Vol. 173, No. 17

Nucleotide Sequences of the Acinetobacter calcoaceticus benABCGenes for Benzoate 1,2-Dioxygenase Reveal Evolutionary

Relationships among Multicomponent OxygenasesELLEN L. NEIDLE,lt CHRISTOPHER HARTNETT,1t L. NICHOLAS ORNSTON,1* AMOS BAIROCH,2

MONIQUE REKIK,2 AND SHIGEAKI HARAYAMA2

Department of Biology, Yale University, New Haven, Connecticut 06511,1 and Department of MedicalBiochemistry, Faculty of Medicine, University of Geneva, 1211 Geneva 4, Switzerland2

Received 22 January 1991/Accepted 13 June 1991

The nucleotide sequences of the Acinetobacter cakoaceticus benABC genes encoding a multicomponentoxygenase for the conversion of benzoate to a nonaromatic cis-diol were determined. The enzyme, benzoate1,2-dioxygenase, is composed of a hydroxylase component, encoded by benAB, and an electron transfercomponent, encoded by benC. Comparison of the deduced amino acid sequences of BenABC with relatedsequences, including those for the multicomponent toluate, toluene, benzene, and naphthalene 1,2-dioxygen-ases, indicated that the similarly sized subunits of the hydroxylase components were derived from a commonancestor. Conserved cysteine and histidine residues may bind a [2Fe-2S] Rieske-type cluster to the oa-subunitsof all the hydroxylases. Conserved histidines and tyrosines may coordinate a mononuclear Fe(ll) ion. The lessconserved 13-subunits of the hydroxylases may be responsible for determining substrate specificity. Eachdioxygenase had either one or two electron transfer proteins. The electron transfer component of benzoatedioxygenase, encoded by benC, and the corresponding protein of the toluate 1,2-dioxygenase, encoded by xylZ,were each found to have an N-terminal region which resembled chloroplast-type ferredoxins and a C-terminalregion which resembled several oxidoreductases. These BenC and XylZ proteins had regions similar to certainmonooxygenase components but did not appear to be evolutionarily related to the two-protein electron transfersystems of the benzene, toluene, and naphthalene 1,2-dioxygenases. Regions of possible NAD and flavin adeninedinucleotide binding were identified.

The complete degradation of benzoate by aerobic bacteriacan occur by either of two catabolic pathways. In bothreaction sequences, benzoate is converted to a nonaromaticcis-diol, 2-hydro-1,2-dihydroxybenzoate, and then to cate-chol (51) (Fig. 1). Catechol is cleaved between the twohydroxyl groups when benzoate is metabolized via the ortho(or ,B-ketoadipate) pathway, and subsequent reactions yieldthe tricarboxylic acid cycle intermediates succinate andacetyl coenzyme A (59). Catechol cleavage via the metapathway occurs adjacent to the hydroxyl groups, and sub-sequent reactions lead to the formation of pyruvate andacetaldehyde. The ortho- and meta-fission routes do notusually operate concurrently, and regulatory factors deter-mine how benzoate is degraded when genes encoding bothpathways are present in the host cell (39).The genes responsible for the conversion of benzoate to its

corresponding cis-diol have been isolated from two differentsoil bacteria: benABC from the chromosome of Acinetobac-ter calcoaceticus (42) and xylXYZ from TOL plasmid pWWOof Pseudomonas putida (78). The benABC-encoded ben-zoate 1,2-dioxygenase exhibits narrow substrate specificity,showing little or no oxidation of most substituted benzoates.In contrast, the plasmid-encoded meta pathway toluate1,2-dioxygenase, encoded by xylXYZ, catalyzes the hydrox-ylation not only of benzoate but of many 3- and 4-substitutedbenzoates including toluates (78). Benzoate 1,2-dioxygenaseand toluate 1,2-dioxygenase are each multicomponent en-

* Corresponding author.t Present address: Microbiology Department, University of

Texas Medical School, Houston, TX 77225.t Present address: Neurogen Corporation, Branford, CT 06405.

zymes consisting of three different polypeptides of about 53,19, and 38 kDa (19, 21, 42).The similar sizes of the polypeptides and the similarity

among the biochemical reactions catalyzed by the benABC-and xylXYZ-encoded enzymes raise questions about theirevolutionary origins. Benzoate and toluate 1,2-dioxygenasesare structurally related to a family of multicomponent aro-matic ring-hydroxylating dioxygenases (9). These dioxygen-ases contain an aromatic ring hydroxylase component com-posed of two different subunits. The sizes of these a and ,Bsubunits, about 50 and 20 kDa, respectively, are similar indifferent dioxygenases. Studies of benzoate 1,2-dioxygenasefrom the soil bacterium Pseudomonas arvilla C-1 indicatethat the structure of this native enzyme is c3133 (75). Eacha-subunit contains one iron-sulfur cluster [2Fe-2S], whereasthe isolated 1-subunit does not contain any detectable ironor labile sulfide. One mononuclear Fe(II) atom may also beassociated with each hydroxylase component (75).

In addition to the hydroxylase component, the dioxygen-ases described above usually contain one or two electrontransport proteins. The benzoate 1,2-dioxygenase of P.arvilla has a single iron-sulfur flavoprotein exhibiting anNADH-cytochrome c reductase activity that is responsiblefor the electron transfer from NADH to the aromatic ringhydroxylase. This enzyme is a 38-kDa polypeptide with oneiron-sulfur cluster of the [2Fe-2S] type and one molecule offlavin adenine dinucleotide (FAD) (73, 74). The involvementof similar proteins in the electron transfer reactions ofbenzoate 1,2-dioxygenase from A. calcoaceticus and toluate1,2-dioxygenase from P. putida have been suggested by ourprevious genetic studies (19, 21, 42). In the benzene, tolu-ene, and naphthalene dioxygenase systems, however, two

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5386 NEIDLE ET AL.

BENZOATE 1.2-DIOXYGENASE(TOLUATE 1.2-DIOXYGENASE)

CRA DNADH

( RR)HH(R-)

BENZOATE edrectronR - = H transfer

component componentbenAB8 benC(XyxylX) (xI) ND

NADHq ~ oo NADen

COHOOH

(R) OH(R')

DHB R = R = HNAD.-+ DHB DEHYDROGENASE

NADH benDco2O (xylL)

1311 ~ ~~~~NW acetyl CoA and~OH ortho ring cleavage succinate

(R) ' pathway(RP)

CATECHOL=R- H

meta

pathway

acetaldehydeand

pyruvate

FIG. 1. Multicomponent dioxygenases initiate benzoate (1) deg-radation by converting it to 2-hydro-1,2-dihydroxybenzoate (DHB,2). Benzoate and toluate 1,2-dioxygenases are each composed of atwo-subunit hydroxylase, encoded by benAB and xylXY, respec-tively, and an electron transfer oxidoreductase encoded by benCand xyIZ, respectively. DHB is converted to catechol (3) by anNAD-dependent dehydrogenase, the gene product of benD or xylL.The chromosomal ben genes, isolated from A. calcoaceticus, en-code enzymes specific for the substrate R=R'=H. The plasmid-borne xyl genes, isolated from P. putida TOL plasmid pWWO,encode enzymes with broader substrate specificity and can degradecompounds with methyl substitutions at positions R and R'.

subclones were generated from smaller KpnI-KpnI, KpnI-EcoRI, and KpnI-SalI restriction endonuclease fragments ofthe ben DNA. Escherichia coli JM101 supE thi A(lac-proAB)(F' traD36 proAB laqIq ZAM15) (69) was used as a host forrecombinant plasmids and bacteriophages.The Cyclone I Biosystem of International Biotechnolo-

gies, Inc. (New Haven, Conn.) was used to generate nesteddeletions of the insert DNA of recombinant bacteriophages.Using this method, the exposed single-stranded ends ofcloned DNA fragments were progressively digested by T4DNA polymerase. Overlapping clones entirely covering bothstrands of the benABC genes were isolated.DNA sequencing. Procedures described in the "M13 Clon-

ing/Dideoxy Sequencing Instruction Manual" (4a) were fol-lowed for the isolation and propagation of M13 phages andfor the generation of single-stranded DNA sequencing tem-plates. DNA sequence was determined by the dideoxy chaintermination method (56) with commercial kits obtained fromBethesda Research Laboratories, Inc. (Gaithersburg, Md.)and United States Biochemical Corp. (Cleveland, Ohio).a-35S-dATP was purchased from Amersham Corp. (Arling-ton Heights, Ill.). As described previously (41), sequencingreaction mixtures were electrophoretically separated on 8%polyacrylamide gels with 42% urea in Tris-borate-EDTAbuffer. Gels were dried under vacuum with a gel slab drier(model 224; Bio-Rad Laboratories, Richmond, Calif.) beforeautoradiography with Kodak X-Omat film.DNA sequence analysis. Computer-assisted sequence anal-

ysis was done with software packages PC/GENE (Genofit;Intelligenetics; developed by A. Bairoch) and MicroGenie(Beckman). Protein similarities were detected by scanningversion 13 of the SWISS-PROT protein sequence data bank(A. Bairoch; distributed by the EMBL Data Library) with analgorithm based on that of Lipman and Pearson (37). Com-puter-calculated alignments were used to assess the signifi-cance of sequence similarities (40), and secondary structurepredictions were computed by using the conformationalparameters of Chou and Fasman (7).

