Báo cáo khoa học: Functional expression of the quinoline 2-oxidoreductase genes (qorMSL) in Pseudomonas putida KT2440 pUF1 and in P. putida 86-1 Dqor pUF1 and analysis of the Qor proteins doc

Functional expression of the quinoline 2-oxidoreductase genes ( qorMSL )in Pseudomonas putida KT2440 pUF1 and in P. putida 86-1 D qor pUF1 and analysis of the Qor proteins Ursula Frerichs-Deeken 1 , Birgit Goldenstedt 1,2, *, Renate Gahl-Janßen 1 , Reinhard Kappl 3 , Ju¨ rgen Hu¨ ttermann 3 and Susanne Fetzner 1,2, * 1 AG Mikrobiologie, Institut fu ¨ r Chemie und Biologie des Meeres, Carl von Ossietzky Universita ¨ t Oldenburg, Germany; 2 Institut fu ¨ r Mikrobiologie, Westfa ¨ lische Wilhelms-Universita ¨ tMu ¨ nster, Germany; 3 Fachrichtung Biophysik und Physikalische Grundlagen der Medizin, Universita ¨ t des Saarlandes, Homburg/Saar, Germany The availability of a system for the functional expression of genes coding for molybdenum hydroxylases is a prerequisite for the construction of enzyme variants by mutagenesis. For the expression cloning of quinoline 2-oxidoreductase (Qor) from Pseudomonas putida 86 – that contains the molybdo- pterin cytosine dinucleotide molybdenum cofactor (Mo-MCD), two distinct [2Fe)2S] clusters and FAD – the qorMSL genes were inserted into the broad host range vector, pJB653, generating pUF1. P. putida KT2440 and P. putida 86-1 Dqor were used as recipients for pUF1. Whereas Qor from the wild-type strain showed a specific activity of 19–23 UÆmg )1 , the specific activity of Qor purified from P. putida KT2440 pUF1 was only 0.8–2.5 UÆmg )1 , and its apparent k cat (quinoline) was about ninefold lower than that of wild-type Qor. The apparent K m values for quinoline were similar for both proteins. UV/visible and EPR spectroscopy indicated the presence of the full set of [2Fe)2S] clusters and FAD in Qor from P. putida KT2440 pUF1, however, the very low intensity of the Mo(V)-rapid signal, that occurs in the presence of quinoline, as well as metal analysis indicated a deficiency of the molybdenum center. In contrast, the metal content, and the spectroscopic and catalytic properties of Qor produced by P. putida 86-1 Dqor pUF1 were essentially like those of wild-type Qor. Release of CMP upon acidic hydrolysis of the Qor proteins suggested the presence of the MCD form of the pyranopterin cofactor; the CMP contents of the three enzymes were similar. Keywords: quinoline 2-oxidoreductase; molybdenum hydro- xylase; expression cloning; molybdopterin cytosine dinucleo- tide; Pseudomonas sp. Quinoline 2-oxidoreductase (Qor) from Pseudomonas putida 86 catalyses the formation of 1H-2-oxoquinoline (2-hydroxyquinoline) from quinoline [1,2]. Besides quino- line, some quinoline derivatives and the benzodiazines quinazoline and quinoxaline are accepted as substrates [1,3]. Like other enzymes catalysing the hydroxylation of N-heteroaromatic rings at positions that are susceptible to nucleophilic attack, Qor belongs to the family of molyb- denum hydroxylases that introduce an oxygen atom (originating from water) into their substrate according to the following stoichiometry: R-H + H 2 O fi R-OH + 2[e – ]+2H + . Due to a common structure of their molyb- denum center and due to significant amino acid sequence similarity to xanthine oxidases/xanthine dehydrogenases, the molybdenum hydroxylases have also been classified as enzymes belonging to the Ôxanthine oxidase familyÕ [4–7]. Molybdenum hydroxylases basically contain the same type of redox centers constituting an intramolecular electron transport chain, namely a molybdenum ion, that is the site of substrate hydroxylation, two distinct [2Fe)2S] clusters, and – in most cases – FAD [5,8,9]. The molybdenum is bound to the sulfur atoms of the ene-dithiolate function of a unique pyranopterin cofactor. Other coordination positions to the molybdenum are occupied by a sulfido and an oxo ligand, and a catalytically labile )OH group or H 2 Omole- cule [5–7,10–12]. Whereas almost all known xanthine dehy- drogenases contain a pyranopterin derivative, known as molybdopterin (MPT), as the organic part of the