The eutD Gene of Salmonella enterica Encodes a Protein with Phosphotransacetylase Enzyme Activity

The EutD protein of Salmonella enterica is homologous to thecatalytic domain of the phosphotransacetylase (Pta) enzyme.The Pta-like activity level of the EutD enzyme compared favorablyto that of other Pta enzymes. High-pressure liquid chromatographyand mass spectrometry verified that acetyl-coenzyme A was theproduct of the reaction. The EutD protein restored growth ofan S. enterica pta strain on acetate as the source of carbonand energy.

INTRODUCTION

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Introduction

Ethanolamine is used by Salmonella enterica serovar TyphimuriumLT2 (hereafter referred to as S. enterica) as a source of carbon,nitrogen, and energy (8, 21, 22). In principle, the biochemistryof the conversion of ethanolamine to the central metaboliteacetyl-coenzyme A (Ac-CoA) is relatively simple (Fig. 1A). Thefirst step in the catabolism of ethanolamine is catalyzed bythe well-characterized ethanolamine ammonia-lyase, a coenzymeB₁₂-dependent enzyme (2, 3, 5, 9, 10, 24). The products of theethanolamine ammonia-lyase-catalyzed reaction are ammonia andacetaldehyde, whose oxidation to acetate may be coupled to thesynthesis of Ac-CoA (1). There are two possible fates for Ac-CoAin this bacterium. When the cell is not starved for energy,Ac-CoA enters the glyoxylate bypass of the tricarboxylic acidcycle to fuel the energy generation system and serves as a buildingblock for many intermediary and secondary metabolism pathways(16). Under conditions of energy limitation, Ac-CoA is convertedto acetyl-phosphate (Ac-P) and ultimately to acetate by theaction of the phosphotransacetylase (Pta [EC 2.3.1.8]) and acetatekinase (Ack [EC 2.7.2.1]) enzymes. Under these conditions, acetateis excreted and later recaptured (17). In this pathway, theconversion of Ac-P to acetate is coupled to substrate-levelphosphorylation of ADP to yield ATP. The EutE protein has beenproposed to be the enzyme responsible for converting acetaldehydeto Ac-CoA; however, the EutE protein has not been isolated orstudied in detail. Ethanolamine catabolic functions in S. entericaare encoded by the 17-gene eut operon (15, 25). Many of theannotated functions of the eut genes are based exclusively ontheir homology to proteins of known function. For example, thepredicted primary amino acid sequence of the EutD protein is37% identical and 56% similar to that of the catalytic domainof the Pta enzyme of S. enterica and many other prokaryotes(11). However, no experimental support for this annotation hasbeen reported. In this paper, we provide in vivo and in vitroevidence that shows that the EutD protein is a bona fide Ptaenzyme.

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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.

The EutD protein compensates for the lack of Pta activity during growth on acetate.

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The eutd protein compensates...

To investigate whether the EutD protein had Pta activity, theeutD gene was cloned under the control of an arabinose-induciblepromoter and was introduced into a pta acs strain unable togrow on acetate. The acs gene encodes a high-affinity Ac-CoAsynthetase enzyme (EC 6.2.1.1) that activates acetate to Ac-CoAwhen acetate is present at a low concentration in the environment(17, 18). Because the Ack/Pta and Acs systems are the only onesS. enterica has for the conversion of acetate to Ac-CoA, inactivationof both of these systems renders a strain unable to use acetateas a carbon and energy source. Arabinose-induced expressionof eutD in the pta acs/pEUTD2 P_araBAD-eutD⁺ strain (JE7245)restored growth on acetate to a rate comparable to that measuredfor the wild-type strain (Fig. 2) and to the activity associatedwith the Pta enzyme of the methanogenic archaeon Methanosarcinathermophila (Fig. 2).

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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.

EutD converts Ac-P to Ac-CoA in the presence of free CoA.

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Eutd converts ac-p to...

Incubation of the H₆-EutD enzyme with Ac-P and CoA yielded Ac-CoA.Reverse-phase high-pressure liquid chromatography was used toseparate components of the reaction mixture on the basis oftheir hydrophobicity characteristics. The chromatogram of thereaction mixture showed a compound eluting 104 min after injection(Fig. 3A). This compound was identified as Ac-CoA on the basisof its retention time and its mass spectrum (Fig. 3B). The massspectrum of the unknown compound was identical to that obtainedwith authentic, commercially available Ac-CoA. For the sakeof simplicity, only the signals diagnostic of the molecularion are labeled in Fig. 3B. The latter were observed with mass/chargeratios (m/z) of 808.6 (M - 1H), 810.6 (M + 1H), and 846.4 (M- 2H + K), where M is the mass of the molecular ion, H is aproton, and K is a potassium ion. These data confirmed thatEutD had Pta activity.

