Vol. 170, No. 4JOURNAL OF BACTERIOLOGY, Apr. 1988, p. 1709-17140021-9193/88/041709-06$02.00/0Copyright © 1988, American Society for Microbiology

Purification and Properties of Benzoate-Coenzyme A Ligase,a Rhodopseudomonas palustris Enzyme Involved

in the Anaerobic Degradation of BenzoateJOHANNA F. GEISSLER,' CAROLINE S. HARWOOD,2 AND JANE GIBSON'*

Section of Biochemistry, Molecular and Cell Biology, Division of Biological Sciences,' andDepartment of Microbiology,2 Cornell University, Ithaca, New York 14853

Received 1 October 1987/Accepted 18 January 1988

A soluble benzoate-coenzyme A (CoA) ligase was purified from the phototrophic bacterium Rhodopseudo-monas palustris. Synthesis of the enzyme was induced when cells were grown anaerobically in light withbenzoate as the sole carbon source. Purification by chromatography successively on hydroxylapatite,phenyl-Sepharose, and hydroxylapatite yielded an electrophoretically homogeneous enzyme preparation witha specific activity of 25 ,umol/min per mg of protein and a molecular weight of 60,000. The purified enzyme wasinsensitive to oxygen and catalyzed the Mg2' ATP-dependent formation of acyl-CoA from carboxylate and freereduced CoA, with high specificity for benzoate and 2-fluorobenzoate. Apparent Km values of 0.6 to 2 ,uM forbenzoate, 2 to 3 ,uM for ATP, and 90 to 120 ,uM for reduced CoA were determined. The reaction product,benzoyl-CoA, was an effective inhibitor of the ligase reaction. The kinetic properties of the enzyme match thekinetics of substrate uptake by whole cells and confirm a role for benzoate-CoA ligase in maintaining entry ofbenzoate into cells as well as in catalyzing the first step in the anaerobic degradation of benzoate by R. palustris.

Vast quantities of aromatic compounds are found in higherplants as monomeric components of the plant polymer ligninand as various plant secondary products (5, 8). In addition,synthetic organic compounds with aromatic structures areproduced in large amounts for industrial and agriculturalpurposes (18). Many of these synthetic compounds arereleased into the biosphere, and a proportion of them,together with aromatics derived from natural sources, even-tually accumulate in anaerobic environments (18). Severalgroups of anaerobic bacteria including photoheterotrophs,nitrate- and sulfate-respiring anaerobes, and methanogenicbacterial consortia can break down aromatics under anaer-obic conditions, but the degradative pathways followed areincompletely understood (1, 18).The anaerobic degradation of benzoate has received the

most attention, and work by several groups (3, 4, 6, 7, 11, 16)has led to a proposed pathway (Fig. 1). The essentialfeatures of this metabolic sequence are an initial reductiveattack on the benzene nucleus, followed by ,-oxidation ofthe reduced intermediates prior to ring cleavage. The non-sulfur purple phototrophic bacterium Rhodopseudomonaspalustris can grow anaerobically in light with benzoate as itssole carbon source, and Dutton and Evans (3, 4), as well asothers (7, 11), have obtained evidence that coenzyme A(CoA) esters rather than free acids are the immediate sub-strates in the enzymatic sequence mediating benzoate break-down. Previous work from our laboratories (7) has shownthat benzoate is taken up by intact cells of R. palustris withan apparent Km of less than 1 ,uM and that benzoyl-CoA,rather than free benzoate, accumulates intracellularly duringthe first 45 s of uptake. This indicates that thioesterificationis also important for benzoate uptake and predicts that theenzyme responsible for thioester formation should have avery high affinity for benzoate.We show here that benzoyl-CoA formation depends on the

activity of a soluble and very specific benzoate-CoA ligase.

* Corresponding author.

The enzyme has been purified to homogeneity and has thekinetic characteristics required to account for high-affinityuptake of benzoate by whole cells. This is the first enzyme ofthe reductive benzoate catabolic pathway to be purified andcharacterized.