moly- bdenum cofactor [13], Qor [2,14] as well as isoquinoline 1-oxidoreductase [15], quinaldine 4-oxidase [3], nicotinate dehydrogenase [16], isonicotinate and 2-hydroxyisonicoti- nate dehydrogenase [16,17], CO dehydrogenases [18–20] and the aldehyde oxidoreductases belonging to the xanthine oxidase family [11,12,21,22], contain Mo-MPT that is modified by covalent attachment of cytidine monophos- phate to its terminal phosphate group to form molybdenum molybdopterin cytosine dinucleotide (Mo-MCD). Correspondence to S. Fetzner, Institut fu ¨ r Mikrobiologie, Westfa ¨ lische Wilhelms-Universita ¨ tMu ¨ nster, Corrensstr. 3, D-48149 Mu ¨ nster, Germany. Fax: +49 251 83 38388, Tel.: +49 251 83 39824, E-mail: fetzner@uni-muenster.de Abbreviations: Mo-MCD, molybdopterin cytosine dinucleotide form of the molybdenum pyranopterin cofactor; Mo-MGD, molyb- dopterin guanine dinucleotide form of the molybdenum pyranopterin cofactor; MPT, molybdopterin; Qor, quinoline 2-oxidoreductase. Enzymes: Quinoline 2-oxidoreductase; quinoline:acceptor 2-oxido- reductase (hydroxylating) (EC 1.3.99.17). *Present address: Institut fu ¨ r Mikrobiologie, Westfa ¨ lische Wilhelms- Universita ¨ tMu ¨ nster, Germany. (Received 20 November 2002, revised 3 February 2003, accepted 19 February 2003) Eur. J. Biochem. 270, 1567–1577 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03526.x The genes encoding Qor have been cloned and sequenced, some biochemical properties of Qor have been described, and its redox-centers have been characterized by EPR spectroscopy [1,2,23–25]. However, a thorough study of the catalytic mechanism of Qor and other molybdenum hydroxylases should also involve the construction of protein variants carrying distinct amino acid replacements, and their biochemical, spectroscopic, and – if possible – struc- tural characterization. A prerequisite for such a mutagenic approach is the availability of a suitable system for the manipulation and the regulated, functional expression of genes coding for molybdenum hydroxylases. Whereas genes coding for Mo-MPT- or molybdenum molybdopterin guanine dinucleotide- (Mo-MGD-) containing hydroxylases have been expressed successfully in Escherichia coli hosts [26–28], attempts to achieve heterologous functional expression of Mo-MCD-containing enzymes in E. coli failed [23,29] (K. Parschat & S. Fetzner, unpublished results). However, in E. coli, all known molybdoenzymes contain the MGD form of the molybdenum cofactor. Synthesis of Mo-MGD from Mo-MPT and Mg 2+ -GTP is catalyzed by the MobA protein [30–36]. Possibly, E. coli lacks an enzyme that catalyses the formation of Mo-MCD from Mo-MPT, and/or it is not able to integrate the Mo-MCD cofactor into the corresponding apoprotein. In a first attempt to functionally express genes coding for a Mo-MCD-containing hydroxylase in heterologous hosts, the iorAB genes of Brevundimonas diminuta 7, coding for isoquinoline 1-oxidoreductase, were cloned in P. putida KT2440 and in the quinoline degrading strain, P. putida 86. However, the level of Ior synthesis was very low in both expression clones, and only P. putida 86 pIL1 produced Ior protein that was catalytically active [37]. As it is highly desirable to obtain an expression system for molybdenum hydroxylases harboring the MCD cofactor, we tested whether expression of the qorMSL genes from P. putida 86 in P. putida host strains results in the forma- tion of catalytically competent enzyme. Materials and methods Plasmids, bacterial strains and growth conditions Plasmids and bacterial strains used in this work are listed in Table 1. E. coli XL-1 Blue MRF¢ and E. coli S17-1 were grown at 37 °C in Luria–Bertani (LB) broth [38]. P. put- ida 86 was grown in mineral salts medium containing quinoline as the sole carbon source [2], or in LB broth [38], at 30 °C. For the preparation of P. putida cells that are competent for electroporation, TB medium (Terrific broth) [38] was used. When growing P. putida 86-1, streptomycin (500 lgÆmL )1 )wasaddedtotherespectivemedium. DNA techniques Standard recombinant DNA techniques were used for DNA isolation [38,39] and restriction, agarose gel electro- phoresis and cloning [38]. Random digoxigenin labelling of probes was performed using the DIG High Prime Labeling and Detection Kit (Roche Diagnostics). Competent E. coli and P. putida cells for electroporation were generated as described by Dower et al.