Cloning, Expression, and Sequence Analysis of the Three GenesEncoding Quinoline 2-Oxidoreductase, a Molybdenum-containingHydroxylase from Pseudomonas putida 86*

(Received for publication, April 16, 1996, and in revised form, June 11, 1996)

Marcel Blase, Christina Bruntner, Barbara Tshisuaka, Susanne Fetzner, and Franz Lingens‡

From the Institut fur Mikrobiologie (250), Universitat Hohenheim, D-70593 Stuttgart, Germany

The three genes coding for quinoline 2-oxidoreduc-tase (Qor) of Pseudomonas putida 86 were cloned andsequenced. The qor genes are clustered in the transcrip-tional order medium (M) small (S), large (L) and code forthree subunits of 288 (QorM), 168 (QorS), and 788 (QorL)amino acids, respectively. Formation of active quinoline2-oxidoreductase and degradation of quinoline oc-curred in a recombinant P. putida KT2440 clone. Theamino acid sequences of Qor show significant homologyto various prokaryotic molybdenum containing hy-droxylases and to eukaryotic xanthine dehydrogenases.QorS contains two conserved motifs for [2Fe-2S] clus-ters. The binding motif for the N-terminal [2Fe-2S] clus-ter corresponds to the binding site of bacterial and chlo-roplast-type [2Fe-2S] ferredoxins, whereas the aminoacid pattern of the internal [2Fe-2S] center apparently isa distinct feature of molybdenum-containing hydroxy-lases, showing no homology to any other described [2Fe-2S] binding motif. The medium subunit QorM presum-ably contains the FAD, but no conserved sequence areasor described motifs of FAD, NAD, NADP, or ATP bindingwere detected. Putative binding sites of the molybdo-pterin cytosine dinucleotide cofactor were detected inQorL by comparison with “contacting segments” re-cently described in aldehyde oxidoreductase from De-sulfovibrio gigas (Romao, M. J., Archer, M., Moura, I.,Moura, J. J. G., LeGall, J., Engh, R., Schneider, M., Hof,P., and Huber, R. (1995) Science 270, 1170–1176).

Pseudomonas putida 86 utilizes quinoline as sole source ofcarbon, energy, and nitrogen. Degradation proceeds via the“coumarin pathway” (Schwarz et al., 1989; Shukla, 1989). Inthe first step, quinoline 2-oxidoreductase (Qor)1 catalyzes thehydroxylation to 2-oxo-1,2-dihydroquinoline with concomitantreduction of a suitable electron acceptor. The incorporated ox-ygen atom is derived from water. The native enzyme (molecular

mass 300 kDa) consists of three nonidentical subunits of 85 (L),30 (M), and 20 (S) kDa, arranged in a L2M2S2 structure(Bauder et al., 1990; Peschke and Lingens, 1991).Quinoline 2-oxidoreductase contains four redox active cen-

ters that constitute an internal electron transport chain,namely, molybdenum molybdopterin cytosine dinucleotidewith a monooxo-monosulfido-type molybdenum center, two dis-tinct [2Fe-2S] clusters discernible by their characteristic EPRsignals, and FAD (Bauder et al., 1990; Hettrich et al., 1991;Peschke and Lingens, 1991; Tshisuaka et al., 1993).Here we report the cloning, expression, sequencing, and com-

parative sequence analysis of the qor genes encoding quinoline2-oxidoreductase from P. putida 86. Based on alignments withother molybdenum-containing hydroxylases of both prokary-otic and eukaryotic origin, putative cofactor binding motifs arediscussed.

EXPERIMENTAL PROCEDURES

Bacterial Strains and Growth Conditions—Growth of P. putida 86,which had been isolated from soil of a coal tar refining factory (Ruet-gerswerke, Castrop-Rauxel, Germany) by selective enrichment on quin-oline as carbon source (Schwarz et al., 1988), was described previously(Tshisuaka et al., 1993). Escherichia coli S17-1 (Simon et al., 1983) wasused to multiply the cosmid and for the mobilization of recombinantcosmids. For the construction of the genomic library, E. coli ED 8654(Borck et al., 1976; Murray et al., 1977; Sambrook et al., 1989) was usedas the host strain. Both E. coli strains were cultured in LB medium(Sambrook et al., 1989) at 37 °C. E. coli clones containing recombinantcosmid DNA were grown in the presence of tetracycline (15 mg/ml). Inorder to investigate expression of the qor genes, recombinant cosmidDNA in E. coli S17-1 was transferred in P. putida mt-2 KT2440 (Bag-dasarian et al., 1981). P. putidamt-2 KT2440 was grown in LB mediumat 30 °C, and recombinant clones of strain KT2440 were cultured inmineral salt medium (Fetzner et al., 1989) with catechol (1 mM) as asource of carbon and tetracycline (50 mg/ml) at 30 °C. E. coli TG2(Benen et al., 1989) was the host of recombinant pUC18 and pUC19plasmid DNA (Vieira and Messing, 1982; Norrander et al., 1983). Suchclones were cultured at 37 °C on LB medium with ampicillin (100mg/ml). E. coliMV1190 (Vieira and Messing, 1987) grew on M9 mineralsalt medium (Miller, 1972) in the presence of vitamin B1 at 37 °C. E.coli MV1190 with recombinant M13mp18 or M13mp19 DNA (Nor-rander et al., 1983) was cultured in 2 3 YTmedium (Miller, 1972) in thepresence of ampicillin (100 mg/ml).Plasmids and Cosmid—In order to construct a genomic library of P.

putida 86, the plasmid pCIB 119 was used. Plasmid pCIB 119 is adouble cosmid and was a kind gift of Dr. Stephen T. Lam (Ciba-Geigy,Research Triangle Park, NC). It was constructed by cloning a fragmentfrom plasmid c2XB (Bates and Swift, 1983) containing double cos sitesinto the broad-host range plasmid pRK 290 (Ditta et al., 1980). Plasmidvectors used for subcloning and sequencing were pUC18 or 19 andM13mp18 or 19 (Norrander et al., 1983; Vieira and Messing, 1987),respectively.DNA Techniques—Genomic DNA of P. putida 86 was isolated accord-

ing to Davis et al. (1980). Cosmid DNA was isolated by the method ofClewell and Helinski (1969). Plasmid DNA and recombinant cosmidsand plasmids were isolated by the methods of Kieser (1984) or byalkaline lysis (Sambrook et al., 1989). Agarose gel electrophoresis, DNArestriction, DNA ligation, and dephosphorylation of DNA fragments