MATERIALS AND METHODS

Organism and growth conditions. The strain of R. palus-tris, the growth medium (JGR), and the conditions forphototrophic growth that we used have been describedpreviously (7). For large-scale culture, cells were grown at28 to 33°C in a 10-liter carboy that was filled to the top withJGR medium supplemented with 6 mM benzoate. The car-boy was illuminated with two 60-W incandescent lamps, andthe medium was stirred slowly. This yielded about 15 g (wetweight) of cells at the end of growth.

Preparation of cell extracts. Cells were harvested by cen-trifugation for 10 min at 20,000 x g and were washed oncewith 20 mM triethanolamine-HCl buffer (pH 7.5). The cellpaste was stored at -18°C. All subsequent procedures werecarried out at 0 to 4°C. All buffers were filter sterilized bypassage through nitrocellulose membrane filters (pore size,0.45 ,m) prior to use and supplemented with 0.1 mMphenylmethylsulfonyl fluoride at the time of use. The cellswere thawed, suspended in 20 mM triethanolamine-HCI (pH7.5) (1:10, wt/wt), passed twice through a French pressurecell (American Instrument Co., Silver Spring, Md.) with 86MPa at the orifice, and centrifuged twice for 10 min at 18,000x g to remove unbroken cells and cell debris. The superna-tant was termed the crude cell extract. Ultracentrifugation ofthe crude cell extract at 200,000 x g for 1 h sedimented aparticulate membrane fraction. The supernatant from thisstep was incubated with 1% (wt/wt) streptomycin sulfate for1 h on ice and then centrifuged for 10 min at 18,000 x g. Theresulting orange supernatant was termed the soluble proteinfraction.

Assays of benzoyl-CoA ligase activity. Standard assayswere performed at 30°C with 20 mM triethanolamine-HCl

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Q%'OH I Q, CH

IIII

IIBenzoic acid y Ic Benzoic acid

Cytoplasmic membrane

O SCOA"C'

Benzoyl CoA

6H \0 SCoA

Cyclohexane -

carboxyl CoA

2H

O\ SCoAC ,SCoA

Acetyl*CoA 4- 4 (,C=O _

CoAPimelyl di-CoA

oI /SCoA&02H

L-uxocycionexane-carboxyl - CoA

o"I SCoA

OOH

O SCoA

H20

2-Hydroxycyclohexane. e6-CycIohelene-carboxyl- CoA curboxyI-CoA

FIG. 1. Proposed pathway of anaerobic benzoate metabolism in R. palustris.

buffer (pH 8.0). The two assay systems used gave compara-ble results for the specific activity of the enzyme, differing by<:15%. Catalytic activities are given in units, defined asmicromole of substrate converted per minute.

(i) Isotopic assay (system 1). The isotopic assay was similarto assays of fatty acid-CoA ligase activities (9). In addition tothe buffer, the reaction mixture contained 2.5 mM MgCl2,0.5 mM ATP, 0.25 mM reduced CoA (CoASH), and[14C]benzoate (4 ,uM in standard assays; 0.1 to 0.2 ,uCi) in afinal volume of 0.5 ml. After equilibration at 30°C, thereaction was initiated by addition of 10 to 20 ,ul (10 to 100,uU) of the enzyme. The reaction was stopped, usually after2 min, by addition of 50 ,ul of 50% (wt/vol) trichloroaceticacid. The mixture was chilled in ice, and 1 ml of 0.1 N HCIwas added. Free benzoic acid was removed by extracting theacidified assay mixture twice with 2.5 ml of diethyl ether.The radioactivity in 0.2-ml samples of the aqueous phase in3 ml of Liquiscint (National Diagnostics, Parsippany, N.Y.)was measured with a scintillation spectrometer (LS 100-C;Beckman Instruments, Inc., Fullerton, Calif.). This discon-tinuous assay procedure was suitable for activity measure-ments at all stages of enzyme purification.