Nucleotide sequence accession number. The sequence ofthe A. calcoaceticus 3,353-bp HindIII-EcoRI restrictionendonuclease fragment described in this report has beendeposited with GenBank under accession number M62649.

different proteins are required for the electron transferreaction (2, 16, 17, 60, 61).To better understand the evolutionary relationships

among aromatic ring-hydroxylating dioxygenases, we se-quenced benABC and xylXYZ. The sequencing of the lattergenes is presented elsewhere (20). In this report, we presentthe nucleotide sequence of the A. calcoaceticus benABCgenes. The deduced amino acid sequences of the geneproducts responsible for the hydroxylation of benzoate werecompared with those of enzymes which catalyze similarreactions.

MATERIALS AND METHODS

Bacterial strains, plasmids, and bacteriophages. Recombi-nant plasmids derived from pUC19 (69, 76) carrying wild-type DNA ofA. calcoaceticus BD413 (29) (designated ADP1in our collection) from the chromosomal ben region havebeen previously described (42). The 3.4-kbp insert ofpIB1354 carrying benABC was subcloned into the replicativeform of bacteriophages M13mpl8 and M13mpl9 (76). Similar

RESULTS

Nucleotide sequence of benABC genes. The benABC geneswere previously localized to a 3.4 kbp HindIII-EcoRI restric-tion endonuclease fragment cloned from the A. calcoace-ticus ADP1 chromosome (42). The nucleotide sequence ofthis 3,353-bp DNA fragment was determined and, as shownin Fig. 2, it contained three open reading frames. Computeranalysis by three different methods (12, 32, 57) suggestedthat each open reading frame would be likely to encode aprotein. The first open reading frame, corresponding tobenA, started at nucleotide 347 (Fig. 2). An ATG transla-tional start codon was preceded by the putative ribosomebinding sequence GGAG (58). Translation would yield aprotein product of 52,079 Da, the size of the predictedbenA-encoded hydroxylase subunit (42).The second open reading frame, corresponding to benB,

started at nucleotide 1729. An initiation codon at this posi-tion was preceded by the ribosome binding sequenceGGAG. The deduced molecular mass of the benB product,20,073 Da, was in agreement with previous in vitro analysis(42). The ATG initiation codon of benB overlapped with the

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VOL. 173, 1991 A. CALCOACETICUS benABC SEQUENCE 5387

AAGCTTTOT CCTCMCCAC AGCCACAAA TAGCGGAAT GTCTAGTT CAIT?AAA TACTCTAG GTATTTATT ATACMAAAA TOTOTTTGAA CTTMTTAAA ATCTT AOGATAMC 130AAGCAMAGM CACAAGAAGA AGGCAGGGGC TTGMCCCATT AAATGCTTTC TTCAATTTGG AAAMTGOAAA GCTGAMTGG ATATTCGT?T TATTT TTCTOCCGT? AAGTAMACAT TTTrATGCGTT 260GCGTTGTT?A ATTGAATGTT TGACTAGMC CAOCGTTT?G CTCTGOCCTAk GACAAGTTTC T TTGAATG7TWIW AAGGAT

ATG CCA COT ATT CCC GTC ATT AAT ACT AGC CALT CTTMet Pro Arg Ile Pro Val Ile Asn Thr Sor Hiis Leu

GAC CGA ATT GALT GAA CTG CTT GTA GAC AAT ACC GAA ACA GGT GAA TTT AAG TTA CAT CGT TCT GTA TT? ACA GAT CAG GCA CT? TTT GAT CTT GMA ATG AMA TAC 487Asp Arq Ile Asp Glu Lou Loeu Val Asp Asn Thr Glu Thr Gly Glu Phe Lys Lau His Arg Ser Val Phe Thr Asp Gin Ala LOU Ph. AsP Leu Glu Met Lys TyrIATT TTC GMA OGG AAT TOG GTT TAT TTG OCT CAT GMA AGC CAG AT? CCC AAC AAC AAC GAC TAT TAC ACC ACTT AT?GCAAC C ATTT ATGCCO 592

Ile Phe Glu Gly Asn Trp Val Tyr Lou Ala His Glu Ser Gin Ile Pro Asn Asn Asn Asp Tyr Tyr Thr Thr Tyr Ile Gly Arg Gin Pro Ile Lou Ile Ala ArgAAT CCT AMC GGT GAA CTC AA.C GCC MTG AT? AAC GCA TOT TCA CAT CGT GOT GCA CAG CTG CTC GOT CAT AAG CGT GGT AAT AMG ACC ACA TMT ACT TOC CCA TT? 697Asn Pro Asn Gly Glu Lou Asn Ala Met Ile Asn Ala Cys Ser His Arg Gly Ala Gin Leu Lru Gly His Lys Arg Gly AE[ Lys Thr Thr Tyr Thr Cys Pro PheCAT GGC TGG ACC TTC ART ARC TCA OGA AAM T?G T?G AMG GTM ARAT CCA AMC GAT GCT GGT TAT TCA GAT TGT TTT AMT CMG GAC GOT TCC CMC GAC T?A AMA 802His Gly Trp Thr Ph. Asn Asn Ser Gly Lys Leu Leu Lys Val Lys Asp Pro Ser Asp Ala Gly Tyr Ser Asp Cy2 Ph. Aon Gin Asp Gly Ser His Asp Leu LysAMG GTG GCG CGT T?T GMA MGT TAT AMA GGT TT? T?A TIT? GGC ACT CTG AMT CCT GTA GAT CCG TCA CTG CM GMG T?T TTO GMG GM ACC ACC AMA AT? ATC GAC 907Lys Val Ala Arq Ph. Giu Ser Tyr Lys Gly Ph. Lou Ph. Gly Sor Lou Asn Pro Val Asp Pro Ser Leu Gin Glu Phe Lou Gly Gin Thr Thr Lys Ile Ile Asp

ATG AT? GTC GGG CMA TCC GAT CMG GGC CT? GMA GTA CTG CGT GGT GTT TCG MCc TM MCc TAT GMA OGA AMc TGG AMG TTO MCC GCA GMA AMC GGA WCA GAT GMC 1012Met Ilie Val Gly Gin Sor Asp Gin Gly Lou Glu Val Lou Arq Gly Val Ser Thr Tyr Thr Tyr Glu Gly Asn Trp Lys Lou Thr Ala Glu Aou Gly Ala Asp Gly

TAT CAT GTT TCG GCG GTG CMC TOG AMC TAT GCA GCCAMC MCG CM CAT CGT AMa GMA AMA CM GCA GOT OT ACx MTT CGC GCG MTG AGC GCG GC TCG, TOG GGG 1117 benATyr His Val Ser Ala Val His Trp Asn Tyr Ala Ala Thr Thr Gin His Arg Lys Glu Lys Gin Ala Gly Asp Thr Ilie Arg Ala Met Ser Ala Gly Ser Trp GiyAMA CAT GGT GGC GGT TCA TAT GGA T?T GMA CAT GGT CAT MTG T?G CTC TGG MAc CM TGG GG AAT CCG GMA GMz cGa CCA AM IT?T CC? AMA GCAGCGMG TMT 1222