* This work was supported by the Deutsche Forschungsgemeinschaftand by the Fonds der Chemischen Industrie. The costs of publication ofthis article were defrayed in part by the payment of page charges. Thisarticle must therefore be hereby marked “advertisement” in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.Dedicated to Prof. Dr. P. Renz on the occasion of his 60th birthday.‡ To whom correspondence should be addressed. Tel.: 49-711-459-

2222; Fax: 49-711-459-2238.1 The abbreviations used are: Qor, quinoline 2-oxidoreductase; EPR,

electron paramagnetic resonance; PAGE, polyacrylamide gel electro-phoresis; aa, amino acid(s); D, small aliphatic amino acids; F, hydro-philic amino acids; C, acid or acid amide; q, basic amino acids; V,charged amino acids; P, aliphatic amino acids; Q, aromatic amino acids;S, hydrophobic amino acids; C, conserved cysteine; dots (z) indicate thatthis amino acid exists only once in this aligned position; hyphen (-) noamino acid at this position; numbers in inferior positions indicate thenumber of this amino acid in this aligned position; bp, base pair(s); kb,kilobase pair(s).

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 271, No. 38, Issue of September 20, pp. 23068–23079, 1996© 1996 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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were done according to the instruction manuals of the manufacturers(Boehringer Mannheim; Pharmacia Biotech Inc.) or by standard proce-dures (Sambrook et al., 1989). DNA fragments were isolated fromagarose gels according to Tautz and Renz (1983) or by the instructionmanual of the Geneclean-II kit (Dianova GmbH, Hamburg, Germany).Transformation of E. coli TG2 with recombinant pUC18 or pUC19 DNAand transformation of E. coli MV1190 with recombinant cosmid DNAwas carried out using the CaCl2 method of Mandel and Higa (1970).Conjugation of recombinant E. coli S17-1 clones with P. putida mt-2KT2440 was performed according to Sambrook et al. (1989).Construction of a Genomic Library—Genomic DNA of P. putida 86

was partially digested with Sau3AI. DNA fragments of about 14–29 kbin size were isolated from an agarose gel and treated with alkalinephosphatase from shrimp (U. S. Biochemical Corp.). The dephosphoryl-ated DNA fragments were ligated into the cosmid, and the resultinggenomic library in E. coli ED8654 was screened using an oligonucleo-tide designated “corg” as a probe.Hybridization—Colony blotting was performed as described by Grun-

stein and Hogness (1975). Colonies on nylon membranes (Hybond-N,Amersham Corp.) were subjected to cell lysis, and the released DNAwas denatured with alkali and transferred onto the nylon membranes.Colony hybridization using corg as a DNA probe was performed toidentify recombinant clones in the genomic library. Corg was an 8-folddegenerate 17-mer (ATG-ATG-AA(A/G)-CA(T/C)-GA(A/G)-GT) deducedfrom the N-terminal protein sequence (Met-Met-Lys-His-Glu-Val) of thelarge subunit (Peschke and Lingens, 1991), which was labeled with theDIG-39-end-labeling kit as described by the supplier (Boehringer Mann-heim). Hybridization according to Sambrook et al. (1989) was carriedout at 44 °C overnight. The membranes were stringently washed twicefor 10 min at 49 °C in 1 3 SSC containing 0.1% SDS. Immunologicaldetection was performed with the DIG luminescent detection kit (Boeh-ringer Mannheim).Subcloned gene coding DNA fragments in pUC18 as well as in pUC19

plasmid vectors were identified by agarose gel electrophoresis, South-ern blotting, and hybridization (Sambrook et al., 1989) with both thecorg probe and a second DNA probe named “cork.” The latter was adigoxigenin-labeled 32-fold degenerated 17-mer (ATG-CA(A/G)-GCX-CA(C/T)GA(A/G)-GA) deduced from the N-terminal protein sequence(Met-Gln-Ala-His-Glu-Glu) of the small subunit (Peschke and Lingens,1991). The hybridization with corg was done as described above, thehybridization with cork was carried out at 49 °C for 18 h, and stringentwashes were performed with 1 3 SSC and 0.1% SDS at 49 °C.Expression of Quinoline 2-Oxidoreductase—Recombinant P. putida

mt-2 KT2440 clones that showed positive hybridization signals wereassayed for expression of the quinoline 2-oxidoreductase genes by test-ing for growth on mineral salt medium with quinoline as the only sourceof carbon, energy, and nitrogen. In the presence of succinate as anadditional source of carbon, cometabolic conversion of quinoline wasinvestigated. Growth was monitored by measuring the optical densityat 600 nm. The conversion of quinoline to 2-oxo-1,2-dihydroquinolinewas followed by recording UV-visible spectra in the range of 250 to 400nm. The decrease of absorbance at 299 and 323 nm was indicative forconsumption of quinoline and 2-oxo-1,2-dihydroquinoline, respectively(Bauder et al., 1990).Enzyme Assay—The activity of quinoline 2-oxidoreductase in cell free

extracts of recombinant P. putida KT2440 clones was determined spec-trophotometrically, measuring the substrate-dependent reduction ofp-iodonitrotetrazolium violet as described by Bauder et al. (1990). Cell-free extracts which showed enzymatic activity were separated by non-denaturing PAGE using 10% resolving and 4% stacking gels in the high

pH system (Hames, 1990), and gels were immersed in a standardenzyme assay solution. Quinoline 2-oxidoreductase was visible on thepolyacrylamide gel as red band due to p-iodonitrotetrazolium violet-formazan formation.DNA Sequencing and Sequence Analysis—DNA fragments to be se-

quenced were inserted into the vectors M13mp18 as well as inM13mp19, and E. coliMV1190 was transfected with them (Sambrook etal., 1989). DNA sequences were determined using the dideoxy-mediatedchain termination procedure (Sanger et al., 1977) on single strandedtemplates with a-35S-dATP and M13 universal primers. Overlappingsingle DNA fragments which included all three genes of the quinoline2-oxidoreductase as well as flanking regions were isolated and se-quenced in both directions. The sequencing reaction was performedaccording to the Deaza-G/A-T7-sequencing kit instruction manual(Pharmacia). Vertical PAGE and analysis of the 6% polyacrylamide gelwas performed according to Sambrook et al. (1989). Computer analysisof qor/Qor and sequence comparisons with other molybdenum-contain-ing hydroxylases were performed with the GENMON program (Gesell-schaft fur Biotechnologische Forschung mbH, Braunschweig, Germany)and the HUSAR 4.0 program package (EMBL, Heidelberg, Germany)which includes the GCG software version 7.1 of the University ofWisconsin (Devereux et al., 1984). The programs used were BFASTA tosearch for similar sequences and CLUSTAL W for the multiple se-quence alignments of QorS, M, and L.Nucleotide Sequence Accession Number—The DNA sequence pre-

sented in this report, encoding QorS, QorM, and QorL, has been depos-ited in the EMBL Nucleotide Sequence Library, Heidelberg, Germany,under accession no. X98131.