(ui) Coupled enzymatic assay (system 2). The coupled enzy-matic assay monitored the rate of AMP formation by ben-zoate-CoA ligase by coupling the reaction via adenylatekinase, pyruvate kinase, and lactic dehydrogenase to theoxidation of NADH, which was measured at 340 nm in aspectrophotometer (DU-7; Beckman) (16). Two moles ofNADH was oxidized per mole ofAMP formed. The reactionmixture, slightly modified from that used previously (16),contained 2.5 mM MgCl2, 0.5 mM ATP, 0.25 mM CoASH,the substrate (0.25 mM in standard assays), 10 mM KCl, 10mM phosphoenol pyruvate, 0.35 mM NADH, and approxi-mately 2 U of each of the auxiliary enzymes pyruvate kinase,lactic acid dehydrogenase, and adenylate kinase in a totalvolume of 1 ml. The cuvette containing the assay mixturewas allowed to equilibrate at 30°C, and the reaction wasinitiated by addition of 1 to 10 mU of benzoate-CoA ligase.This continuous assay system could not be used for crudecell extracts and soluble protein fractions owing to interfer-ence by high endogenous ATPase activities.

Assay of acyl-CoA transferase activities. System 1 wasmodified by substituting 1 mM acetyl-CoA or succinyl-CoA

as a potential CoA donor for [14C]benzoate; ATP and freeCoASH were omitted from the reaction mixtures.

Purification of benzoate-CoA ligase. For benzoate-CoAligase purification, 1.5% streptomycin sulfate was added tothe crude cell extract prior to ultracentrifugation.

(i) First hydroxylapatite chromatography. The soluble pro-tein fraction (100 ml) was applied at a flow rate of 1 ml/minto a hydroxylapatite column (diameter, 2.5 cm; 100 ml) thathad been equilibrated with 20 mM potassium phosphatebuffer (pH 7.5). The loaded column was washed with 300 mlof the same buffer. The ligase was eluted with 300 ml of 80mM potassium phosphate buffer (pH 7.5).

(ii) Phenyl-Sepharose chromatography. The fraction withligase activity was applied directly to a column (diameter,2.5 cm; 50 ml) containing phenyl-Sepharose-Sepharose CL4B 200 (1:10; wt/wt), which had been equilibrated with 20mM triethanolamine-HCl buffer (pH 7.5). The column waswashed with 150 ml of the same buffer and then with 300 mlof the buffer containing 5 mM L-phenylalanine. The ligasewas eluted with 300 ml of 20 mM triethanolamine-HCl buffer(pH 7.5) supplemented with 5 mM phenylalanine and 0.1 mMeach ATP and MgC12.

(iii) Second hydroxylapatite chromatography. The fractionwith ligase activity was applied directly to a second hydrox-ylapatite column (diameter, 1 cm; 10 ml) that had beenequilibrated with 20 mM potassium phosphate buffer (pH7.5). After the column had been washed with 60 ml of 80 mMpotassium phosphate buffer, the ligase was eluted with 20 mlof 200 mM potassium phosphate buffer and concentrated byultrafiltration (30-kilodalton [kDa] cutoffmembrane; AmiconCorp., Lexington, Mass.). Protein was determined by themethod of Bradford (2). The results of a typical purificationare shown in Table 1.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis(8% polyacrylamide gels) was carried out at roomn tempera-ture with the discontinuous buffer system of Laemmli (13).Polypeptides were visualized with Coomassie blue.

Size exclusion HPLC. Native proteins were separated bygel filtration high-pressure liquid chromatography (HPLC) atroom temperature on a Zorbax GF-250 column (Du PontCo., Wilmington, Del.) with 0.2 M potassium phosphatebuffer (pH 7.5) as the mobile phase. The flow rate was 0.5

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BENZOATE-CoA LIGASE FROM RHODOPSEUDOMONAS PALUSTRIS

TABLE 1. Purification of benzoate-CoA ligasea

StepVol Total Sp.tTotal FoldStep (ml) protein Sp.c activity Recovery Fo(mg) (Um)(U)Reoeyprfctn

Soluble protein 100 100 0.05 4.8 100Hydroxylapatite (1st); 300 60 0.1 5.7 119 2

80 mM PiPhenyl-Sepharose; 300 0.26 18 3.6 74 286MgCl2-ATP

Hydroxylapatite 10 0.05 25 1.3 26 526(2nd); 200 mM Pia Purification starting material: 10 g (wet weight) of cells.

ml/min, and the A254 of the effluent was recorded. Fractionsof 0.25 ml were collected and assayed for enzyme activity byusing assay system 1. Molecular mass standards (20 ,ug each)were thyroglobulin (443 kDa), 3-albumin (200 kDa), alcoholdehydrogenase (150 kDa), bovine serum albumin (66 kDa),and carbonic anhydrase (29 kDa).