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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.

Isolation and initial characterization of the EutD Pta activity.

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Isolation and Initial...

Basic kinetic parameters of the Pta activity were obtained withhomogeneous, N-terminally tagged H₆-EutD protein purified usingNi-affinity chromatography. H₆-EutD protein was overproducedin Escherichia coli strain BL21( ${lambda}$ DE3) harboring plasmid pEUTD8(pET15b eutD⁺) after induction with isopropyl-ß-D-thiogalactopyranoside(250 µM). Cells were broken open using a French press(Spectronic Unicam) at 1.034 x 10⁴ kPa. Cell debris was pelletedby centrifugation at 39,191 x g in an Avanti J-25I centrifuge(Beckman-Coulter) for 40 min at 4°C with a J25.50 rotor,and the clarified cell extract was loaded onto a 1-ml His-Bindnitrilotriacetic acid affinity chromatography column (Novagen)which was developed per the manufacturer's instructions. Fractionscontaining H₆-EutD protein were dialyzed overnight at 4°Cwith 1 liter of 50 mM Tris-Cl buffer (pH 7.2) containing 2 mMdithiothreitol, 10 mM EDTA, and 20 mM KCl. EutD protein wasdialyzed using the buffer described above without EDTA and with10% (vol/vol) glycerol as cryoprotectant. H₆-EutD protein (>95%homogeneous) was stored at -80°C until used. Assay conditionsand detection of product formation were as described previously(20) except that the final volume of the reaction mixture was1 ml, the reaction was started by the addition of substrate(i.e., potassium lithium Ac-P [1 µmol]), and the reactionwas performed at 30°C. The purity and the concentrationof the H₆-EutD protein were established as previously described(7, 19, 23) (data not shown). Pseudo-first-order kinetics yieldedapparent K_m values of 46 µM for free CoA and 129 µMfor Ac-P. The k_cat value calculated per micromole of EutD monomerwas 1,927 s^-1, and the catalytic efficiency (k_cat/K_m) valuesfor free CoA and Ac-P were 4.2 x 10⁴ and 1.5 x 10⁴, respectively.A pH activity profile for the H₆-EutD enzyme showed maximalactivity at pH 7.2, with an approximately 90% loss of activitymeasured at pH 6.5 and 25% of the activity lost at pH 7.8 (datanot shown). A thermal stability analysis showed that >50%of the enzyme's activity was lost after a 5-min incubation at25°C, with >90% of the activity lost at >45°C(data not shown).

EutD Pta activity is a key difference between ethanolamine and 1,2-propanediol catabolism in S. enterica.

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Eutd pta activity is...

Despite the fact that ethanolamine and 1,2-propanediol catabolismsin S. enterica occur via very similar biochemical reactions(Fig. 1) (6, 15), no evidence has been reported for the existenceof a structural or functional homolog of Pta encoded by thepropanediol utilization (pdu) operon. The catabolism of bothcompounds requires the assembly of a carboxysome-like structure,with increasing evidence supporting the hypothesis that bothcompounds are catabolized inside the carboxysome-like structure(12, 13, 15, 25). Why does ethanolamine catabolism require EutDin addition to the housekeeping Pta enzyme? Why is an additionalPta activity not required for 1,2-propanediol catabolism? Answersto these questions will shed light on the physiological restrictionsconfronted by S. enterica during the catabolism of these compounds.

ACKNOWLEDGMENTS

This work was supported by NIH grant RO1-GM40313 to J.C.E.-S.

We thank J. G. Ferry (Penn State University) for plasmid pML702and V. J. Starai for helpful discussions.

FOOTNOTES

* Corresponding author. Mailing address: Department of Bacteriology, University of Wisconsin—Madison, 264 Enzyme Institute, 1710 University Ave., Madison, WI 53726-4087. Phone: (608) 262-7379. Fax: (608) 265-7909. E-mail: escalante{at}bact.wisc.edu.

REFERENCES

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