Lys Hiis Gly Gly Gly Ser Tyr Gly Ph. Giu His Gly His net Lou Lou Trp Thr Gin Trp Gly Asn Pro Giu Asp Arg Pro Ann Ph. Pro Lys Ala Ala Gin TyrMCC GMA AMA TTC GGT GCT GCA ATG TCG AMA TOG MTG MTC GMA CCC TcA CGT AMc T?G TGT T?A TAT ccA AMc GTG TMC CTG ATG GAT CMG IT?T GOT TCC CM AT? 1327Thr Glu Lys Ph. Gly Ala Ala Met Ser Lys Trp Met Ile Glu Arq Ser Arg Asn Lou Cys Lou Tyr Pro Asn Val Tyr Lou Het Asp Gin Ph. Gly Ser Gin IleCGT GT? T?A COT CCA AT? TCG GTC AAT AMA MC GMA GTC McC AT? TMc TGT AT? GCG CCT GTA GOT GMA GCA CCC GM GCC COT GCA CGC COT MTr C= CMG TMT 1432 1Arq Val Lou Arg Pro Ile Ser Val Asn Lys Thr Giu Val Thr Ile Tyr Cys Ile Ala Pro Vai Gly Glu Ala Pro Glu Ala Mrg Ala Arg Mrg Ile Mrg Gin ITyrGMA GAT TTC TTT MRT GCA TCT GG ATMG GCG MCG CCA GMC GAT CT? GMG GM T?G CCT cGc TGT CMG GCT GGT TAT GCA GG ATC GMA CTG GM TGG AMC GMC ATG 1537Glu Asn Ph. Ph. Asn Ala Sor Gly Not Ala Thr Pro Asp Asp Lou Glu Glu Lou Pro Arg Cys Gin Ala Giy Tyr Ala GLy Ile Glu Lou Gin Trp Asn Asp M4etTGC CGC GGA TCA AMA CAT TGG AT? TMT GGA CCA GAT GAT 0CC GCT MAT GMA ATC GOA T?A AMA CCG OCT MTT MGT GOT MTT AMA ACT GMA GMC GM GOC ITOG TMT 1642Cys Arg Gly Sor Lys His Trp Ile Tyr Giy Pro Asp Asp Ala Ala Asn Giu Ile Gly Loy Lys Pro Ala Ile Ser Gly Ile Lys Thr Glu Asp GLu GLy Lou TyrT?G WCA CMG CAT CMA TMC TOG CTC AMA MCT MTG AMG CMA GCG MTT OCT 0CG GMA AMA GMA T?T MA TCG COT CAGMB_ AMC GCATGAAT GCT MAC GCA CTT T?A 1749Lou Ala Gin His Gin ['yr Trp Lou Lys Ser Met Lys Gin Ala Ile Ala Ala Glu Lys Giu Ph. Ala Sor Arg Gin Gly Gin Azi Al-%End

MetAsn Ala Thr Ala Lou LouGAC MCC AIC MGC ATC GMA CMG AT? MOC CMG TT? T?G VTAT C GAO 0CC GOT T?T T?A GAT OAT GAG CAA TOG GAT GMc TOG cTT? GM TOT TMT GCA CCT CM 0CC TCA 1857Asp Thr Ile Ser Ilie Glu Gin Ile Ser Gin Ph. Lou Tyr Ser Giu Ala Arg Phe Lou Asp Asp Giu Gin Trp Asp Asp Trp Lou Glu Cys Tyr Ala Pro Gin Ala SerTT? TOO ATG CCC 0CC TOO GMC OAT AMc GM CMG CTC MCT GM AMRCC CM MCT GM MTT TCG CTGOAMT TAT TAT CCA CAT CCC CMA GOT CT? GMA OAT CGAL OTA TT? 1965Ph. Trp flat Pro Ala Trp Asp Asp Asn Asp Gin Lou Thr Glu Asn Pro Gin Thr Gle Ile Ser Lou Ile Tyr Tyr Pro Asp Mgq Gin Gly Lou Glu Asp Mgq Vai Ph.e

COO MTT AMA MC GMG COT TCA TCC GCG MCC MTG CCA OAT MCG CCT MCG GCA CMC MAT MTT MGC AAT ATC GM OTT GMA TCA COT GAT GO;C CT? CMP ATC MCA OTA CCT 2073 benBMgq Ile Lys Thr Glu Mgq Ser Ser Ala Thr Not Pro Asp Thr Mgq Thr Ala His Asn Ilo Ser Asn Ilie Glu Val Glu Ser Mrg Asp Gly Lou Gin Ile Thr Val MgrqTT? AMc TOG MT MG CTC MGT TTc COC TMT AMA AMC MT TMC MGC TMT T?T CCC MTG TcA CCC TAT OTA MTC GAT T?C TCA GOT GMA CMA CCA AMA ATC T?G MGC AMG 2181IPh. Asn Trp Asn Thr Lou Ser Ph. Mgq Tyr Lys Asn Ser Tyr Sor Tyr Phe Gly Not Ser Mgq Try Vai Ile Asp Ph. Ser Gly Giu Gin Pro Lys Ile Lou Ser Lys

TMT GTG MTG CT? AMG MT GMC TAT MTT AAT CMA OTC AT? OAT MTT TAT CAT AT? TAA, ATCAGTTTA, C cTaTcMh AGGACACT? GACTOMCG- T?T ATO MGT T?A TAkT T?G 2295Tyr Val Met Lou Lys A.w[ Asp Tyr Ile Asn Gin Val Ilie Asp Ilie Tyr His Ile End Met Ser Lou Tyr Lou

AMT AGG AT? CCT 0CC MTG TCA AMC CAT CMh GTA GCA CTT CMA T?T GMA CAT GOC GTT?AMC CCT T?T MTC TGC MTC OCT CM GOT GM MCC T?A TCC GAkT GCA GCA TMC 2403Asn Mgq Ile Pro Ala Met Ser Asn His Gin Val Ala Lou Gin Ph. Giu Asp Giy Val Thr Mgq Ph. Ile Cys Ilie Ala Gin Giy Glu Thr Lou Sor Asp Ala Ala Tyr

COT CMG CMA ATC MAT AT? CC ATMG GMC TOC COT GMA CCC GMG TOT GOT MCC TGC CCT OCT TT'? TOT GMA TCG GCC AMC TMT GMC ATO CCT GMk GMk AAT TMC AT? GMA 2511Mrg Gin Gin Ilie Asn Ile Pro Met Asp Cys Mrg Giu Giy Giu Cys Giy Thr Cys Mrg Ala Ph. Cys Giu Ser Giy Asn Tyr Asp Met Pro Giu Asp Asn Tyr I1e Glu

OAT GCA CTC MCC CCA GM GMA 0CC CMG CMG GC TMC GM' T?G OCA TOT CM TOC C? CCA MCT TCA OAT OCT OTA TI'? CM MTT CMG GCG TCT TCT GMG OTA TOT AMA 2619Asp Ala Lou Thr Pro Glu Giu Ala Gin Gin Gly Tyr Val Lou Ala Cys Gin Cys Mgq Pro 'Thr Ser Asp Ala Val Pho Gin Ilie Gin Ala Ser Ser Glu Val Cys Lys

MCC AMG AT? CAT CMC IT?T GMA GOC MCG TTO 0CC CCG OTT GMA MRT CTA TCC OAT TCICCMCTC MCC TT'? CAT MTT CMG CTC OAT GMC GOT CMG CCC GOAT AT? CAT TTT 2727Thr Lys Ile His His Ph. Giu Gly Thr Lou Ala Mgq Val Giu Asn Lou Ser Asp Ser Thr Ilie Thr Ph. Asp Ile Gin Lou Asp Asp Gly Gin Pro Asp Ile His Pho

CTO GCA 000 CMG TAT GTC AMC GTO MCG CTG CCC CCC MCC McG GMA MCA CCC TCC TAT TCG TI'? MC TCA CMh CCA CCC MAT C= T?A MCC 000 TMCMOT GT? COT MT 2835 be-nCLoeu Ala Gly Gin Tyr Vai Asn Val Thr loeu Pro Giy Thr Thr Giu Thr Mgq Ser Tyr Ser Ph. Ser Ser Gin Pro Giy Asn Mgq Lou Thr Gly Ph. Val Vai Mrg Asn