Isoelectric focusing. The purified enzyme was applied toServalyt Precote gels, pH 3 to 10 (Serva, Heidelberg, Fed-eral Republic of Germany) and focused for 4 h at constantpower of 4 W (0.1 to 2 kV) at approximately 10°C.Amino acid composition and N-terminal sequence. Amino

acid composition and N-terminal sequence analyses wereperformed at the Amino Acid Analysis Facility, Departmentof Chemistry, Cornell University. The amino acid composi-tion was determined with a Waters Analyzer (MilliporeWaters Chromatography Division, Milford, Mass.) afterprotein samples had been hydrolyzed with 6 N HCI undernitrogen (65 min at 150°C). The N-terminal sequence deter-mination was carried out with a gas phase analyzer (470 A;Applied Biosystems, Foster City, Calif.) as described previ-ously (10).

Reverse-phase HPLC. Substrate solutions or assay mix-tures were analyzed by HPLC (Beckman) on a C18 reverse-phase column (Biosil ODS 5-S; Bio-Rad Laboratories, Rich-mond, Calif.) with H20 and acetonitrile (each supplementedwith 0.1% [vol/vol] trifluoroacetic acid or H3PO4) as solventsA and B, respectively. The gradient was from 2% to 40osolvent B in 38 min at a flow rate of 1 ml/min. The A210 of theeffluent was recorded.

Reagents. Chemicals of reagent grade were obtained fromMallinckrodt, Inc., St. Louis, Mo.; biochemicals and en-zymes were from Sigma Chemical Co., St. Louis, Mo.,except cyclohexanecarboxylic acid (Aldrich Chemical Co.,Inc., Milwaukee, Wis.), cyclohexenecarboxylic acids (A1,A3) (Frinton Laboratories, South Vineland, N.J.), and 1,4-dihydrobenzoic acid (Alfa Products, Danvers, Mass.). Thepurities of all compounds used as enzyme substrates werechecked by optical spectroscopy and reverse-phase HPLC;all were free of contamination except for 1,4-dihydroben-zoate, which contained approximately 5% benzoate. Hy-droxylapatite (Bio-Gel HTP) and the dye reagent for proteindetermination were obtained from Bio-Rad; pyruvate ki-nase, lactic dehydrogenase in 50% glycerol, and adenylatekinase in ammonium sulfate were from Boehringer Mann-heim Biochemicals, Indianapolis, Ind. [7-14C]benzoic acid(56 mCi/mmol) was purchased from ICN PharmaceuticalsInc., Irvine, Calif.

RESULTSEnzyme activities in cell extracts. Crude cell extracts of R.

palustris grown photoheterotrophically on benzoate con-verted radioactively labeled benzoate to a product which,

unlike the free acid, was hydrophilic under acidic conditions(assay system 1). This activity was strictly dependent onadded CoASH, and less than 5% of the initial rate obtainedwith the complete assay was observed when ATP wasomitted from the reaction mixture. Over 90% of the activityremained in the soluble protein fraction after removal of thecell membranes.When acetyl-CoA or succinyl-CoA was substituted for

free CoASH in the reaction mixture, with either crudeextracts or soluble protein fractions at pH 7.5 or 8.0,formation of a hydrophilic derivative of [4(C]benzoate wasbelow 5% of that observed in complete assay system 1.Benzoate derivatization could therefore be attributed to theactivity of a soluble benzoate-CoA ligase, rather than anacyl-CoA transferase.

