A Specialized Citric Acid Cycle Requiring Succinyl-Coenzyme A (CoA):Acetate CoA-Transferase (AarC) Confers Acetic Acid Resistance on the Acidophile Acetobacter aceti

The characteristic ability of acetic acid bacteria to aerobically oxidize ethanol to acetic acid has been harnessed for millennia to produce vinegar. Acetic acid permeates cell membranes at low pH and generally inhibits bacterial growth at low millimolar concentrations. The strains used for vinegar production, however, thrive in near-molar concentrations. This remarkable resistance results from the combined contributions of several molecular mechanisms. This study examines the inherent acid stability of proteins from the industrial vinegar-production strain Acetobacter aceti 1023 and the process by which cytoplasmic acetic acid is overoxidized to carbon dioxide. Acetate overoxidation in A. aceti strain 1023 is catalyzed by a variant citric acid cycle: CAC) that lacks succinyl-coenzyme A: CoA) synthetase. The acetic-acid-resistance protein succinyl-CoA:acetate CoA-transferase: SCACT, AarC) circumvents this deficiency and bypasses substrate-level phosphorylation and/or adenylation of acetate. Continuous acetate dissimilation by this specialized CAC is dependent upon only favorable oxidation of reduced cofactors. Biphasic growth of A. aceti strain 1023 in yeast extract-peptone-dextrose-ethanol medium is accompanied by distinct stages of acetate production, conservation, and depletion. Acetate is initially accumulated as ethanol is oxidized in the first log phase, transiently maintained in the first stationary phase, and ultimately consumed in the second log phase. The high levels of AarC and SCACT activity present prior to the acetate depletion phase suggest that regulation of CAC enzyme levels is not the only mechanism by which premature acetate overoxidation is avoided. Acetate catabolism may be further minimized during ethanol oxidation by the maintenance of a low cytoplasmic acetate concentration. A. aceti employs multiple means of active acetic acid efflux which may be driven by the energetically favorable oxidation of ethanol. Selective pressure to function in a primary metabolic role increased the specificity and catalytic rapidity of AarC relative to other class I CoA-transferases. Catalysis relies upon a novel oxyanion hole configuration composed partly of the distal amide nitrogen of CoA to stabilize tetrahedral oxyanion intermediates. Structural alignments suggest this mechanism is employed by all class I enzymes. Favorable hydrogen-bonding and electrostatic interactions between the protein and the diphosphate moiety of CoA induce a protein conformational change that was previously predicted to accelerate CoA transfer. This motion is influenced by an auxiliary binding site that preconcentrates carboxylate substrates

Functional analysis of the acetic acid resistance (aar) gene cluster in Acetobacter aceti strain 1023

Vinegar production requires acetic acid bacteria that produce, tolerate, and conserve high levels of acetic acid. When ethanol is depleted, aerobic acetate overoxidation to carbon dioxide ensues. The resulting diauxic growth pattern has two logarithmic growth phases, the first associated with ethanol oxidation and the second associated with acetate overoxidation. The vinegar factory isolate Acetobacter aceti strain 1023 has a long intermediate stationary phase that persists at elevated acetic acid levels. Strain 1023 conserves acetic acid despite possessing a complete set of citric acid cycle (CAC) enzymes, including succinyl-CoA:acetate CoA-transferase (SCACT), the product of the acetic acid resistance (aar) gene aarC. In this study, cell growth and acid production were correlated with the functional expression of aargenes using reverse transcription-polymerase chain reaction, Western blotting, and enzyme activity assays. Citrate synthase (AarA) and SCACT (AarC) were abundant in A. aceti strain 1023 during both log phases, suggesting the transition to acetate overoxidation was not a simple consequence of CAC enzyme induction. A mutagenized derivative of strain 1023 lacking functional AarC readily oxidized ethanol but was unable to overoxidize acetate, indicating that the CAC is required for acetate overoxidation but not ethanol oxidation. The primary role of the aar genes in the metabolically streamlined industrial strain A. aceti 1023 appears to be to harvest energy via acetate overoxidation in otherwise depleted medium

Metal stopping reagents facilitate discontinuous activity assays of the de novo purine biosynthesis enzyme PurE

The conversion of 5-aminoimidazole ribonucleotide (AIR) to 4-carboxy-AIR (CAIR) represents an unusual divergence in purine biosynthesis: microbes and nonmetazoan eukaryotes use class I PurEs while animals use class II PurEs. Class I PurEs are therefore a potential antimicrobial target; however, no enzyme activity assay is suitable for high throughput screening (HTS). Here we report a simple chemical quench that fixes the PurE substrate/product ratio for 24 h, as assessed by the Bratton-Marshall assay (BMA) for diazotizable amines. The ZnSO4 stopping reagent is proposed to chelate CAIR, enabling delayed analysis of this acid-labile product by BMA or other HTS method