OTO CCC CMh GOT AMA ATO MOC GMK TMT T?KAGM TGOT CM CC AMA GCA OGC GMC AMA MTG MC T?T MCT GGA CCA TI'? GOT MaT T?T TMT CTO COT OAT OTC AMG COT 2943Val Pro Gln Giy Lys Met Ser Glu Tyr Lou Ser Val Gin Ala Lys Ala Gly Asp Lys Met Ser Ph. Thr Giy Pro Ph. Giy Sor Ph. Tyr Lou Mgc Asp Val Lys MgrqCCT CrG CTC ATO CTO OCT CCC GOT MCG OGG ATC OCA CCG T?T T?A TCC MTG T?G CMA GTA CT? GM Cm AMA GCC MGT GMG CAT CCA OTA CGA CTG OTG IT?T CCC OTA 3051Pro Val Lou Met Lou Ala Gly Gly Thr Gly Ile Ala Pro Phe Leu Ser Met Leu Gin Val Lou Glu Gin Lye Giy Ser Giu His Pro Val Mgq Lou Val Ph. Gly Val

AMC CMA OAT TOT GMT CTO GGTOC CT? GMA CMA CTC OAT GCA CTT CM CMG AMA CTA CCA TOO T?T GMA TAT CCT MC GTG OTm GCA CAT GCA GM MCT CMA CAT GMA 3159Thr Gln Asp Cys Asp Lou Val Ala Lou Giu Gin lou Asp Ala Lou Gin Gin Lys Lou Pro Trp Ph. Giu Tyr Mgq Thr Val Vai Ala His Ala Giu Ser Gin Htis Glu

COT AMh GOT TMC GTO MG GOT CAT ATC GMA TMT GMC TOO CTA MT GcC GOT GMA OTT CAT OTG TAT cTG TOC GGA CCG CI' CCT MTG OTM GMA GCG OTM CCC MC TGG 3267Mrg Lys Gly Tyr Val Thr Giy His Ile Glu Tyr Asp Trp Lou Asn Giy Giy Giu Val Asp Val Tyr Lou Cys Giy Pro Val Pro Met Val Glu Ala Val Mgq Ser Trp

CTO OAT MCG CMA GOT AT? CMA CCC 0CC AMC TT'? T?A T?T GMA AMA T?C TCT 0CC AMC TMA CTCAAAACCC AOACCOATATOAMT?Lou Asp Thr Gln Giy Ilie Gin Pro Ala Asn Ph. Lou Ph. Glu Lys Ph. Ser Ala Asn End

FIG. 2. DNA sequence of the 3,353-bp chromosomal A. calcoaceticus Hindlll-EcoRI restriction endonuclease fragment carrying thebenABC genes. The open reading frame corresponding to each gene is marked by broken lines, and within each open reading frame,nucleotides are arranged as codons above the deduced amino acid sequence. Numbers indicate the nucleotide positions of adjacent bases.Possible ribosome binding sites are underlined at positions 336, 1718, and 2273. A second possible initiation codon and ribosome binding sitefor the translation of benC are found at positions 2299 and 2311, respectively.

TGA stop codon of benA. This arrangement, which may be the third open reading frame, benC. At positions 2281 andinvolved in translational coupling, has been observed previ- 2311, potential initiation codons were preceded by theously with catabolic genes, in particular those, like benA and ribosome binding sequences GAGG and AGGA, respec-benB, encodingz nonidentical enzymatic subunits (44). tively. Sequence comparisons described below suggLestedTwo possible translational initiation sites were found for that benC starts at position 2281. Translational initiation at

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5388 NEIDLE ET AL. J. BACTERIOL.

BenA MPRIPVINTSHLDRIDELLVDNTETGEFKLHRSVFTDQALFDLEMKYIFEGNWVYLA H E

XylX MTMHLGLDYIDSLVEEDENEGIYRCKREMFTDPRLFDLEMKHIFEGNWIYLA H E

BnzA(P1) MNQTDTSPIRLRRSWNTSEIEALFDEHAGRIDPRIYTDEDLYQLELERVFARWWLLLG H E

TodC1 MNQTDTSPIRLRRSWNTSEIEALFDEHAGRIDPRIYTDEDLYQLELERVFARSWLLLG H E

NdoB MNYNNKILVSESGLSQKHLIHGDEELFQHELKTIFARNWLFLT H D* * * * * * I.±

BenA SQIPNNNDYYTTYIGRQPILIARNPNGELNAMINA C S H RGAQLLGHKRGNKTTYT C PF H G

XylX SQIPEKNDYYTTQMGRQPIFITRNKDGELNAFVNA C S H RGATLCRFRSGNKATHT C SF H G

BnzA(P1) TQIRKPGDYITTYMGEHPVVVVRQKDASIAVFLNQ C R H RGMRICRADAGNAKAFT C SY H G

TodCl TQIRKPGDYITTYMGEDPVVVVRQKDASIAVFLNQ CIR H RGMRICRADAGNAKAFT C SY H G

NdoB SLIPAPGDYVTAKMGIDEVIVSRQNDGSIRAFLNV CIR H RGKTLVSVEAGNAKGFV C SY H G

* ** * * *i.* LI** ** * **BenA WTFNNSGKLLKVKDPSDAGYSDCFNQDGSHDLKKVARFESYKGFLFGSLNPVDPSLQEFLXylX WTFSNSGKLLKVKDPKGAGYPDSFDCDGSHDLKKVARFASYRGFLFGSLREDVAPLEEFLBnzA(P1) WAYDTAGNLVNVPYEAES-FA-CLNKKEWSPLK--ARVETYKGLIFANWDENAVDLDTYLTodCl WAYDTAGNLVNVPYEAES-FA-CLNKKEWSPLK--ARVETYKGLIFANWDENAVDLDTYLNdoB WGFGSNGELQSVPFEKDL-YGESLNKKCLG-LKEVARVESFHGFIYGCFDQEAPPLMDYL

* * * * ** ** * * *

BenA GETTKIIDMIVGQSDQGLEVLRGVSTYTYEGNWKLTAEN-GADG Y H VS AV H WNYAATTQXylX GESRKVIDMVVDQSPEGLEVLRGSSTYVYEGNWKVQVEN-GADG Y H VS TV H WNYAATQQ

BnzA(P1) GEAKFYMDHMLDRTEAGTEAIPGVQKWVIPCNWKFAAEQFCSDM Y H AGTTS H LSGILAGL

TodCl GEAKFYMDHMLDRTEAGTEAIPGVQKWVIPCNWKFAAEQFCSDM Y H AGTTS H LSGILAGL

NdoB GDAAWYLEPMFKHS-GGLELVGPPGKWVIKANWKAPAENFVGDA Y H VG WT H ASSLRSGE

* ** *** * *** *Li

BenA HRKEKQAGDT----------IRAMSAGSWGKHGGGS Y GFEHGHMLLWTQWGNPEDRPNFP

XylX QRKLRDAGDD----------IRAMTASSWGGDGGGF Y SFENGHQMVWARWGDPKNRPLFABnzA (P1) PEDLEMADLA--PP-TVGKQ----YRASWGGHGSGF Y VGDPNLMLAIMGPKVTSYWTEGP

TodC1 PEDLEMADLA--PP-TVGKQ----YRASWGGHGSGF Y VGDPNLMLAIMGPKVTSYWTEGP

NdoB SIFSSLAGNAALPPEGAGLQMTSKYGSGMGVLWDG- Y SGVHSADLVPELMAFGGAKQERL

BenA KAAEYTEKFGAAMSKWMIERSRNLCLYPNVYLMDQFGSQI-RVLRPISVNKTEVTIYCIA

XylX ERDRLASEFGEARADWMIGVSRNLCLYPNLYLMDQFGSQL-RITRPLSVDRTEITIYCIA

BnzA (P1) ASEKAAERLGSVERGSKLMVEHMTVFPTCSFLP---GINTVRTLASARAERGEVWAFTVVTodCl ASEKAAERLGSVERGSKLMVEHMTVFPTCSFLP---GINTVRTWiPRGPNEVEVWAFTVVNdoB NKEIGDVRARIYRSHLNCTVFPNNSMLTCS------GVFKVWNPIDANTTEVWTYAIVEK

BenA PVGEAPEARARRIRQYEDFFNASGMATPDDLEELPRCQAGYAGIELEWNDMCRGSKHWIYXylX PKGETPR-RARRVRQYEDFFNVSGMATPDDLEEFRACQEGFAGGGM--NDMSRGAKHWIEBnxA (P1) DADAPDDIKEEFRAR-LRTFSPVACSSRTTGRT-GSRSSTSCEATSRSRPFNAEM-----