Product identification. The single radioactive hydrophilicproduct that was formed when the soluble protein fractionwas assayed with [14C]benzoate coeluted in reverse-phaseHPLC with authentic benzoyl-CoA. After incubation of thisreaction mixture at pH 12 for 5 min at 70°C, all radioactivitycoeluted with benzoic acid, as would be expected from thealkali lability characteristic of acyl-CoA thioester bonds (17).

Formation of AMP (as opposed to ADP) as a product inthe ligase reaction was demonstrated indirectly. With puri-fied benzoate-CoA ligase, no reaction was observed with thecoupled enzymatic assay (system 2) in the absence ofadenylate kinase, which catalyzes the reversible formationof ADP from ATP and AMP. Furthermore, an exact 2:1stoichiometry of NADH oxidation and benzoate conversionwas also demonstrated, in agreement with the formation ofAMP and PPi as the products.

Purification of benzoate-CoA ligase. The procedure de-scribed above resulted in a 500-fold purification of theenzyme, with an overall yield of about 25% (Table 1). Finalspecific activities in the eluate from the second hydroxylap-atite column ranged from 21 to 27 U/mg of protein.An unusual step in the purification scheme was chroma-

tography on a mixture of phenyl-Sepharose and Sepharose,which brought about a 200- to 300-fold purification. In theexploratory stages of this work, a number of hydrophobicinteraction matrices were tested, and the enzyme was foundto bind with high affinity to L-phenylalanine agarose. Activ-ity was not released from the column by low concentrationsof benzoate (5 ,uM), sufficient to saturate the enzyme inassay system 1, by 0.1 mM ATP or MgCl2 alone. However,0.1 mM Mg2+-0.1 mM ATP effectively eluted a proteinfraction with high specific activity (10 to 20 U/mg), and up tohalf of the total protein in this eluate was a polypeptide ofmolecular weight 60,000. Binding of the enzyme to phenyl-alanine agarose appeared to be very sensitive to the liganddensity of the gel, and the gel was abandoned in favor of thephenyl-Sepharose-Sepharose mixture. Washing the loaded

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1712 GEISSLER ET AL.

phenyl-Sepharose column with phenylalanine solutions elut-ed much of the contaminating protein, but the ligase wasreleased only when Mg2+-ATP was also included. Thesefindings suggest that binding to the matrices involved spe-

cific affinities rather than hydrophobic interactions and that aconformation change was needed to release the enzyme.

Stability of the purified enzyme. The ligase was not sensi-tive to oxygen, since specific activities at each stage of thepurification were not increased when buffers were deaeratedby autoclaving, and all solutions were supplemented with 1mM.4ithiothreitol and maintained under argon. The concen-trated purified enzyme (0.1 mg of protein per ml) lost activityat 0°C, with a half time of about 8 days. No significantimprovement in enzyme stability was observed when the pHof the triethanolamine hydrochloride buffer was varied be-tween 6.5 and 8.0 or when 0.1 mM EDTA or 1 mMdithiothreitol was added; addition of 50% (vol/vol) glyceroldid not stabilize activity. The enzyme could be stored inliquid nitrogen for at least several months; an activity loss ofabout 20% was probably caused by freezing and thawing.

Physicochemical properties of the enzyme. The purifiedprotein migrated as a single band on sodium dodecyl sulfate-polyacrylamide gels (Fig. 2) with a calculated molecularweight of 60,000 (± 2,000). The approximate molecularweight of the native enzyme as determined by the appear-ance of enzymatic activity in eluate fractions from sizeexclusion HPLC was 58,000, which indicates that the en-

zyme is a monomer. Isoelectric focusing of the nativeenzyme showed a single band at about pH 5.2 (not shown).The amino acid composition of the benzoate-CoA ligase is

given in Table 2. The N-terminal sequence was Asn-Ala-Ala-Val-Pro.pH dependence. Rates of benzoyl-CoA formation were

measured at different pH values of the triethanolamine-HClbuffer with isotopic assay system 1. A broad pH optimumwas observed between pH 8.4 and 8.9. Residual activitieswere 75% at pH 8.0 and 10% at pH 7.0. No reaction wasobserved at pH 6.5.