Functional dissection of the bipartite active site of the class I coenzyme A (CoA)-transferase succinyl-CoA:acetate CoA-transferase

Coenzyme A (CoA)-transferases catalyze the reversible transfer of CoA from acyl-CoA thioesters to free carboxylates. Class I CoA-transferases produce acylglutamyl anhydride intermediates that undergo attack by CoA thiolate on either the internal or external carbonyl carbon atoms, forming distinct tetrahedral intermediates less than 3 Å apart. In this study, crystal structures of succinyl-CoA:acetate CoA-transferase (AarC) from Acetobacter aceti are used to examine how the Asn347 carboxamide stabilizes the internal oxyanion intermediate. A structure of the active mutant AarC-N347A bound to CoA revealed both solvent replacement of the missing contact and displacement of the adjacent Glu294, indicating that Asn347 both polarizes and orients the essential glutamate. AarC was crystallized with the nonhydrolyzable acetyl-CoA (AcCoA) analogue dethiaacetyl-CoA (1a) in an attempt to trap a closed enzyme complex containing a stable analogue of the external oxyanion intermediate. One active site contained an acetylglutamyl anhydride adduct and truncated 1a, an unexpected result hinting at an unprecedented cleavage of the ketone moiety in 1a. Solution studies confirmed that 1a decomposition is accompanied by production of near-stoichiometric acetate, in a process that seems to depend on microbial contamination but not AarC. A crystal structure of AarC bound to the postulated 1a truncation product (2a) showed complete closure of one active site per dimer but no acetylglutamyl anhydride, even with acetate added. These findings suggest that an activated acetyl donor forms during 1a decomposition; a working hypothesis involving ketone oxidation is offered. The ability of 2a to induce full active site closure furthermore suggests that it subverts a system used to impede inappropriate active site closure on unacylated CoA

Crystal Structures of <i>Acetobacter aceti</i> Succinyl-Coenzyme A (CoA):Acetate CoA-Transferase Reveal Specificity Determinants and Illustrate the Mechanism Used by Class I CoA-Transferases

Coenzyme A (CoA)-transferases catalyze transthioesterification reactions involving acyl-CoA substrates, using an active-site carboxylate to form covalent acyl anhydride and CoA thioester adducts. Mechanistic studies of class I CoA-transferases suggested that acyl-CoA binding energy is used to accelerate rate-limiting acyl transfers by compressing the substrate thioester tightly against the catalytic glutamate [White, H., and Jencks, W. P. (1976) <i>J. Biol. Chem. 251</i>, 1688–1699]. The class I CoA-transferase succinyl-CoA:acetate CoA-transferase is an acetic acid resistance factor (AarC) with a role in a variant citric acid cycle in <i>Acetobacter aceti</i>. In an effort to identify residues involved in substrate recognition, X-ray crystal structures of a C-terminally His₆-tagged form (AarCH6) were determined for several wild-type and mutant complexes, including freeze-trapped acetylglutamyl anhydride and glutamyl-CoA thioester adducts. The latter shows the acetate product bound to an auxiliary site that is required for efficient carboxylate substrate recognition. A mutant in which the catalytic glutamate was changed to an alanine crystallized in a closed complex containing dethiaacetyl-CoA, which adopts an unusual curled conformation. A model of the acetyl-CoA Michaelis complex demonstrates the compression anticipated four decades ago by Jencks and reveals that the nucleophilic glutamate is held at a near-ideal angle for attack as the thioester oxygen is forced into an oxyanion hole composed of Gly388 NH and CoA N2″. CoA is nearly immobile along its entire length during all stages of the enzyme reaction. Spatial and sequence conservation of key residues indicates that this mechanism is general among class I CoA-transferases

Shocks de Precios Relativos e Inflación: La Mediana Ponderada como Medida de Inflación Subyacente en Chile

Relative price shocks with zero mean can affect measured inflation in the presence of non-convex adjustment costs, if core inflation is different from zero or the distribution of the shock is asymetric. In such cases, central tendency measures of the shock's distribution are a better measure of core inflation. The purpose of this paper is to calculate and evaluate the properties of the weighted median as an indicator of core inflation in Chile. This paper analyzes the cross sectional distribution of price changes in Chile. It is shown that this distribution is very asymmetric, and that the degree of asymmetry is an important explanatory variable of actual inflation. In comparison to CPI and more traditional measures of core inflation, the weighted median is more correlated to past innovations in monetary aggregates, and constitutes a better predictor of future inflation.