyl-CoA node in Escherichia coli

Coenzyme A (CoA) and its thioester derivative acetyl-Coenzyme A (acetyl-CoA) participate in over 100 different reactions in intermediary metabolism of microorganisms. Earlier results indicated that overexpression of upstream rate-limiting enzyme pantothenate kinase with simultaneous supplementation of precursor pantothenic acid to the culture media increased intracellular CoA levels significantly ( approximately 10-fold). The acetyl-CoA levels also increased ( approximately 5-fold) but not as much as that of CoA, showing that the carbon flux from the pyruvate node is rate-limiting upon an increase in CoA levels. In this study, pyruvate dehydrogenase was overexpressed under elevated CoA levels to increase carbon flux from pyruvate to acetyl-CoA. This coexpression did not increase intracellular acetyl-CoA levels but increased the accumulation of extracellular acetate. The production of isoamyl acetate, an industrially useful compound derived from acetyl-CoA, was used as a model reporter system to signify the beneficial effects of this metabolic engineering strategy. In addition, a strain was created in which the acetate production pathway was inactivated to relieve competition at the acetyl-CoA node and to efficiently channel the enhanced carbon flux to the ester production pathway. The synergistic effect of cofactor CoA manipulation and pyruvate dehydrogenase overexpression in the acetate pathway deletion mutant led to a 5-fold increase in isoamyl acetate production. Under normal growth conditions the acetate pathway deletion mutant strains accumulate intracellular pyruvate, leading to excretion of pyruvate. However, upon enhancing the carbon flux from pyruvate to acetyl-CoA, the excretion of pyruvate was significantly reduced.     

Acetyl-coenzyme A synthetase in the thermophilic, acetate-utilizing methanogen Methanothrix sp. strain CALS-1

Abstract There is considerable evidence that acetyl-CoA synthetase (acetate thiokinase, ACS, EC 6.2.1) is responsible for acetate activation in the mesophilic acetotrophic methanogen Methanothrix soehngenii . If the pyrophosphate produced by ACS is simply cleaved, two high-energy phosphodiester bonds are expended in acetate activation. Hi High ACS activity (2–4 μmol min⁻¹ mg protein⁻¹) was present in cell-free extracts of the thermophile Methanothrix sp. strain CALS-1. The 23-fold purified enzyme had a molecular mass near 165 kDa and a subunit molecular mass near 78 kDa, suggesting that the enzyme is a homodimer. The temperature optimum for ACS was near 70°C and the apparent K _m values were 2–4 mM for acetate and 5.5 mM for MgATP. Coenzyme A at concentrations greater than 0.2 mM inhibited ACS, while acetyl-CoA was not inhibitory. AMP and pyrophosphate inhibited ACS with K _i values of 5 mM and 1.5 mM respectively. Other divalent cations could replace Mg²⁺, with Mn²⁺ showing the highest activity. Activity with ITP was 20% of that with ATP, and other nucleotides tested were considerably less active. Since Methanothrix sp. strain CALS-1 has an active soluble pyrophosphatase, it appears that it uses the same energetically costly method for acetate activation as M. soehngenii .     

An energy-conserving pyruvate-to-acetate pathway in Entamoeba histolytica. Pyruvate synthase and a new acetate thiokinase.

Under anaerobic conditions, cells of Entamoeba histolytica grown with bacteria produce H2 and acetate while cells grown axenically produce neither. Aerobically, acetate is produced and O2 is consumed by amebae from either type of cells. Centrifuged extracts, 2.4 x 106 x g x min, from both types of cells contain pyruvate synthase (EC 1.2.7.1) and an acetate thiokinase which, together, form a system capable of converting pyruvate to acetate. Pyruvate synthase catalyzes the reaction: pyruvate + CoA leads to CO2 + acetyl-CoA + 2E. Electron acceptors which function with this enzyme are FAD, FMN, riboflavin, ferredoxin, and methyl viologen, but not NAD or NADP. The amebal acetate thiokinase catalyzes the reaction acetyl-CoA + ADP + Pi leads to acetate + ATP + CoA. For this apparently new enzyme we suggest the trivial name acetyl-CoA-synthetase (ADP-forming). Extracts from axenic amebae do not contain hydrogenase, but extracts from cells grown with bacteria do. It is postulated that in bacteria-grown amebae electrons generated at the pyruvate synthase step are utilized anaerobically to produce H2 via the hydrogenase and that the acetyl-CoA is converted to acetate in an energy-conserving step catalyzed by amebal acetyl-CoA synthetase. Aerobically, cells grown under either regimen may utilize the energy-conserving pyruvate-to-acetate pathway since O2 then serves as the ultimate electron acceptor.     

Catalytic coupling of the active sites in acetyl-CoA synthase, a bifunctional CO-channeling enzyme.