Kinetics. Michaelis-Menten-type saturation kinetics were

1 2 3 4 5

S__~~~~~~~~~.4...'^. OY..:

K Da

66

4 5

3 6

2 4

^ '~~~~~20

FIG. 2. Sodium dodecyl sulfate-polyacrylamide gel electropho-resis analysis of the fractions obtained during the purification ofbenzoate-CoA ligase. Lanes: 1,5, molecular mass standards (bovineserum albumin [66 kDa], egg albumin [45 kDa], glyceraldehyde-3-phosphate dehydrogenase [36 kDa], carbonic anhydrase [29 kDa],trypsinogen [24 kDa], and trypsin inhibitor [20 kDa]; 2, solubleprotein fraction; 3, fraction eluted from first hydroxylapatite col-umn; 4, fraction eluted from second hydroxylapatite column.

TABLE 2. Amino acid composition of benzoate-CoA ligase

Amino acid Recovery No. of Mol %(pmol)a residues/polypeptidebo'

Asxc 948 44 8Glxc 1,042 45 8Ser 588 27 5Gly 1,199 36 7His 231 13 2Arg 827 29 5Thr 731 36 7Ala 1,337 66 12Pro 813 35 6Tyr 352 20 4Val 846 42 8Met 187 10 2Cys 36 2 <1Ile 327 23 4Leu 1,111 48 9Phe 485 23 4Lys 460 22 4a Amount of each amino acid (picomoles) in actual experiment; tryptophan

was not recovered. Assuming an average molecular weight of 110 per aminoacid, the sample contained 21 pmol of the polypeptide (Mr, 60,000), i.e., 545residues per polypeptide.

b Amino acid residues per polypeptide after correction for loss duringhydrolysis by companson with an RNase A standard subjected to the sameprocedures.

Asn and Asp, and Gln and Glu were not distinguished.

obtained with benzoate, ATP, and CoASH. No substrateinhibition occurred with up to 0.25 mM benzoate, 1 mMATP, or 1 mM CoASH, but higher concentrations of thesecomponents reduced the reaction rate. The apparent kineticconstants Vmax and Km for the purified enzyme were deter-mined from Lineweaver-Burk plots (Table 3). Very similarapparent Km values for benzoate (0.6 ,uM) and for ATP (3,uM) were measured with unpurified enzyme in the solubleprotein fraction, whereas the value for CoASH was slightlyhigher (250 ,uM).

Product inhibition. Addition of the reaction products AMPand PP1 (<0.1 mM) had no effect on the initial rates of theligase reaction (assay system 1), but 0.1 mM benzoyl-CoAcaused about 80% inhibition. As would be expected fromthis observation, the reaction rate decreased progressivelyduring prolonged benzoate turnover in the coupled enzy-matic assay (system 2) (Fig. 3). Consumption of CoASHduring the period shown was less than 10% of the total addedinitially, and benzoate and ATP remained at saturatingconcentrations throughout the experiment. This indicatedthat benzoyl-CoA inhibited the reaction with a K, of about 40,uM under the given conditions (Fig. 3, insert). Identicalreaction courses were observed when initial benzoate con-centrations were 50 to 250 ,uM, as long as residual benzoatewas at saturating concentrations (.5 ,uM). Varying ATP

TABLE 3. Kinetic constants of benzoate-CoA ligasea

Substrate KKm Vm.(p.M) (U/mg)

Benzoate 0.6-2 27CoASH 90-120 18bATP 2-3 27

a Initial rates of benzoyl-CoA formation were measured by assay system 1.Concentrations of unvaried substrates were 4 ,M benzoate, 250 FM CoASH,and 500 ,uM ATP; MgCl2 concentration was 2.5 mM.

b The enzyme solution had decreased in activity by ca. 30% followingstorage for 5 days.