RESULTS

Cloning and Expression of Quinoline 2-OxidoreductaseGenes—792 E. coli ED8654 clones of a genomic DNA library ofP. putida 86 in plasmid pCIB 119 were screened for the threegenes of quinoline 2-oxidoreductase using a digoxigenin labeledmixed oligonucleotide in colony hybridization. The DNA probecorresponded to six amino acids of the N terminus of the largesubunit QorL. Four clones harboring inserts about 30 kb in sizeshowed positive hybridization signals. However, attempts todetect catalytically active quinoline 2-oxidoreductase in the E.coli ED8654 clones failed. The recombinant cosmid DNA ofeach of the four clones was isolated and transferred to compe-tent P. putida mt-2 KT2440. To check for expression of quino-line 2-oxidoreductase genes in the four recombinant P. putidaKT2440 clones, cometabolic conversion of quinoline in the pres-ence of succinate was tested. However, such a conversion onlyoccurred with one clone, designated 13/42. In addition, onlyclone 13/42 grew on mineral salt medium with quinoline as theonly source of carbon, energy and nitrogen. Spectrophotometricanalysis of the culture supernatant of clone 13/42 showed that2-oxo-1,2-dihydroquinoline was formed from quinoline. How-ever, 2-oxo-1,2-dihydroquinoline accumulated only transientlyand was consumed further. Since P. putida mt-2 KT2440 doesnot utilize 2-oxo-1,2-dihydroquinoline, this result indicatesthat not only the qor genes, but also genes encoding furtherenzymes of the “coumarin pathway” of quinoline degradation

FIG. 1. Restriction map and sequencing strategy of the 4585-bp DNA fragment carrying the qor genes of P. putida 86. The arrowsindicate the extent and direction of each sequencing reaction. The boxes indicate the positions and the direction of transcription of the three openreading frames qorM, qorS, and qorL.

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(Schwarz et al., 1989; Shukla, 1989) are located on the recom-binant cosmid of clone 13/42. The specific activity of quinoline2-oxidoreductase in cell-free extracts of clone 13/42 and of wild-type P. putida 86 was 0.37 unit/mg protein and 0.72 unit/mgprotein, respectively. In native PAGE, crude extract of clone13/42 was separated, and after staining for activity (usingp-iodonitrotetrazolium violet as cosubstrate), Qor was visibleas a red band. Thus, only clone 13/42 contained the completethree genes of quinoline 2-oxidoreductase and was able to syn-thesize the active enzyme. The recombinant cosmid DNA ofclone 13/42 was digested with EcoRI, followed by hybridizationwith the two DNA probes corg and cork. A DNA fragment of11.5 kb in size was isolated from an agarose gel, ligated inpUC19, and transformed in E. coli TG2. Fig. 1 shows therestriction map of a 5-kb area of this 11.5-kb fragment whichcontains the complete three genes of quinoline 2-oxidoreduc-tase as well as flanking regions.Sequencing—We used the 5-kb DNA fragment shown in Fig.

1 for sequencing. The nucleotide sequences of the three quino-line 2-oxidoreductase genes and flanking regions were deter-mined in both directions. The resulting nucleotide sequenceshown in Fig. 2 is 4585 bp in size, starting at a HincII restric-tion site and ending at a SphI site. In this sequence, threecomplete gene-coding regions were identified by the computerprograms FRAMES and GENMON. These open readingframes, designated qorS, qorM, and qorL, were arranged in thetranscriptional order (59)-qorM-qorS-qorL-(39) (see Fig. 1). Up-stream of qorM and downstream of qorL no further gene-coding

FIG. 2. Nucleotide sequence of the 4585-bp HincII/SphI frag-ment from P. putida 86 containing the three genes coding forQor.Deduced amino acid sequences are shown below the correspondingDNA sequences and are separately numbered. Underlined amino acid-sequences are identical to those determined by Edman degradation.

FIG. 2—continued

Potential ribosome binding sites (marked as SD, boldface, double-un-derlined), start codons (marked as qorM, qorS, qorL, boldface) and stopcodons (three asterisks) are indicated.

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FIG. 3. Amino acid alignment of QorS with 16 corresponding subunits or domains of other molybdenum-containing hydroxylases.The comparison was carried out with the CLUSTAL W program. Asterisks (*) indicate amino acid residues that are conserved in all the alignedsequences, dots (z) indicate positions where all residues are similar. Diamonds (l) indicate cysteine residues conserved in all [2Fe-2S]-containingprotein subunits or domains. The numbering system above the sequences refers only to the amino acid arrangement in this figure and not to anyof the sequences. Numbers at the end of each line correspond to the amino acid number of the respective protein subunit or domain. Here and inFigs. 4 and 5, the abbreviations are as follows: RatS,M, L, xanthine dehydrogenase (XDH) of R. norvegicus (Amaya et al., 1990);MauS,M, L, XDHof Mus musculus (Terao et al., 1992); MenS, M, L, XDH of Homo sapiens (Ichida et al., 1993); HuhS, M, L, XDH of G. gallus (EMBL accession:

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open reading frames were detected on the 4585-bp segmentusing the programs CODONPREFERENCE, FRAMES, andTESTCODE.

qorM begins with the start codon ATG at position 685 (Fig. 2)and is 867 bp in length. It ends with the stop codon TGA atposition 1551. The corresponding protein consists of 288 aa,

FIG. 4. Amino acid alignment of QorMwith 13 corresponding subunits or domains of other molybdenum-containing hydroxylases.Symbols and abbreviations are given in the legend of Fig. 3. Plus (1) indicates 13 identical amino acids and one different amino acid in one column.