TodCl DADAPDDIKEEFRRQTLRTFSAGGVFEQDDGENWVEIQHILRGHKARSRPFNAEM-----

NdoB DM--PEDLKRRLADSVQRTFGPAGFWESDDNDNMETASQNGKKYQSRDSDLLSNL-----

BenA GPDDAANEIGLKPAISGIKTEDEGLYLAQHQYWLKSMKQAIAAEKEFASRQGENAXylX GPDEGAKEIDLHPKLSGVRSEDEGLFVMQHKYWQQQMIKAVKREQDRLIHAEGVBnzA (P1) SMDQTVDNDPVYPGRISNNVYSEEAARGLYAHWLRMMTSPDWDALKATRTodCl SMDQTVDNDPVYPGRISNNVYSEEAARGLYAHWLRMMTSPDWDALKATRNdoB GFGEDVYGDAVYPGVVGKSAIGETSYRGFYRAYQAHVSSSNWAEFEHASSTWHTELTKTTDR

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FIG. 3. The a-subunits of the hydroxylase components of five multicomponent aromatic ring dioxygenases appear to be homologous.BenA, XylX, BnzA (P1) (27), TodC1 (79), and NdoB (34) are approximately 50-kDa components of the benzoate, toluate, benzene, toluene,and naphthalene 1,2-dioxygenases, respectively. Positions at which amino acid residues of all proteins are identical are indicated by asterisks.Conserved histidines and tyrosines which may coordinate mononuclear Fe(II) atoms are enclosed by boxes. Two conserved cysteines are alsoenclosed by boxes, and these residues together with the closest conserved histidines may bind a Rieske-type [2Fe-2S] center. Gaps introducedinto the amino acid sequences for the purpose of alignment are indicated by dashes.

this position would yield a 38,787-Da protein, in goodagreement with the predicted size of 38 kDa (42).Codon usage and G+C content of benABC. The G+C

content of the benA, -B, and -C genes, 45, 41, and 47%,respectively, reflected the characteristic 38 to 47% valuefound in Acinetobacter species (28). Similar codon usagewas observed among the three genes and was much like thatfound in previously sequenced A. calcoaceticus structuralgenes (6, 8, 13, 14, 41, 45, 54). The most marked preferencefor particular codons was observed for arginine, for whichtwo of six possible codons predominated. The CGT andCGC codons were used 50 to 63% and 13 to 30% of the time,respectively. The bias toward using preferred codons wasfound in E. coli to be a measure of gene expression levels (1).

Although relatively few genes have been sequenced fromAcinetobacter species, a comparison of codon usage inbacteria of this genus with that found in E. coli (1) suggestedthat the general patterns of codon usage are similar. Differ-ences included a bias toward using the GCA codon foralanine and the CAA codon for glutamine by Acinetobacterspecies. The E. coli preference for the CTG (leucine) codonwas not observed in benABC. A comparison of codons usedfor proline showed that the E. coli bias toward CCG wasreplaced by one in Acinetobacter species for CCA. The TCCcodon for serine was used in fewer than 10% of the serineresidues by Acinetobacter species but not by E. coli.

Choice of related sequences for comparison with the de-duced amino acid sequences of BenABC. The deduced aminoacid sequences of the protein components of the A. calco-aceticus benzoate 1,2-dioxygenase system were comparedwith those of other aromatic ring-hydroxylating dioxygena-ses for which sequence data are available. These include the

BenAXylXBnzA(P1)TodClNdoB

R.n.R.c.S. c.P.d.B.j.

TodBlNdoABnzC (P3)

MINAFVNAFLNQFLNQFLNV

LIGVMLGVMLGIMIGVVIGI

CCCC

SiSRRR

HHHHH

RGAQLLGHKRGNKTTYTRGATLCRFRSGNKATHTRGMRICRADAGNAKAFTRGMRICRADAGNAKAFTRGKTLVSVEAGNAKGFV

C T H LGCVPIAN-AGCFGGYYC T H LGCVPMGDLSGDFGGWFC T H LGCVPIGE-AGDFGGWFC T H LGCVPIGDGAGDFGGWFCITH LGCIPIAHE-GNYDGFF* * * *

benzene, naphthalene, toluene, and toluate dioxygenases.Irie et al. (27) determined the nucleotide sequence of achromosomal DNA fragment of P. putida carrying fourgenes, bnzABCD, encoding the benzene 1,2-dioxygenasesystem. The bnzABCD gene products have been designatedP1 to P4, and we use these designations parenthetically.Sequence data for three components of naphthalene 1,2-dioxygenase, encoded by ndoABC, were derived from DNAof P. putida NCIB 9816 carrying a naphthalene-degradingplasmid (34). The nucleotide sequences of the todCJC2BAgenes for toluene 1,2-dioxygenase have been determinedfrom chromosomal DNA of P. putida Fl (79). In addition,we have determined the nucleotide sequence of the TOLplasmid pWWO xylXYZ genes encoding toluate 1,2-dioxy-genase (20).

In the hydroxylase components, the greatest degree ofsimilarity was found in the N-terminal region of the a-sub-units. The amino acid sequences of BenA, XylX, BnzA (P1)(27), TodCl (79), and NdoB (34) were all found to be similar(Fig. 3). Pairwise comparisons showed that 62% of thealigned BenA and XyIX residues were identical, whereascomparison of the BenA sequence with each of the others inFig. 3 revealed 20 to 24% identity. These values reflected theaveraging of approximately 30% identity in the N-terminalregions of the protein sequences and 10% identity in theC-terminal regions. Each of these proteins is an approxi-mately 50-kDa subunit of a dioxygenase hydroxylase com-ponent. Two cysteine residues and two histidine residues,conserved among all the hydroxylase subunits, were foundto align with conserved residues in Rieske iron-sulfur pro-teins (Fig. 4). As discussed below, these residues may binda Rieske-type [2Fe-2S] cluster (53).

C

IC

CCCC

*

PF HSF HSY HSY HSY H

PC HPC HPC HPC HPC H

*

GGGGG

GIGIGIG

*

VQDT C T H GDWALSDGYLDGDIVE TL H FGTDNL C T H GSARMSDGYLEGREIE C PL H QGVQDT C T H GDWALSDGYLDGDIVE C TL H FG

* * * * ***** * * * * * *

MULTICOMPONENTDIOXYGENASES:HYDROXYLASEa-SUBUNITS

RIESKEIRON-SULFURPROTEINS

MULTICOMPONENTDIOXYGENASES:nFERREDOXIN"COMPONENTS

FIG. 4. Two conserved cysteines and histidines may coordinate [2Fe-2S] clusters. Similar alignments of these residues, enclosed in boxes,suggested that the aromatic dioxygenase subunits, BenA, XylX, BnzA (P1) (27), TodCl (79), and NdoB (34), are Rieske-type iron-sulfurproteins. The small iron-sulfur proteins, TodBl, NdoA, and BnzC (P3), of the toluene, naphthalene, and benzene dioxygenases, respectively,also have cysteines and histidines similarly aligned with those of the Rieske iron-sulfur proteins of Rattus norvegicus (R.n.) (43), Rhodobactercapsulatus (R.c.) (10), S. cerevisiae (S.c.) (3), Paracoccus denitrificans (Pd.) (35), and Bradyrhizobium japonicum (B.j.) (66). Residuesidentical in all aligned positions are indicated by asterisks.