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BENZOATE-CoA LIGASE FROM RHODOPSEUDOMONAS PALUSTRIS

2 3TIME, min

FIG. 3. Time course of the benzoate-CoA ligase reaction. Thereaction of purified benzoate-CoA ligase (in the presence of 0.25 mMbenzoate, 0.5 mM ATP, and 0.75 mM CoASH) was coupled toNADH oxidation as described in the text. The plot shows thecontinuous time course of a single experiment over approximately15 min. The first arrow indicates initiation of the reaction by enzymeaddition, and the second arrow indicates consumption of about 10%of the CoASH (75 ,uM). A Dixon plot of the rate of reaction versus

concentration of benzoyl-CoA is given in the insert.

levels within the saturation range from 50 ,uM to 1 mM was

also without influence on the progressive inhibition pattern.Benzoyl-CoA thus did not appear to compete with benzoateor ATP for a common binding site.

Substrate specificity. A selection of carboxylic acids weretested as potential substrates (Table 4), and only carbox-ylates with close steric resemblance to benzoate were activein the ligase reaction. 2-Fluorobenzoate was the most effec-tive analog; the reaction rate was the same as with benzoate,and the enzyme was saturated at less than 25 ,uM. Substitu-ents at the ortho position of the aromatic ring decreased thecatalytic rate slightly less than if present at the meta or para

position. There was no correlation between the ability of a

compound to serve as a substrate in the ligase reaction andits ability to support photoheterotrophic growth of R. palus-tris (Table 4; 7a).

In the isotopic assay system I (0.1 mM ATP), Mg2+ couldbe replaced by Mn2+. Relative rates with various concentra-tions ofMg2+ were 100% (2.5 mM) and 96% (0.25 mM). WithMn2+, rates (relative to 2.5 mM Mg2+) of 17% (2.5 mM) and106% (0.25 mM) were observed. The enzyme was specificfor ATP, and no reaction was observed when GTP (10 ,uM to1 mM) was substituted for ATP.

DISCUSSION

Activation through CoA ester formation plays an essentialrole in fatty acid degradation in bacteria (15), and a similarrole of thioester formation is apparent in the anaerobicpathway of benzoate utilization. Inducible benzoate-CoAligase activities in bacteria grown anaerobically with ben-

zoate as the sole carbon source have been described (11, 16),and the in vivo formation of benzoyl-CoA from benzoate bywhole cells of R. palustris has also been demonstrated (7).We report here the purification and characterization of abenzoate-CoA ligase that was obtained from R. palustris andthat catalyzes the formation of benzoyl-CoA from benzoateand free CoASH.The enzyme was active with only a small number of

structurally related organic acids. Significant activity at lowsubstrate concentrations (25 ,uM) was observed only withbenzoate and the sterically very similar fluorobenzoates.Compounds such as cyclohexanecarboxylate and A'-cyclo-hexenecarboxylate, which are thought to be metabolized bythe same general pathway as benzoate (Fig. 1), were rela-tively poor substrates for this enzyme. Various benzoatederivatives with modified C1 side chains and with singlesubstituents on the aromatic nucleus can support anaerobicgrowth of R. palustris cells (Table 4; 7a), but were also notsubstrates for the benzoate-CoA ligase.A high affinity of the ligase for benzoate was shown by an

apparent Km of about 1 ,uM. This is in accord with in vivofindings showing high-affinity uptake of benzoate by wholecells and supports our previous suggestion (7) that benzoatethioesterification plays an important role in maintaining adownhill concentration gradient of benzoate across the cellmembrane. In this way the ligase maintains the entry ofbenzoate into cells, and its activity would be especiallyimportant in environments containing very low concentra-tions of this substrate. The enzyme activity remained whollyin the supernatant fractions after centrifugal removal ofmembranes from crude cell extracts. This indicates that

