The eutD gene of Salmonella enterica encodes a protein with phosphotransacetylase enzyme activity

Abstract

The EutD protein of Salmonella enterica is homologous to the catalytic domain of the phosphotransacetylase (Pta) enzyme. The Pta-like activity level of the EutD enzyme compared favorably to that of other Pta enzymes. High-pressure liquid chromatography and mass spectrometry verified that acetyl-coenzyme A was the product of the reaction. The EutD protein restored growth of an S. enterica pta strain on acetate as the source of carbon and energy.

FIG. 1.

Biochemical steps for the conversion of ethanolamine and 1,2-propanediol to the corresponding acyl-CoA derivative. (A) Ethanolamine is converted to Ac-CoA in two steps. EutD and Ack then convert Ac-CoA to acetate, which is excreted. (B) Similarly, 1,2-propanediol is converted from Pro-CoA to propionate by Pta and Ack. Propionate is eventually excreted.

FIG. 2.

EutD can restore growth of a pta acs strain on 50 mM acetate. Growth kinetics was analyzed using a 96-well microtiter plate (Becton Dickinson) and a computer-controlled Ultra microplate reader (Bio-Tek Instruments) equipped with KC4 software. The temperature of the incubation chamber was set at 37°C. Each well of the plate contained 198 μl of fresh medium, which was inoculated with 2 μl of an overnight culture of S. enterica grown on nutrient broth medium. Growth was monitored according to increases in the absorbance at 650 nm. Data were collected every 15 min; cultures were shaken for 800 s between readings. Plasmids carrying wild-type alleles of the M. thermophila pta (pML702 pta⁺) or S. enterica eutD (plasmid pEUTD2 P_araBAD-eutD⁺) genes were introduced into the strain. Expression of the eutD gene was induced by the presence of arabinose in the medium (250 μM). Strains were grown on a minimal medium (4) supplemented with MgSO₄ (1 mM), l-methionine (0.5 mM), and acetate (50 mM). pBAD30, cloning vector; peutD⁺, pEUTD2.

FIG. 3.

Ac-CoA is the product of the EutD-catalyzed reaction. (A) Elution profile of components of a EutD-catalyzed reaction. Components of the reaction mixture were separated (as described previously) (14) using a Prodigy 5μ ODS-2 column (Phenomenex) (250 by 4.60 mm; 5 μm), a computer-controlled high-pressure liquid chromatography system equipped with a model 600 quaternary solvent delivery system (Waters, Milford, Mass.), and a model 996 Plus photodiode array detector. Elution of materials from the column was monitored at 260 nm. The column was first developed isocratically with acetonitrile and water (1.75/98.25) containing 0.2 M ammonium acetate for 20 min at a rate of 1 ml/min and then switched to a 100-min convex gradient (Waters curve 3) with acetonitrile and water (10/90) containing 0.2 M ammonium acetate. The column was maintained at 35°C, and the flow rate was kept constant at 1 ml/min. A reaction mixture containing authentic Ac-CoA (Sigma) but lacking EutD was processed in parallel and used to determine the retention time for Ac-CoA under the conditions used. (B) Mass spectrometry. Fractions containing the EutD reaction product were concentrated using a vacuum and a SpeedVac concentrator (Thermo Savant), resuspended in 50% acetonitrile, and analyzed by ion electrospray mass spectrometry (negative mode) at the University of Wisconsin—Madison Biotechnology Center. The labeled peaks identified prominent ions of the molecular ion. The fragmentation pattern of the sample was identical to that of authentic Ac-CoA purchased from Sigma (data not shown). amu, atomic mass units.