GGXDHY); MelS, M, L, XDH of D. melanogaster (Keith et al., 1987; Lee et al., 1987); ObsS, M, L, XDH of D. pseudoobscura (Riley, 1989); CalS,M, L, XDH of Calliphora vicina (Houde et al., 1989); BomS, M, L, XDH of B. mori (EMBL accession: BMXG); AspS, M, L, XDH of Aspergillusnidulans (Glatigny and Scazzocchio, 1994);MopS, L, aldehyde dehydrogenase of D. gigas (Thoenes et al., 1994; Romao et al., 1995; EMBL accessionno.: X77222); CoS, M, L, carbon monoxide dehydrogenase of Oligotropha carboxidovorans (Schubel et al., 1995); CuS, M, L, carbon monoxidedehydrogenase of Pseudomonas thermocarboxydovorans C2 (Pearson et al., 1994); NdhS, M, L, nicotine dehydrogenase of A. nicotinovorans(Grether-Beck et al., 1994); QorS, M, L, quinoline 2-oxidoreductase of P. putida 86 (this report); KuS, M, quinaldic acid 4-oxidoreductase ofPseudomonas sp. AK-2 (M. Sauter, unpublished results); IorS, isoquinoline 1-oxidoreductase of Pseudomonas diminuta 7 (Lehmann et al., 1995);AdhS, aldehyde dehydrogenase of A. polyoxogenes (Tamaki et al., 1989). S is small,M is medium, and L is large subunit or corresponding domain.

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and its exact molecular mass is 30,650 Da, which agrees withthe molecular mass of 30 kDa determined by SDS-PAGE(Bauder et al., 1990). The N-terminal amino acid sequence ofthe medium subunit determined by Edman degradation2 to-tally matches with the amino acid sequence derived from thenucleotide sequence. The potential ribosome-binding site islocated 14 bp upstream of the start codon, from position 671 to680 (AAGTAGGTGA).qorS starts at position 1538, 14 bp upstream of the end of

qorM and in another reading frame, so there is a 14-bp over-lapping region. The stop codon TGA is located at position 2050,and the length of qorS is 513 bp. The corresponding proteinconsists of 168 aa and has a molecular mass of 18,012 Da,coinciding with the mass of 20 kDa as estimated previously bySDS-PAGE (Bauder et al., 1990). The N-terminal amino acidsequence of the small subunit determined by Edman degrada-tion (Peschke and Lingens, 1991) totally coincides with theamino acid sequence derived from the nucleotide sequence. Theputative ribosome binding site (AAGGAGCT) (position 1526–1533) is located 12 bp upstream of the start codon ATG.qorL, the coding region of the large subunit, starts with the

codon ATG at position 2044 and ends at position 4410. So the2367-bp region is translated to a 788-aa peptide with a molec-ular mass of 84,113 Da, which agrees with the result (85 kDa)of previous analysis by SDS-PAGE (Bauder et al., 1990). Thepotential ribosome-binding site (AGGAG) (position 2032–2036)is located 12 bp upstream of the start codon ATG. The deducedN-terminal amino acid sequence totally agrees with the 16 aaof the N terminus determined by Edman degradation.The G 1 C content of qorS, qorM, and qorL is 62.82%, which

matches with the G 1 C content of 62.5% reported for thegenome of P. putida biovar A (Palleroni, 1984).Multiple Alignments—The amino acid sequences of the three

subunits of quinoline 2-oxidoreductase derived from the nucle-otide sequence were compared with the corresponding subunitsof other molybdenum-containing hydroxylases which possess a

monooxo-monosulfido-type molybdenum center (Figs. 3-5).QorS shows about 25% homology3 to the protein sequences

aligned in Fig. 3. 25 aa (14.9%) are identical and 17 aa aresimilar4 (10.1%) in all compared protein sequences. Eight ofthese 25 conserved aa are cysteines, and always four of themare arranged in two distinct motifs as described previously forseveral molybdenum-containing hydroxylases (Wootton et al.,1991; Hughes et al., 1992a, 1992b; Lehmann et al., 1995). Thefirst four cysteines in QorS are in the positions 48, 53, 56, and68, and the corresponding motif (C-X4-C-G-X-C-Xn-C) is typicalfor the consensus binding site of bacterial and plant-type [2Fe-2S] ferredoxins. Instead of n 5 11 in all prokaryotic molybde-num-containing hydroxylases, there is n 5 21 in all describedeukaryotic xanthine dehydrogenases and n 5 29 in most bac-terial and plant ferredoxins. The [2Fe-2S] cluster coordinatedby this motif is assumed to correspond to one of the Fe/Scenters observed by EPR spectroscopy (Hughes et al., 1992a,1992b; Tshisuaka et al., 1993). In all described sequences, thesecond four cysteines are arranged in the motif (C-G-X-C-X31-C-X-C) (cysteines in QorS are in the positions 107, 110, 142,and 144), and this motif presumably is the binding site of theother type of [2Fe-2S] center.QorM probably harbors the FAD cofactor, but no conserved

motif of a putative FAD binding site was found. The compari-son of five medium subunits of various prokaryotic molybde-num-containing hydroxylases with corresponding domains ofeight eukaryotic xanthine dehydrogenases shows only six iden-tical aa (2.1%) and 26 aa which are similar (9.0%) (Fig. 4).Thus, the degree of homology among all sequences aligned isonly 11.1%. Furthermore, none of the potential FAD-, NAD- orATP-binding motifs known shows any homology to the mediumsubunits or domains, respectively. However, it may be remark-able to note that four of these six conserved aa are glycine

2 B. Tshisuaka, unpublished results.

3 “Homology” refers to identical (conserved) plus similar amino acids.4 “Similar” amino acids are physicochemically related as defined by

Dayhoff (1972) and Taylor (1986) and correspond to groups D, F, C, q,V, P, Q, S.