Acetogenic bacteria contain acetyl-CoA synthase (ACS), an enzyme with two distinct nickel-iron-sulfur active sites connected by a tunnel through which CO migrates. One site reduces CO2 to CO, while the other synthesizes acetyl-CoA from CO, CoA, and the methyl group of another protein (CH3-CP). Rapid binding of CO2 and a two-electron reduction activates ACS. When CoA and CH3-CP bind ACS, CO is rerouted through the tunnel to the synthase site, and kinetic parameters at the reductase site are altered. Under these conditions, the rates of CO2 reduction and acetyl-CoA synthesis are synchronized by an ordered catalytic mechanism.     

Cofactor engineering of intracellular CoA/acetyl-CoA and its effect on metabolic flux redistribution in Escherichia coli

Coenzyme A (CoA) and its thioester derivatives are important cofactors participating in over 100 different reactions in intermediary metabolism of microorganisms. The time profiles of intracellular CoA and acetyl-CoA levels were studied in an aerobic batch reactor. The CoA level starts at a high value and falls off gradually over the exponential and stationary growth phases, reaching negligible levels at the end of 24h. The acetyl-CoA level, on the other hand, increases initially reaching a maximum and decreases gradually reaching negligible levels after 24h. Overexpressing one of the upstream rate-controlling enzyme the pantothenate kinase with simultaneous supplementation of the precursor pantothenic acid to the culture medium increased the intracellular CoA/acetyl-CoA levels. It was found that supplementation of the precursor pantothenic acid is essential to increase CoA/acetyl-CoA levels. A 10-fold increase in CoA level was observed upon this overexpression in complex medium. Acetyl-CoA levels also increased (5-fold) but not as much as CoA, leaving much of the CoA in free unacetylated form. The increase in intracellular CoA/acetyl-CoA levels led to an increase in carbon flux to the acetate production pathway leading to formation of more acetate in complex medium, whereas no such change in metabolite redistribution was observed in minimal medium.     

Catabolic and anabolic enzyme activities and energetics of acetone metabolism of the sulfate-reducing bacterium Desulfococcus biacutus.

Acetone degradation by cell suspensions of Desulfococcus biacutus was CO2 dependent, indicating initiation by a carboxylation reaction, while degradation of 3-hydroxybutyrate was not CO2 dependent. Growth on 3-hydroxybutyrate resulted in acetate accumulation in the medium at a ratio of 1 mol of acetate per mol of substrate degraded. In acetone-grown cultures no coenzyme A (CoA) transferase or CoA ligase appeared to be involved in acetone metabolism, and no acetate accumulated in the medium, suggesting that the carboxylation of acetone and activation to acetoacetyl-CoA may occur without the formation of a free intermediate. Catabolism of 3-hydroxybutyrate occurred after activation by CoA transfer from acetyl-CoA, followed by oxidation to acetoacetyl-CoA. In both acetone-grown cells and 3-hydroxybutyrate-grown cells, acetoacetyl-CoA was thioyltically cleaved to two acetyl-CoA residues and further metabolized through the carbon monoxide dehydrogenase pathway. Comparison of the growth yields on acetone and 3-hydroxybutyrate suggested an additional energy requirement in the catabolism of acetone. This is postulated to be the carboxylation reaction (delta G(o)' for the carboxylation of acetone to acetoacetate, +17.1 kJ.mol-1). At the intracellular acyl-CoA concentrations measured, the net free energy change of acetone carboxylation and catabolism to two acetyl-CoA residues would be close to 0 kJ.mol of acetone-1, if one mol of ATP was invested. In the absence of an energy-utilizing step in this catabolic pathway, the predicted intracellular acetoacetyl-CoA concentration would be 10(13) times lower than that measured. Thus, acetone catabolism to two acetyl-CoA residues must be accompanied by the utilization of teh energetic equivalent of (at lease) one ATP molecule. Measurement of enzyme activities suggested that assimilation of acetyl-CoA occurred through a modified citric acid cycle in which isocitrate was cleaved to succinate and glyoxylate. Malate synthase, condensing glyoxylate and acetyl-CoA, acted as an anaplerotic enzyme. Carboxylation of pyruvate of phosphoenolpyruvate could not be detected.     