TABLE 4. Substrate specificity of benzoate-CoA ligase

% Relative GrowthSubstrate (concn [>.M]) velocitya substrateb

Benzoate (50-250) 100 +2-Fluorobenzoate (25-250) 1024-Fluorobenzoate (250) 722-Chlorobenzoate (250) 10A1-Cyclohexenecarboxylate (250) 13 +A3-Cyclohexenecarboxylate (250) 54 +Nicotinate (250) 12Picolinate (250) 452-Hydroxybenzoate (250) 54-Hydroxybenzoate (250) 1 +3-Methylbenzoate (250) 14-Methylbenzoate (250) 13-Chlorobenzoate (250) 1Cyclohexanecarboxylate (250) 1 +1,4-DihydrobenzoateC (250) 1 +Benzoylformate (250) <1 +Phenylacetate (250) <1trans-Cinnamate (250) <1 +4-Coumarate (250) <1 +Pimelate (250) <1 +Succinate (250) <1 +Anthranilate (250) <1Hexanoate (250) <1 NDOctanoate (250) <1 NDDecanoate (250) <1 ND

a Relative velocities of acyl-CoA formation were determined with purifiedenzyme (ca. 10 mU) in assay system 2.

b Symbols: +, Substrate supports growth under phototrophic conditions;-, substrate does not support growth; ND, function as growth substrate notdetermined.

I Relative rate after consumption of the contaminating benzoate.

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benzoate thioesterification occurs in the cytoplasmic com-partment of the cell and follows, rather than accompanies,passage of the substrate across the cytoplasmic membrane.In contrast to benzoate, the affinity of the ligase for CoA wasonly moderate and was comparable to that of acetyl-CoAligase from mitochondria (14). This may have physiologicalsignificance in preventing sequestration of the entire intra-cellular CoA pool when benzoate serves as the growthsubstrate.The formation of benzoyl-CoA appeared to be catalyzed

exclusively by the benzoate-CoA ligase, since we detectedno transferase activity which could use acetyl-CoA or suc-cinyl-CoA as a CoA donor for benzoate. Specific transferfrom pimelyl-diCoA, a postulated intermediate in the ringopening sequence, or from still other acyl-CoAs, has notbeen eliminated. However, the specific activity of benzoate-CoA ligase in crude cell extracts was adequate to account forthe rate at which whole cells could take up and convertbenzoate to benzoyl-CoA (7).Enzymes described under the general grouping of car-

boxylate:CoASH ligases (AMP forming) (EC 6.2.1.1 to EC6.2.1.17) which utilize aromatic carboxylates as substratesare quite varied in terms of substrate specifity. Medium-chain fatty acid CoA ligases from mammalian mitochondriahave been described which have very broad specifity andcan activate several aromatic acids, including benzoate, inaddition to fatty acids (12, 14). These enzymes are regardedas part of mammalian detoxification pathways for aromaticcompounds. Aromatic carboxylate-CoA ligases that are spe-cific for hydroxycinnamic acids have been described forplants (5), in which they play a central role in lignin andflavonoid biosynthetic pathways (5, 8). Similar enzymeshave also been found in fungi and in Pseudomonas putida (5,19). The basic properties of this subgroup of CoA ligases arevery similar: the enzymes are specific for a small number ofhydroxylated and/or methoxylated cinnamate derivativesand do not activate benzoate or hydroxybenzoates.

In view of its specificity for benzoate and its role inanaerobic aromatic degradation, it appears justified to con-sider benzoate-CoA ligase from R. palustris to be a novelenzyme distinct from other described ligases. Furthermore,it is, to our knowledge, the first acyl-CoA ligase that reactswith aromatic substrates to be purified to homogeneity. Afew of the mammalian and plant enzymes and the P. putidaenzyme have been partially purified (5, 19).The narrow substrate specificity of the benzoate-CoA

ligase is particularly interesting because R. palustris canutilize a somewhat wider range of aromatic and alicycliccompounds for anaerobic growth than can other bacteria sofar described. Biochemical and genetic evidence suggeststhat these compounds are also metabolized via metabolicroutes involving thioesters. This study indicates that addi-tional CoA ligases must be present in cells to activate diversecompounds.

ACKNOWLEDGMENTS

This work was supported in part by grant DE-FG02-86ER13495from the Department of Energy and by a grant from the CornellBiotechnology Program, which is sponsored by the New York StateScience and Technology Foundation and a consortium of industries.

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