FIG. 4—continued

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residues, which were reported to be involved in FAD binding(Wierenga et al., 1986; Hanukoglu and Gutfinger, 1989; Egginket al., 1990).QorL, the large subunit, probably contains the binding site(s)

of the pterin molybdenum cofactor, as proposed for other largesubunits of prokaryotic molybdenum-containing hydroxylasesand for to the C-terminal domains of the eukaryotic xanthinedehydrogenases (Wootton et al., 1991; Grether-Beck et al.,1994; Pearson et al., 1994; Glatigny and Scazzocchio, 1995;Schubel et al., 1995). The comparison of nine eukaryotic xan-thine dehydrogenases and five prokaryotic hydroxylases (QorL

included) shows 47 aa (6.0%) which are absolutely conservedand 50 positions (6.3%) where the aligned amino acids aresimilar (Fig. 5), corresponding to a homology of 12.3% betweenthe several sequences.

DISCUSSION

The qorM, S, and L genes encoding the first enzyme of the“coumarin pathway” of quinoline degradation by P. putida 86were cloned and sequenced. Expression of the qorM, S, and Lgenes and formation of catalytically active quinoline 2-oxi-doreductase was achieved in recombinant P. putida mt-2

FIG. 5. Amino acid alignment of QorL with 13 corresponding subunits or domains of other molybdenum-containing hydroxylases.Symbols and abbreviations are explained in the legends of Fig. 3 and Fig. 4. Arrows (1) indicate the position of the three putative molybdopterinbinding segments designated MoCoI, MoCoII, and MoCoIII as well as the two putative dinucleotide binding segments designated MoCoIV andMoCoV (Romao et al., 1995).

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KT2440 clone 13/42. Attempts to detect the active enzyme inthe corresponding E. coli ED8654 clone possessing the samerecombinant cosmid failed. It is known that Pseudomonas pro-motors as a rule are poorly recognized by E. coli RNA polym-erase, because they show little homology to consensus se-quences of E. coli promotors (Frantz and Chakrabarty, 1986;Jeenes et al., 1986). Another possible cause for the lack ofenzyme formation in E. coli may be the difference in the G1Ccontent of Pseudomonas (67–68%) (Palleroni, 1984) and E. coli

(50%) (Brenner, 1984), which may affect the codon-anticodoninteraction in the E. coli host (Frantz and Chakrabarty, 1986;Soldati et al., 1987). However, if gene expression in E. coli tookplace, it might result in formation of inactive Qor that lacks themolybdenum molybdopterin cytosine dinucleotide cofactor. E.coli may be unable to synthesize the cytosine dinucleotide,since up to now, molybdenum enzymes in E. coli have beenfound to exclusively contain the molybdopterin guanine dinu-cleotide form of the pterin molybdenum cofactor (Rajagopalan

FIG. 5—continued

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and Johnson, 1992).P. putida mt-2 KT2440 13/42 utilized both quinoline and

2-oxo-1,2-dihydroquinoline, indicating that apart from theqorM, S, and L structural genes, further genes of the catabolicpathway are localized on the 30-kb DNA insert.The sequenced DNA stretch was 4584 bp in size, and it

contained three open reading frames. Based on comparisonswith N-terminal amino acid sequences known and based on thegene expression studies, these three open reading frames wereidentified as the structural genes encoding the small (QorS),medium (QorM), and large (QorL) subunit of quinoline 2-oxi-

doreductase. The transcriptional order of these three genes,59-qorM-S-L-39 (Fig. 1), corresponded to all other describedstructural genes of molybdenum-containing prokaryotic hy-droxylases with LMS or L2M2S2 structure.We presume that both iron-sulfur centers are localized on the

small, the FAD on the medium, and the pterin molybdenumcofactor as well as the substrate binding site on the largesubunit, as discussed for the subunits or corresponding do-mains of other molybdenum-containing hydroxylases (Woottonet al., 1991; Grether-Beck et al., 1994; Pearson et al., 1994;Thoenes et al., 1994; Glatigny and Scazzocchio, 1995; Lehmann

FIG. 5—continued

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et al., 1995).The amino acid sequences of eight small subunits of different

prokaryotic enzymes and nine N-terminal sequences of eukary-otic molybdenum-containing hydroxylases showed a high de-gree of homology (Fig. 3). The degree of homology of the smallsubunit of quinoline 2-oxidoreductase and other prokaryoticenzymes compared ranged from 67.09%, with quinaldic acid4-oxidoreductase from Pseudomonas sp. AK-2,5 to 53.75% withaldehyde dehydrogenase from Acetobacter polyoxogenes (Ta-maki et al., 1989). The degree of homology between the N-

terminal amino acid sequence of the compared eukaryotic xan-thine dehydrogenases and the small subunit of quinoline2-oxidoreductase ranged from 53.57%, with Drosophilapseudoobscura (Riley, 1989), to 50.60%, with Gallus gallus(EMBL accession: GGXDHY, unpublished) and Bombyx mori(EMBL accession: BMXG, unpublished). Among all amino acidsequences compared, there were at least eight conserved cys-teines which are assumed to coordinate the two different [2Fe-2S] centers detected by EPR studies (Bray, 1975; Bray et al.,1991; Hughes et al., 1992b; Tshisuaka et al., 1993). The bindingmotif of the N-terminal [2Fe-2S]-center is homologous to thesignature sequence of bacterial and chloroplast-type [2Fe-2S]5 M. Sauter, unpublished results.

FIG 5—continued

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ferredoxins (CX4CX2CXnC) (n is 11 for prokaryotic molybde-num hydroxylases (Wootton et al., 1991; Hughes et al., 1992a),21 for eukaryotic molybdenum hydroxylases, and 29 or, excep-tionally, 27–32 for bacterial and plant ferredoxins). The multi-ple sequence alignment shows significant homology from posi-tion 16–68 (positions referring to quinoline 2-oxidoreductase),which includes the motif described above at position 48–68.This consensus sequence is ((P/S)NGX6–7(VPA)FFVX2LX3(P/S)RX3–4L(D/F)GX(q/V)XG(C/Dz)FX(D/F)XCG(A/Sz)C(T/Sz)(V/Iz)X(P/S)FX3–13(P/S)X(A/S)C) (Fig. 3).The binding motif for the internal [2Fe-2S] center, since it