AMP-forming acetyl-CoA synthetase from the extremely halophilic archaeon Haloarcula marismortui: purification, identification and expression of the encoding gene, and phylogenetic affiliation

Halophilic archaea activate acetate via an (acetate)-inducible AMP-forming acetyl-CoA synthetase (ACS), (Acetate + ATP + CoA Acetyl-CoA + AMP + PP_i). The enzyme from Haloarcula marismortui was purified to homogeneity. It constitutes a 72-kDa monomer and exhibited a temperature optimum of 41°C and a pH optimum of 7.5. For optimal activity, concentrations between 1 M and 1.5 M KCl were required, whereas NaCl had no effect. The enzyme was specific for acetate (100%) additionally accepting only propionate (30%) as substrate. The kinetic constants were determined in both directions of the reaction at 37°C. Using the N-terminal amino acid sequence an open reading frame — coding for a 74 kDa protein — was identified in the partially sequenced genome of H. marismortui. The function of the ORF as acs gene was proven by functional overexpression in Escherichia coli. The recombinant enzyme was reactivated from inclusion bodies, following solubilization in urea and refolding in the presence of salts, reduced and oxidized glutathione and substrates. Refolding was dependent on salt concentrations of at least 2 M KCl. The recombinant enzyme showed almost identical molecular and catalytic properties as the native enzyme. Sequence comparison of the Haloarcula ACS indicate high similarity to characterized ACSs from bacteria and eukarya and the archaeon Methanosaeta. Phylogenetic analysis of ACS sequences from all three domains revealed a distinct archaeal cluster suggesting monophyletic origin of archaeal ACS.     

Crystal structure of DltA. Implications for the reaction mechanism of non-ribosomal peptide synthetase adenylation domains

DltA, the D-alanine:D-alanyl carrier protein ligase responsible for the initial step of lipoteichoic acid D-alanylation in Gram-positive bacteria, belongs to the adenylation domain superfamily, which also includes acetyl-CoA synthetase and the adenylation domains of non-ribosomal synthetases. The two-step reaction catalyzed by these enzymes (substrate adenylation followed by transfer to the reactive thiol group of CoA or the phosphopantheinyl prosthetic group of peptidyl carrier proteins) has been suggested to proceed via large scale rearrangements of structural domains within the enzyme. The structures of DltA reported here reveal the determinants for D-Ala substrate specificity and confirm that the peptidyl carrier protein-activating domains are able to adopt multiple conformational states, in this case corresponding to the thiolation reaction. Comparisons of available structures allow us to propose a mechanism whereby small perturbations of finely balanced metastable structural states would be able to direct an ordered formation of non-ribosomal synthetase products.     

Demonstration of carbon-carbon bond cleavage of acetyl coenzyme A by using isotopic exchange catalyzed by the CO dehydrogenase complex from acetate-grown Methanosarcina thermophila.

The purified nickel-containing CO dehydrogenase complex isolated from methanogenic Methanosarcina thermophila grown on acetate is able to catalyze the exchange of [1-14C] acetyl-coenzyme A (CoA) (carbonyl group) with 12CO as well as the exchange of [3'-32P]CoA with acetyl-CoA. Kinetic parameters for the carbonyl exchange have been determined: Km (acetyl-CoA) = 200 microM, Vmax = 15 min-1. CoA is a potent inhibitor of this exchange (Ki = 25 microM) and is formed under the assay conditions because of a slow but detectable acetyl-CoA hydrolase activity of the enzyme. Kinetic parameters for both exchanges are compared with those previously determined for the acetyl-CoA synthase/CO dehydrogenase from the acetogenic Clostridium thermoaceticum. Collectively, these results provide evidence for the postulated role of CO dehydrogenase as the key enzyme for acetyl-CoA degradation in acetotrophic bacteria.     

Applicability of CoA/acetyl-CoA manipulation system to enhance isoamyl acetate production in Escherichia coli

Coenzyme A (CoA) and its thioester derivatives are important precursor molecules for many industrially useful compounds such as esters, PHBs, lycopene and polyketides. Previously, in our lab we could increase the intracellular levels of CoA and acetyl-Coenzyme A (acetyl-CoA) by overexpressing one of the upstream rate-controlling enzymes pantothenate kinase with a concomitant supplementation of the precursor pantothenic acid to the cell culture medium. In this study, we showed that the CoA/acetyl-CoA manipulation system could be used to increase the productivity of industrially useful compounds derived from acetyl-CoA. We chose the production of isoamyl acetate as a model system. Isoamyl acetate is an important flavor component of sake yeast and holds a great commercial value. Alcohol acetyl transferase (AAT) condenses isoamyl alcohol and acetyl-CoA to produce isoamyl acetate. The gene ATF2, coding for this AAT was cloned and expressed in Escherichia coli. This genetic engineered E. coli produces isoamyl acetate, an ester, from intracellular acetyl-CoA when isoamyl alcohol is added externally to the cell culture medium. In the current study, we showed that in a strain bearing ATF2 gene, an increase in intracellular CoA/acetyl-CoA by overexpressing panK leads to an increase in isoamyl acetate production. Additionally, the cofactor manipulation technique was combined with more traditional approach of competing pathway deletions to further increase isoamyl acetate production. The acetate production pathway competes with isoamyl acetate production for the common intracellular metabolite acetyl-CoA. Earlier we have shown that acetate pathway deletion (ackA-pta) increases isoamyl acetate production. The acetate production pathway was inactivated under elevated CoA/acetyl-CoA conditions, which lead to a further increase in isoamyl acetate production.