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BenB MNATALLDTISIEQISQFLYSEARFLDDEQWDDWLE-CYAPQASFWMPAWDDN---DQLTENPQTEISLIYYPDRQGLEDXylY MTISYEAVRDFLYREARYLDDKQWESWLE-MYAPDATFWMPAWDDR---DQLTEDPQSQISLIWYGNRSGLEDNdoC MMINIQEDKLVSAHDAEEILRFFNCHDSALQQEATTLLTQEAHLLDIQAYRAWLEHCVGSEVQYQVISRELRAASERR--YKLNEAMNVYNENFQQLKVBnzB(P2) MIDSANRADVFLRKPAPVAPELQHEVEQFYYWEAKLLNDRRFEEWFAL-LAEDIHYFMPIRTTRIMRDSRLEYSGSREYAHFDDDATMMKGTodC2 MIDSANRADVFLRKPAPVAPELQHEVEQFYYWEAKLLNDRRFEEWFAL-LAEDIHYFMPIRTTRIMRDSRLEYSGSREYAHFDDDATMMKG

** * *

BenB RVFRIKTERSSATMPDTRTAHNISNIEVESRDGLQITVRFNWNTLSFRYK---NSYSYFGMSRYVI--DFSGEQPKILSKYVMLKNDYINQVIDIYHIXylY RVFRIKTERSSATIPDTRTSHNISNLELLEQSDGVCKLRYNWHTMNYRYK---TVDHFFGTNFCTL--DTCGETPLITAKKVVLKNDYIRQVIDVYHVNdoC RVEHQLDPQNWGNSPKLRFTRFITNVQAAMDVNDKELLHIRSNVILHRARRGNQVDVFYAAREDKWKRGEGGVR-KLVQRFVDYPERILQTHNLMVFLBnzB(P2) RLRKITSDVSWSENPASRTRHLVSNVMI-VGAEAEGEYEISSAFIVYRNRLERQLDIFAGERRDTLRRNTSEAGFEIVNRTILIDQSTILANNLSFFFTodC2 RLRKITSDVSWSENPASRTRHLVSNVMI-VGAEAEGEYEISSAFIVYRNRLERQLDIFAGERRDTLRRNTSEAGFEIVNRTILIDQSTILANNLSFFF

* * * * *

FIG. 5. Alignment of the P-subunits of the aromatic hydroxylase components. BenB, XylY, NdoC (34), BnzB (P2) (27), and TodC2 (79)are the approximately 20-kDa proteins of the benzoate, toluate, naphthalene, benzene, and toluene dioxygenases, respectively. Nine residuesmarked by asterisks are identical in all sequences. Dashes indicate sequence gaps.

Amino acid sequence similarities were also discoveredamong BenB, XylY, NdoC (34), BnzB (P2) (27), and TodC2(79) (Fig. 5). In the aligned sequences of BenB and XylY,56% of the amino acids were identical. In pairwise com-parisons of BenB with each of the other sequences shownin Fig. 5, 16 to 18% of the amino acids were identical.Although these values are relatively low, the sequencesimilarities were shown to be significant by the method ofNeedleman and Wunsch (40), with similarity scores above4.2. Each of these proteins is an approximately 20-kDan-subunit of a hydroxylase, and nine residues, marked byasterisks in Fig. 5, were found to be invariant among theprotein sequences.The BenC and XylZ electron transfer components were

compared with both ferredoxins and oxidoreductases. BenCwas similar to XylZ (49% identical residues) but not to anycomponents of the aromatic dioxygenases with two-proteinelectron transfer systems. Homology was detected, how-ever, between BenC and XylA, a protein component ofxylene monooxygenase (62) (Fig. 6). XylA may be involvedin the transfer of electrons from NADH to XylM, thehydroxylase component of this two-subunit monooxygen-ase. The BenC and XylA sequence similarities extendedthroughout the entire length of the proteins; 28% of thealigned residues were identical.As shown in Fig. 6 and 7, the N-terminal region of BenC

was found to resemble chloroplast-type ferredoxins. Resi-dues important for ferredoxin structure and function (4, 49)were conserved in BenC, including the four cysteine resi-dues which coordinate [2Fe-2S] clusters. In addition, thisferredoxinlike region of BenC was similar to the C-terminalregion of VanB, a 33-kDa protein involved in vanillatedemethylation (5) (Fig. 6). In this region, 24% of the alignedVanB and BenC amino acid residues were identical, includ-ing those, like the four cysteines, which are conservedamong chloroplast-type ferredoxins.The C-terminal region of BenC was found to be homolo-

gous to the C-terminal regions of ferredoxin-NADP+ re-ductases from spinach (30) and from Spirulina species (77).In the alignment shown in Fig. 6, 20% of the BenC C-termi-nal residues were identical to those of a ferredoxin reduc-tase. A stretch of roughly 130 amino acids between positions190 and 320 of BenC was also found to resemble a similarlysized region of two other oxidoreductases: NADPH-cy-tochrome P-450 reductase isolated from Saccharomycescerevisiae (72), pigs (18), rats (50), and rabbits (31); andNADPH-sulfite reductase isolated from E. coli and Salmo-

nella typhimurium (46, 48). In this region, the identitybetween BenC amino acid residues and those of each alignedoxidoreductase was 21 to 37%. The regions of strongesthomology, shown in Fig. 8, may be involved in NAD(P) andFAD binding.

DISCUSSION

Nonaromatic cis-diols are common intermediates in thedegradation of a wide variety of aromatic compounds. Suchdiols are usually formed by reactions catalyzed by multicom-ponent dioxygenases. All the hydroxylating dioxygenasesstudied to date have been composed of a hydroxylasecomponent, usually composed of nonidentical a- and p-sub-units, and a one- or two-protein electron transfer system (9).In this study, we compared the sequences encoding A.calcoaceticus benzoate 1,2-dioxygenase with those of the P.putida pWWO TOL plasmid encoding toluate 1,2-dioxygen-ase. In addition, comparisons were made with sequences forfunctionally related enzymes.The hydroxylase components of various multicomponent

dioxygenases were found to be similar. The sizes of the a andP subunits in the different dioxygenases discussed areroughly 50 and 20 kDa, respectively. A comparison of theamino acid sequences of the benzoate, toluate, benzene,toluene, and naphthalene 1,2-dioxygenases suggested thatthe a-subunits and the P-subunits are each derived fromcommon ancestors. The conservation of amino acid se-quences was strongest in the N-terminal region of thea-polypeptides. This region may be responsible for com-mon hydroxylase functions such as recognition and activa-tion of two oxidizable carbons of the aromatic ring, recep-tion of electrons from the electron transfer components,binding to the P-subunit, and binding and activation ofoxygen.The binding of oxygen, which usually involves metal

cofactors, may be mediated by mononuclear Fe(II). Onesuch iron was shown to be associated with each a-subunit ofthe benzoate 1,2-dioxygenase from P. arvilla (75). The ironatom is likely to be coordinated by tyrosine and histidineresidues of the polypeptide. Five invariant histidines andtwo invariant tyrosines were detected in the alignment of thefive a-subunits (Fig. 3). Some of these residues may beinvolved in iron binding.The arrangement of two of the invariant histidines with

two conserved cysteine residues (Fig. 4), however, sug-

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VanB 214 aa-YFAAPVQPAGDARAFEGRLARSGLTLQVP--AERSVAQVLDDAGVCIPLACEQGICGTCLFrdox ATYKVTLINEEEGINAILEVADDQTILDAGEEAGLDLPSSCRAGSCSTCAXylA KKISGLFVPPPESTVSVRGQGFQFKVPRGQTILESALHQGIAFPHDCKVGSCGTCKBenC MSLYLNRIPAMSNHQVALQFEDGVTRFICIAQGETLSDAAYRQQINIPMDCREGECGTCRXylZ MTHKVATDFEDGVTRFIDANTGETVADAAYRQGINLPLDCRDGACGACK

* * *

VanB TRVLDGEPEH-----RDSFLTDAERARNDQFTPCCSRARSACLVLDLFrdox GKLVSGAAPN---QDDQAFLDDDQLAA-GWVMTCVAYPTGDCTIMTHQESEVL ---------

XylA YKLISGRVNE--LTSSAMGLSGDLYQS-GYRLGCQCIPKEDLEIELDTVLGQALVPIETSAL--BenC AFCESGNYDMPEDNYIEDALTPEEAQQ-GYVLACQCRPTSDAVFQIQASSEVCKTKIHHFEG--XylZ CFAESGRYSL-GEEYIEDALSEAEAEQ-GYVLTCQMRAESDCVIRVPAASDVCKTQQAGYQA--FNR1 AKTDIPVNIYI--FNR2 QIASDVEAPPPAPAKVEKHSKMEEGITVNKFK--

**

XylA ISQKRLHFEAHDIVEMEVVPD-------------KQIAFYPGQYADVECAECSA-----BenC TLARVENLSDSTITFDIQLDDGQ------------PDIHFLAGQYVNVTLPGTTE-----XylZ AISNVRQLSESTIALSIKSASL-------------NQLAFLPGQYVNLQVPGSDQ-----FNR1 YKPKNPYIGKCLSNEELVREGGTGTVRHLIFDISGGDLRYLEGQSIGIIPPGTDNNGKPHFNR2 FKPKTPYYGRCLLNTKITGDDAPGETWHMVFS-HEGEIPYREGOSVGVIPDGEDKNGKPH