has not been described in any other iron-sulfur protein, appar-ently is a distinct feature of the molybdenum containing hy-droxylases of pro- and eukaryotic organisms where it is anabsolutely conserved motif (CX2CX31CXC) which contains thefour other conserved cysteines. It is part of an area from posi-tion 82 to 140 (positions refer to Qor) which also shows signif-icant homology to the other aligned sequences. This consensussequence is (T(V/Iz)E(G/D)(P/S)FFX3–5(H/S/N)(P/A)(P/S)QX2-SX6QCG(F/Y)CF(D/F)GX(P/S)(M/Vz/Az)X4(L/Iz)LX5(P/Iz)X3F(P/S)X3SF(G/Kz)X0 –2(N/Sz)(L/Az)CRC(T/Gz/Mz)(G/Tz)YX2(I/Lz))(Fig. 3). In both iron-sulfur centers it is presumed that all fourconserved cysteines are ligands of the two iron atoms (Woottonet al., 1991; Hughes et al., 1992a). The other homologous aminoacids may be functional for the correct folding of the protein tobind the iron-sulfur centers, or they may participate in bindingof the pterin molybdenum cofactor or the FAD. In this contextit is important to note that it was not feasible to define distinctbinding motifs for the pterin molybdenum cofactor or FAD onthe other subunits of quinoline 2-oxidoreductase (see below).In analogy to the medium domain or subunit of other molyb-

denum-containing hydroxylases described, the medium sub-unit QorM should contain the binding site for FAD (Amaya etal., 1990; Wootton et al., 1991; Hughes et al., 1992a; Grether-Beck et al., 1994; Pearson et al., 1994). This assumption isbased on the fact that all flavin-containing hydroxylases havegot a medium subunit and that the homodimeric enzymeswhich lack FAD do not possess a medium subunit (Fetzner andLingens, 1993; Lehmann et al., 1994, 1995). Moreover, alde-hyde oxidoreductase (MOP) from Desulfovibrio gigas, whichlacks both FAD and aa sequences corresponding to the mediumsubunits and which is the only molybdenum containing hydrox-ylase whose crystal structure is determined, does not corre-spond in its structural features to any known FAD bindingsubunits or domains (Romao et al., 1995). The sequence align-ment of these medium subunits of various prokaryotic molyb-denum-containing hydroxylases and the corresponding do-mains of eukaryotic xanthine dehydrogenases (Fig. 4) shows ahigh degree of homology between them, which ranged from56.18%, with nicotine dehydrogenase of Arthrobacter nicotino-vorans (Grether-Beck et al., 1994), to 40.97%, with xanthinedehydrogenase from Rattus norvegicus (Amaya et al., 1990). Itis important to note that in 13 out of 14 aligned sequences, only11 aa residues were absolutely or nearly conserved and that 4of these 11 aa were glycine residues, which generally arethought to be important in FAD binding. Several differentFAD-, NAD-/NADP-, and ADP-binding motifs were describedby Rice et al. (1984), Wierenga et al. (1986), Eggink et al. (1990)and Hanukoglu and Gutfinger (1989). All these FAD bindingmotifs comprise G-rich sequences, such as (GXGX2GX3A) or(GXGX2GX3G). A similar motif, (GXGX2AX3A), is a site forNADP-binding. None of these or other known binding motifswere detected in QorM or in the sequences of nine eukaryoticxanthine dehydrogenases and five prokaryotic molybdenum-containing hydroxylases. Nevertheless, there are studies onDrosophila melanogaster mutants with xanthine dehydrogen-

ase variants suggesting that the medium domain contains theFAD binding site: Hughes et al. (1992a) prepared point muta-tions that were localized in the area between amino acid resi-dues 348 and 357 (positions refer to xanthine dehydrogenase ofD. melanogaster). In these flavin mutants, the electron transferbetween the molybdenum center and FAD was blocked(Hughes et al., 1992a), indicating that the altered area some-how is correlated with FAD binding. This area of xanthinedehydrogenase is part of the medium domain and correspondsto the amino acid positions 113–122 in QorM. Its alignmentwith nine eukaryotic and five prokaryotic molybdenum-con-taining enzymes shows significant homologies. This consensussequence described before (bold letters) and the conservedflanking areas (normal letters) are: AX2Q(P/S)(74/V)X2(D/F)-X2(D/F)(GFSX[inb]2(D/F)F(D/F)XF)(C/F)XF (Fig. 4). How-ever, strong evidence that FAD was present in these FADmutants of D. melanogaster (Hughes et al., 1992a) suggestedthat the altered amino acids are not the only residues involvedin FAD binding. Data on FAD binding in prokaryotic molybdo-iron/sulfur-flavoproteins are not yet available. Thus, the modeof FAD binding in enzymes belonging to the family of molyb-denum-containing hydroxylases is still unknown.The large subunit of quinoline 2-oxidoreductase, QorL, prob-

ably contains the molybdenummolybdopterin cytosine dinucle-otide and the substrate binding site. The 14 compared aa se-quences show significant homology, ranging from 63.2%, withnicotine dehydrogenase from A. nicotinovorans (Grether-Becket al., 1994), to 41.3%, with xanthine dehydrogenase from R.norvegicus (Amaya et al., 1990). The comparison in Fig. 5 shows46 positions which were absolutely conserved. 37.0% of theseamino acids are glycines, and 15.2% are alanine residues.Up to now, the only molybdenum-containing hydroxylase

whose crystal structure was determined is aldehyde oxi-doreductase (MOP) from the sulfate reducer D. gigas (Romao etal., 1995). Each subunit of the homodimeric enzyme containstwo different [2Fe-2S] centers and molybdenum molybdopterincytosine dinucleotide. The C-terminal domain of the 907-aasubunit of MOP shows significant homology to QorL (42.0%)and to all other large subunits or corresponding domains listedin Fig. 5. Romao et al. (1995) detected three molybdopterinbinding segments and two dinucleotide binding segments inMOP. The first molybdopterin-contacting segment, which wasalready discussed in previous reports to be involved in bindingthe molybdenum cofactor (Grether-Beck et al., 1994; Pearson etal., 1994; Lehmann et al., 1995; Schubel et al., 1995), is themost conserved sequence of the five segments detected in MOPby Romao et al. (1995). In QorL, the corresponding segmentstarts at position 252 and ends at position 258. The consensussequence of all compared sequences is: (GG(G13/T1)FG(G9/N2/Q2/Y1)K) (Fig. 5). Based on its homology to the consensussequence (GXGXXG), this segment was discussed as bindingsite for the pyrophosphate moiety of a dinucleotide (Amaya etal., 1990; Pearson et al., 1994; Schubel et al., 1995), but noprokaryotic enzyme contains the last glycine residue of the(GXGXXG) motif. This agrees to the observation that the sub-stitution of the first glycine residue by glutamic acid in thecorresponding region of xanthine dehydrogenase from D. mela-nogaster did not cause significant diminutions in xanthine de-hydrogenase electrontransfer activities, indicating that thestructural change only is a subtle one, probably not affectingbinding of the molybdenum cofactor (Hughes et al., 1992a).However, Romao et al. (1995) showed that, in MOP, thissegment contacts molybdopterin by use of Phe-421 and Gly-422, which are absolutely conserved in all aligned sequences(Fig. 5).The second molybdopterin binding segment ranged from po-

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sitions 99 to 106 in MOP. Among the aligned sequences, it isnot as much conserved as the first segment. The consensussequence is: (A(F11/Y3)R(C2/-12)(G12/S2)(F11/V2/Y1)(G12/R2)(G8/A2/V2/Y1/F1)(P10/T4)(Q10/E2/A2)(G9/W1/V1/S1/A2)(M9/L1/V2/T1/Q1)) (Fig. 5). The third and last molybdopterin binding segmentis a very short one, and it is absolutely conserved except for thefirst position. The consensus sequence of position 506–509 (inQorL) is: (M6/I3/H1/Q3/S1)GQG) (Fig. 5). It is described byRomao et al. (1995) that in MOP the pyrophosphate moiety ofthe molybdopterin cytosine dinucleotide is hydrogen-bonded toNe atoms of a tryptophan residue (Trp-650) and of Gln-655, thelatter corresponding to Gln-508 in QorL.The two dinucleotide binding segments found in MOP also

show some homology to the other 14 aligned sequences. Thefirst one spans the residues 693–698 (MOP), corresponding topositions 542–547 in QorL, and the consensus sequence is:((S9/T1/G1/L2/F1)(P8/G4/S1/A1)(T12/S1/A1)(A9/Y3/I1/G1)(A10/G4)S) (Fig. 5). The second one starts at position 867 and ends at872 (QorL: 741–746), and the consensus sequence of all alignedsequences is: ((V11/I1/M2)(G13/A1)E(P9/S4/L1)(P12/G1/A1)(L11/H1/P1/M1)) (Fig. 5).Using site-directed mutagenesis, we plan to investigate pu-

tatively crucial amino acids of these five amino acid segmentsproposed to be involved in binding the molybdenum molybdo-pterin cytosine dinucleotide in quinoline 2-oxidoreductase. Fur-ther studies also are needed to characterize the substratebinding site of quinoline 2-oxidoreductase and to elucidatethe mode of FAD binding in molybdo-iron/sulfur-flavoproteinsbelonging to the family of molybdenum-containing hydrox-ylases (oxotransferases).

Acknowledgments—We thank Dr. J. Altenbuchner, Stuttgart, Ger-many, for synthesis of oligonucleotides. We also thank Prof. Dr. Karl-Heinz van Pee, Dresden, Germany, and Martin Lehmann for introduc-tion into the methods of genetic engineering and for discussions.

REFERENCES

Amaya, Y., Yamazaki, K., Sato, M., Noda, K., Nishino, T., and Nishino, T. (1990)J. Biol. Chem. 265, 14170–14175

Bagdasarian, M., Lurz, R., Ruckert, B., Franklin, F. C. H., Bagdasarian, M. M.,Frey, J., and Timmis, K. N. (1981) Gene (Amst.) 16, 237–247

Bates, P. F., and Swift, R. A. (1983) Gene (Amst.) 26, 137–146Bauder, R., Tshisuaka, B., and Lingens, F. (1990) Biol. Chem. Hoppe-Seyler 371,

1137–1144Benen, J. A. E., van Berkel, W. J. H., van Dongen, W. M. A. M., Muller, F., and de

Kok, A. (1989) J. Gen. Microbiol. 135, 1787–1797Borck, K., Beggs, J. D., Brammar, W. J., Hopkins, A. S., and Murray, N. E. (1976)

Mol. Gen. Genet. 146, 199–207Bray, R. C. (1975) in The Enzymes (Boyer, P. D., ed.), pp 303–394, Academic Press,

New YorkBray, R. C., Turner, N. A., LeGall, J., Barata, B. A. S., and Moura, J. J. G. (1991)

Biochem. J. 280, 817–820Brenner, D. J. (1984) in Bergey’s Manual of Systematic Bacteriology (Krieg, N. R.,

and Holt, J. G., eds) Vol. 1, pp. 408–516, Williams & Wilkins, BaltimoreClewell, D. B., and Helinski, D. R. (1969) Proc. Natl. Acad. Sci. U. S. A. 62,

1159–1166Davis, R. W., Botstein, D., and Roth, J. R. (1980) A Manual for Genetic Engineer-

ing: Advanced Bacterial Genetics, pp 120–123, Cold Spring Harbor Laboratory,Cold Spring Harbor, NY

Dayhoff, M. O. (1972) Atlas of Protein Sequence and Structure (Dayhoff, M. O., ed),Vol. 5, National Biomedical Research Foundation, Silver Spring, MD

Devereux, J., Haeberli, P., and Smithies, O. (1984) Nucleic Acids Res. 12, 387–395Ditta, G., Stanfield, S., Corbin, D., and Helinski, D. R. (1980) Proc. Natl. Acad. Sci.

U. S. A. 77, 7347–7351Eggink, G., Engel, H., Vriend, G., Terpstra, P., and Witholt, B. (1990) J. Mol. Biol.

212, 135–142

Fetzner, S., Muller, R., and Lingens, F. (1989) Biochem. Biophys. Res. Commun.161, 700–705

Fetzner, S., and Lingens, F. (1993) Biol. Chem. Hoppe-Seyler 374, 363–376Frantz, B., and Chakrabarty, A. M. (1986) in The Bacteria (Gunsalus, I. C.,

Sokatch, J. R., Ornston, L. N., eds), Vol. X, pp. 295–323, Academic Press,London

Glatigny, A., and Scazzocchio, C. (1995) J. Biol. Chem. 270, 3534–3550Grether-Beck, S., Igloi, G. L., Pust, S., Schilz, E., Decker, K., and Brandsch, R.