FAD-ppi binding region of FNR

* ** * * *

XylA -V RSYSFSA PPQPDGSLSFHVRLVP--------------GGVFSFWLFGGDRTFATLTLRBenC -T RSYSFSS QPGNRLTG-FVVRNVP--------------QGKMSEYLSVQAKAGDKMSFTXylZ -T RAYSFSS LQKDGEVS-FLIRKLP--------------GGLMSSFLTSLAKVGDSVSLAFNR1 KL RLYSIAS TRHGDHVDDTVVSLCVRQLEYKHPETGETVYGVCSTYLCNL-EAGADVAITFNR2 KL RLYSIAS SALGDFGDAKSVSLCVKRLIYTN-DAGETIKGVCSNFLCDL-KPGAEVKLT

FAD-isoalloxazine ring-binding NADP-ppi binding region of FNR

XylA APYGQFGL--HE SNATMVCVAGGTGLAPIKCVLQSMTQAQRE RD-----VLLFFGA-RQQBenC GPFGSFYL--RD VKRPVLMLAGGTGIAPFLSMLQVLEQKGSE HP------VRLVFG-VTQXylZ GPLGAFYL--RE IKRPLLLLAGGTGLAPFTAMLEKIAEQGGE HP------LHLIYG-VTHFNR1 GPV LLPED EDATIIMMATGTGIAPFRAFLWRIFKEQHE DYKFKGLAWLFFGIPYSPFNR2 GPVGKEMLMPKD PNATIIMLGTGTGIAPFRSFLWKMFFEKHD DYKFNGLAWLFLGVPTSS

NAD(P)-ribose binding region

* *

XylA RDLYCLDEIEALQLDWGGRFELI-PVLSEESSTSSWKGKRGMVTEYFKEY--EYLTGQPYBenC DCDLVALEQLDALQQKLPWFEYR-TVVAHAESQHERKGYVTGHIEY------DWLNGGEVXylZ DHDLVEMDKLEAFAARIPNFSYS-ACVASPDSAYPQKGYVTQYIEP------KQLNGGEVFNR1 NILYQQE-LEELQEEFPENFRLTLAISREQQNPEGGKMYIQDRIKENADQLWELIQKPNTFNR2 SLLYKEE-FEKMKEKAPDNFRLDFAVSREQTNEKGEKMYIQTRMAQYAVELWEMLKKDNT

* ** *

Xy1A EGYLCGPPPMVDAAETELVRLGVARELVFADRFYNRPPCBenC DVYLCGPVPMVEAVRSWLDTQGIQPANFLFEKFSANXy1Z DIYLCGPPPMVEAVSQYIRAQGIQPANFYYEKFAASAFNR1 HTYICGLKGMEGGIDEGMSAAAGKFDVDWSDYQKELKKKHRWHVETYFNR2 YVYMCGLKGMEKGIDDIMYSLAAAEGIDWIEYKRQLKKAEQWNVEVY

FIG. 6. Alignment of BenC and XylZ oxidoreductases with related sequences. The N-terminal portions of the proteins resemblechloroplast-type ferredoxins such as Aphanothece sacrum II (Frdox) (25) and a region at the C terminus of a Pseudomonas vanillatedemethylase component (VanB) (5). The C-terminal regions of BenC and XylZ resemble ferredoxin-NADP+ oxidoreductases from Spirulinaspecies (FNR1) (77) and spinach (FNR2) (30). XylA, a component of xylene monooxygenase (62), is similar throughout the length of theproteins. Boxes indicate possible FAD-isoalloxazine ring- and NAD(P)-ribose-binding regions. Portions of FNR1 and -2 which may bind theFAD-PPi moiety are underlined. Portions of FNR1 and -2 which may bind the NADP-PPi moiety are underlined and italicized. Asterisksindicate identical residues in the aligned sequences, and dashes indicate gaps.

gested that these particular residues are involved in the by four cysteines in proteins such as ferredoxins, whereascoordination of a Rieske-type (53) iron-sulfur cluster. The coordination is mediated by two cysteines and two histidinesbiochemical study of benzoate 1,2-dioxygenase demon- in Rieske iron-sulfur proteins (11, 15). These small (roughlystrated that one [2Fe-2S] iron-sulfur center is associated with 20-kDa) electron transport proteins are characterized by aeach oa-subunit (75). Iron-sulfur center binding is mediated relatively high redox potential (67).

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* * * * * '* .* * * * * ,*l *BenC 40 AYRQQINIPMD C REGE C GT C RAFCESGNYDMPEDNYIEDALTPEEAQQGYVLA C QCRPTSDAVFQIQASSE 110Xy1Z 29 AYRQGINLPLD C RDGA C GA C KCFAESGRYSL-GEEYIEDALSEAEAEQGYVLT C QMRAESDCVIRVPAASD 98ferl 31 AEEQGIELPYS C RAGA C ST C AGKVLSGTIDQSEQSFLDD----DQMGAGFLLT C VAYPTSDCKVQTHAEDD 96fer2 36 AEEQGYDLPFS C RAGA C ST C AGKLVSGTVDQSDQSFLDD----DQIEAGYVLT C VAYPTSDVTIQTHKEED 102fer3 198 AEEQGIDLPYS C RAGA C ST C AGKVVSGTVDQSDQSFLDD----DQIAAGFVLT C VAYPTSDVTIETHKEED 264fer4 30 AEEEGIELPYS C RAGA C ST C AGKVTEGTVDQSDQSFLDD----EQMLKGYVLT C IAYPESDCTILTHVEQE 96fer5 30 AEEAGLDLPYS C RAGA C ST C AGTITSGTIDQSDQSFLDD----DQIEAGYVLT C VAYPTSDCTIKTHQEEG 96fer6 30 AEEAGLDLPYS C RAGA C ST C AGKITSGSIDQSDQSFLDD----DQIEAGYVLT C VAYPTSDCTIQTHQEEG 96fer7 30 AEEAGLDLPYS C RAGA C ST C AGKIKSGTVDQSDQSFLDD----DQIEAGYVLT C VAYPTSDCTIETHKEEL 96fer8 29 AEEAGLDLPYS C RAGA C ST C AGKLVTGTIDQSDQSFLDD----DQVEAGYVLT C VAYPTSDVTIETHKEED 95fer9 28 AEEAGLDLPYS C RAGA C ST C AGKITAGSVDQSDQSFLDD----DQIEAGYVLT C VAYPTSDCTIETHKEED 94ferlO 52 AEAQGYDWPFS C RAGA C AN C ASIVKEGEIDMDMQQILSDE---EVEEKDVRLT C IGSPAADEVIVYNAKHL 119

FIG. 7. The N-terminal regions of the BenC and XylZ electron transfer proteins resemble chloroplast-type ferredoxins (ferl to -10).Conserved cysteines which bind a [2Fe-2S] center are enclosed in boxes. Additional conserved residues are indicated by asterisks, and gapsare marked by dashes. Ferredoxins 1 to 10 are from the following organisms: 1, Bumilleriopsisfiliformis (26); 2, Anabaena variabilis (68); 3,Anacystis nidulans (52); 4, Porphyra umbilicalis (64); 5, Spirulina platensis (23); 6, Spirulina maxima (65); 7, Chlorogloeopsisfritschia (63);8, Aphanizomenon flos-aquae (36); 9, Synechocystis sp. (22); 10, Halobacterium halobium (24).