(1994) Mol. Microbiol. 13, 929–936Grunstein, M., and Hogness, D. S. (1975) Proc. Natl. Acad. Sci. U. S. A. 72,

3961–3965Hames, B. D. (1990) in Gel Electrophoresis of Proteins (Hames, B. D., and

Rickwood, D., eds), 2nd Ed., p. 36, IRL Press, OxfordHanukoglu, I., and Gutfinger, T. (1989) Eur. J. Biochem. 180, 479–484Hettrich, D., Peschke, B., Tshisuaka, B., and Lingens, F. (1991) Biol. Chem.

Hoppe-Seyler 372, 513–517Houde, M., Tiveron, M.-C., and Bregegere, F. (1989) Gene (Amst.) 85, 391–402Hughes, R. K., Doyle, W. A., Chovnick, A., Whittle, J. R. S., Burke, J. F., and Bray,

R. C. (1992a) Biochem. J. 285, 507–513Hughes, R. K., Bennett, B., and Bray, R. C. (1992b) Biochemistry 33, 3070–3083Ichida, K., Amaya, Y., Noda, K., Minoshima, S., Hosoya, T., Sakai, O., Shimizu, N.,

and Nishino, T. (1993) Gene (Amst.) 133, 279–284Jeenes, D. J., Soldati, L., Baur, H., Watson, J. M., Mercenier, A., Reimmann, C.,

Leisinger, T., and Haas, D. (1986) Mol. Gen. Genet. 203, 421–429Keith, T. P., Riley, M. A., Kreitman, M., Lewontin, R. C., Curtis, D., and Chambers,

G. (1987) Genetics 116, 67–73Kieser, T. (1984) Plasmid 12, 19–36Lee, C. S., Curtis, D., McCarron, M., Love, C., Gray, M., Bender, W., and Chovnick,

A. (1987) Genetics 116, 55–66Lehmann, M., Tshisuaka, B., Fetzner, S., Roger, P., and Lingens, F. (1994) J. Biol.

Chem. 269, 11254–11260Lehmann, M., Tshisuaka, B., Fetzner, S., and Lingens, F. (1995) J. Biol. Chem.

270, 14420–14429Mandel, M., and Higa, A. (1970) J. Mol. Biol. 53, 159–162Miller, J. H. (1972) Experiments in Molecular Genetics, Cold Spring Harbor Lab-

oratory, Cold Spring Harbor, NYMurray, N. E., Brammar, W. J., and Murray, K. (1977) Mol. Gen. Genet. 150,

53–61Norrander, J., Kempe, T., and Messing, J. (1983) Gene (Amst.) 26, 101–106Palleroni, N. J. (1984) in Bergey’s Manual of Systematic Bacteriology (Krieg, N. R.,

and Holt, J. G., eds) Vol. 1, pp. 141–219, Williams & Wilkins, BaltimorePearson, D. M., O’Reilly, C. O., Colby, J., and Black, G. W. (1994) Biochim.

Biophys. Acta 1188, 432–438Peschke, B., and Lingens, F. (1991) Biol. Chem. Hoppe-Seyler 372, 1081–1088Rajagopalan, K. V., and Johnson, J. L. (1992) J. Biol. Chem. 267, 10199–10202Rice, D. W., Schulz, G. E., and Guest J. R. (1984) J. Mol. Biol. 174, 483–496Riley, M. A. (1989) Mol. Biol. Evol. 6, 33–52Romao, M. J., Archer, M., Moura, I., Moura, J. J. G., LeGall, J., Engh, R.,

Schneider, M., Hof, P., and Huber, R. (1995) Science 270, 1170–1176Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Labo-

ratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor,NY

Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U. S. A. 74,5463–5467

Schubel, U., Kraut, M., Morsdorf, G., and Meyer, O. (1995) J. Bacteriol. 177,2197–2203

Schwarz, G., Senghas, E., Erben, A., Schafer, B., Lingens, F., and Hoke, H. (1988)Syst. Appl. Microbiol. 10, 185–190

Schwarz, G., Bauder, R., Speer, M., Rommel, T. O., and Lingens, F. (1989) Biol.Chem. Hoppe-Seyler 370, 1183–1189

Shukla, O. P., (1989) Microbios 59, 47–63Simon, R., Priefer, U., and Puhler, A. (1983) Bio/Technology 1, 784–791Soldati, L., Jeenes, D. J., and Haas, D., (1987) FEMS Microbiol. Lett. 42, 163–167Tamaki, T., Horinouchi, S., Fukaya, M., Okumura, H., Kawamura, Y., and Beppu,

T. (1989) J. Biochem. (Tokyo) 106, 541–544Tautz, D., and Renz, M. (1983) Anal. Biochem. 132, 14–19Taylor, W. R. (1986) J. Theor. Biol. 119, 205–218Terao, M., Cazzaniga, G., Ghezzi, P., Bianchi, M., Falciani, F., Perani, P., and

Garattini, E. (1992) Biochem. J. 283, 863–870Thoenes, U., Flores, O. L., Neves, A., Devreese, B., van Beeumen, J. J., Huber, R.,

Romao, M. J., LeGall, J., Moura, J. J. G., and Rodrigues-Pousada, C. (1994) Eur.J. Biochem. 220, 901–910

Tshisuaka, B., Kappl, R., Huttermann, J., and Lingens, F. (1993) Biochemistry 32,12928–12934

Vieira, J., and Messing, J. (1982) Gene (Amst.) 19, 259–268Vieira, J., and Messing, J. (1987) Methods Enzymol. 153, 3–11Wierenga, R. K., Terpstra, P., and Hol, W. G. J. (1986) J. Mol. Biol. 187, 101–107Wootton, J. C., Nicolson, R. E., Cock, J. M., Walters, D. E., Burke, J. F., Doyle, W.

A., and Bray, R. C. (1991) Biochim. Biophys. Acta 1057, 157–185

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Marcel Bläse, Christina Bruntner, Barbara Tshisuaka, Susanne Fetzner and Franz Lingens86

Pseudomonas putida2-Oxidoreductase, a Molybdenum-containing Hydroxylase from Cloning, Expression, and Sequence Analysis of the Three Genes Encoding Quinoline

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