The amino acid alignment shown in Fig. 4 substantiatesthe idea that the hydroxylase a-subunits of the benzoate,toluate, benzene, toluene, and naphthalene dioxygenases areall Rieske-type iron-sulfur proteins. This suggestion hasbeen made previously on the basis of the physical propertiesof some of the dioxygenases (11). Spectroscopic studies ofthe functionally similar phthalate dioxygenase from Pseudo-monas cepacia have clearly shown that in this enzyme, twohistidines coordinate a [2Fe-2S] Rieske-type cluster (15).The specific residues involved in iron-sulfur binding byRieske proteins have not previously been identified, but thesequence alignment presented in Fig. 4 suggests that theywould be the conserved amino acids which are enclosed inboxes in the figure.The low degree of homology detected among the five

,B-subunits of the hydroxylase components suggested thatthese subunits are not directly involved in the commoncatalytic functions of the dioxygenases. The product of thexylY gene, the 1-subunit of toluate 1,2-dioxygenase, appearsto be important for the determination of the substratespecificity of this enzyme (21). Of the nine conserved resi-dues among the ,B-subunits, five are charged amino acids. Itis possible that, as for the association between cytochromesc and b5 (70), the charged amino acids play a role in theassociation between the a- and 13-subunits of the hydroxy-lases.The sizes and numbers of the electron transfer components

Possible NAD-ribose bindingregion of BenC and XylZ

of the multicomponent aromatic oxygenases vary. A compar-ison of the available sequences of electron transfer compo-nents suggested that variation reflects different evolutionaryancestry of the electron transfer proteins. In the naphtha-lene, toluene, and benzene 1,2-dioxygenases, two redoxproteins are involved in the transfer of electrons fromNADH to the hydroxylase component. One is a flavoproteinthat has NADH oxidase activity, and the other is a ferre-doxin, which has one Rieske-type [2Fe-2S] cluster. In thebenzoate and toluate 1,2-dioxygenases, a single protein thatcontains both flavin and a [2Fe-2S] center performs theelectron transfer. BenC and XylZ were each found to have aferredoxinlike N-terminal region and an oxidoreductaselikeC-terminal region (Fig. 6). Neither of these regions appearedto be homologous to their functional counterparts in thetoluene and benzene dioxygenase systems.The benzoate and toluate dioxygenases use a chloroplast-

type ferredoxin for transferring electrons to the terminalhydroxylase. As shown in Fig. 7, the N-terminal regions ofBenC and XylZ are homologous to chloroplast-type ferre-doxins. Many residues in the 12 aligned sequences areidentical, including the four cysteines which coordinate a[2Fe-2S] center in the ferredoxins (49). Similar examples ofa ferredoxinlike region occurring within a larger protein werefound in two monooxygenase components, VanB (5) andXylA (62) (Fig. 6). In VanB, the ferredoxinlike region occursin the C-terminal portion of the protein. Most examples to

Possible FAD-isoalloxazine ringbinding region of BenC and XylZ

BenC 220 KRPVLMLAGGTGIAPFLSMLQVL***:.*:******:.*** **:

XylZ 207 KRPLLLLAGGTGLAPFTAMLEKI BenC 167 RSYSFSSXylZ 153 RAYSFSS

SR(E.c.) 452 ETPVIMIGPGTGIAPFRAFMQQR SR(S.t.) 386 RLYSIAS**:*::****** * :* SR(E.c.) 386 RLYSIAS

SR(S.t.) 452 ETPVIMIGPGTGIAPFRSFMQQR P450R(rat) 454 RYYSIASP450R(pig) 453 RYYSIAS

P450R(pig) 524 TTPVIMVGPGTGVAPFIGFIQER b5R 66 RPYTPIS

P450R(rat) 525 TTPVIMVGPGTGIAPFMGFIQER*: *** **:.* * * ***

VanB 104 SSRSLLFAGGIGITPILAMAQVL:* *.:****** * * :.:

b5R 146 VKSVGMIAGGTGITPMLQVIRAIFIG. 8. Possible NAD- and FAD-binding regions of aromatic dioxygenase flavoproteins BenC, XylZ, BnzD (P4) (27), and TodA (79).

Possible cofactor-binding regions of BenC and XylZ are aligned with those of sulfite reductase of E. coli, [SR(E.c.)] and S. typhimurium[SR(S.t.)] (46), cytochrome P-450 oxidoreductase of pig, [P450R(pig)] (18) and rat [P450R(rat)] (50), vanillate demethylase (VanB) (5), andcytochrome b5 reductase (bSR) (33). An asterisk above a residue indicates identity with the BenC amino acid; a colon indicates similarity.

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A. CALCOACETICUS benABC SEQUENCE 5393

date of ferredoxinlike segments in larger proteins involve"bacterial-type" [4Fe-4S] centers in complex membraneproteins (4).The benzene, naphthalene, and toluene dioxygenases have

separate electron transport proteins that resemble Rieskeiron-sulfur proteins. Each of these dioxygenases has a small12- to 15-kDa electron transfer component containing a

[2Fe-2S] center (27, 34, 38, 79). Previous observations ofredox potential and spectroscopic properties suggested thatthese small proteins were unlike previously characterizedferredoxins (38). Some similarities to Rieske iron-sulfurproteins have been noted (17, 61). As shown in Fig. 4, similaralignments of two cysteines and two histidines in the Rieske-type hydroxylase components, the Rieske iron-sulfur pro-

teins, and the toluene, naphthalene, and benzene "ferre-doxin" components suggest that these electron transportproteins of the dioxygenases are more similar to Rieskeiron-sulfur proteins than to other ferredoxins.The protein components of benzoate and toluate dioxygen-

ases involved in NADH oxidation are dissimilar to those of thebenzene and toluene dioxygenases. The similarities observedbetween the BenC and XylZ C-terminal regions and a

number of oxidoreductases (Fig. 6 and 8) suggested thatthese portions of the proteins are responsible for directinteraction with NADH. One of the oxidoreductases resem-

bling BenC and XylZ is the NADPH-sulfite reductase in-volved in cysteine biosynthesis (46, 48). It is interesting thathomology has previously been found between two regula-tory proteins, one controlling transcription of the genes forsulfite reductase in E. coli and S. typhimurium (47) and theother controlling transcription of genes needed for benzoatemetabolism in A. calcoaceticus (41).Sequence comparisons failed to reveal significant similar-

ity between BenC and the reductase components of thebenzene and toluene dioxygenases that closely resembleeach other (27, 79). We found two regions of roughly 30amino acids in BnzD (P4) and TodA, identical in bothproteins, which fit the consensus sequence (55, 71) for a

secondary structure Pap-fold. These regions, which are

likely to be involved in binding FAD and NAD, start atamino acid positions 3 and 145 in the protein sequences ofBnzD (P4) and TodA. No sequences within BenC or XylZ fitthis consensus sequence, although regions involved in co-

factor binding by these proteins may still have ,Ba-typesecondary structures.The most highly conserved regions in the alignments of

BenC and XylZ with FAD- and NAD(P)-binding oxidoreduc-tases are likely to be those of cofactor binding (Fig. 6 and 8).The regions shown in Fig. 8 which are comparable to that ofBenC near amino acid position 220 have previously beensuggested to play a role in NADP-ribose binding (18, 46, 50).The additional short, highly conserved region depicted inFig. 8 has previously been suggested to be part of a regionwhich binds the FAD isoalloxazine ring in spinach ferre-doxin-NADP+ reductase and cytochrome P-450 reductase(30, 50, 72).The regions of the ferredoxin reductases underlined in Fig.

6 may play a role in binding FAD-PPi and NAD(P)-PP1moieties (50). The underlined ferredoxin reductase se-

quences and those of BenC and XylZ are similar, with sixamino acids identical in all aligned sequences and with 27%of the BenC amino acid residues identical to those of a

reductase. Gaps have been introduced in the sequence

alignments, however, and the role these regions play incofactor binding by BenC or XylZ remains to be determined.Although the sequence of the oxidoreductase of the naph-

thalene dioxygenase is not presently available, biochemicalstudies suggest that this protein is similar to the benzoateand toluate dioxygenase oxidoreductases (16). The sizes ofthese three proteins are similar, and spectroscopic studies ofthe NADH-ferredoxinNAP reductase suggest that it is a flavinprotein with a [2Fe-2S] chloroplast-type ferredoxin. Thischloroplast-type ferredoxin functions in conjunction with theseparate Rieske-type ferredoxinNAP protein. The naphtha-lene dioxygenase system, therefore, appears to be unique inhaving two proteins and three redox centers for the transferof electrons to the terminal hydroxylase.The xylene monooxygenase (62) also has an electron

transport component like those of the benzoate and toluatedioxygenases (Fig. 6). The hydroxylating region of thismonooxygenase, however, was not found to be similar to thehydroxylase components of the dioxygenases which arerelated to each other. It therefore appears that there are atleast two classes of oxygenase components and at least threeclasses of electron transfer components in multicomponentoxygenases. The partnerships of hydroxylase and electrontransfer components seem to have changed during thecourse of evolution.

ACKNOWLEDGMENTS

This research was supported by grants to L.N.O. from the ArmyResearch Office, the Celgene Corporation, and the National ScienceFoundation. Research in the laboratory of S.H. was supported bythe Swiss National Science Foundation.We thank David P. Ballou for helpful discussion.

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