[40]andIwasakiet al.[41], respectively. Construction of P. putida 86-1 D qor A DNA segment containing the qorMSL genes and flanking regions (Ôqor-upÕ,1055bpandÔqor-downÕ, 1898 bp) was inserted into the SmaI restriction site of pUC18 [42], forming pBG1. Competent E. coli XL-1 Blue MRF¢ cells were transformed with pBG1 by electroporation. The qorMSL genes in pBG1 were removed using XhoI, that cleaves 364-bp upstream of the start codon of qorM,and DraIII, that cleaves 8-bp downstream of the stop codon of qorL. After removing the 3¢ overhang and filling the 5¢ overhang of the plasmid with T4 DNA-polymerase, a PCR amplificate of nptII [43], that contained flanking XhoIand DraIII sites, was inserted by blunt-end ligation, resulting in the two constructs, pBG2a and pBG2b (nptII in the same and in the opposite orientation with respect to the deleted qor genes, respectively). E. coli XL-1 Blue MRF¢ was used as host strain for pBG2a and pBG2b. The nptII inserts together with the flanking regions (Ôqor-upÕ and Ôqor-downÕ) were removed from pBG2a and pBG2b using HindIII, and inserted into the HindIII restriction site of pSUP202 [44], resulting in pBG3a and pBG3b. Competent E. coli S17-1 cells were transformed with pBG3a and pBG3b. Mating of E. coli S17-1 pBG3a/3b and P. putida 86-1 was performed as described by Masepohl et al.[45],exceptthatLBplates were used instead of PY plates. P. putida 86-1 transconju- gants were selected for kanamycin resistance and chloram- phenicol sensitivity, indicating replacement of qorMSL in P. putida 86-1 by nptII by double cross-over events. Mutants with nptII in the same orientation (P. putida 86-1 Km-a) as well as mutants with nptII in the opposite orientation (P. putida 86-1 Km-b) with respect to the deleted qor genes were obtained. DNA isolated from these mutants did not hybridize with a DIG-labelled probe for pSUP202, confirming that nptII actually was inserted by double cross-over. However, DNA from these mutants still showed a positive hybridization signal with a DIG-labelled probe for the qor genes (corresponding to the nucleotides 1201–4233 of GenBank accession number X98131), and the P. putida 86-1 kanamycin resistant mutants still formed Qor. PCR analyses confirmed that nptII was replacing one copy of qorMSL and that P. putida 86-1 contains more than one copy of the qor genes and their flanking regions. The plasmid pBG3a was digested with XhoIandDraIII to remove nptII. After the removal of the 3¢ overhang and the filling of the 5¢ overhang of the plasmid with T4 DNA- polymerase, a PCR amplificate of aacC1 [46] was inserted by blunt end ligation, resulting in pBG4a and pBG4b (aacC1 in the same and in the opposite orientation with respect to the deleted qor genes, respectively), that were used to transform E. coli S17-1. Mating of E. coli S17-1 pBG4a/ 4b and P. putida 86-1 Km-a/P. putida 86-1 Km-b yielded three Kan r and Gen r mutants of P. putida 86-1 with a Qor – phenotype. DNA isolated from these three P. putida 86-1 Dqor (Kan r Gen r ) mutants did not hybridize with the probes for pSUP202 and qor. PCR analyses confirmed the complete deletion of the qor genes and showed that all three mutants contained nptII in the same orientation with 1568 U. Frerichs-Deeken et al. (Eur. J. Biochem. 270) Ó FEBS 2003 respect to the deleted qorMSL genes, and aacC1 in the opposite orientation with respect to the deleted second copy of qorMSL. Expression cloning of qorMSL genes Using genomic DNA isolated from wild-type P. putida 86 as template, the qorMSL genes, including the preceding Shine-Dalgarno sequence [23] (GenBank accession number X98131), were amplified using 5¢-GCAGgaattc CTGCTGGTTTTTCGCTTG-3¢ as the forward primer and 5¢-ATAGggatccCTGGTAGACAGGACTCACCC-3¢ as the reverse primer in the Expand Long Template PCR System (Roche Diagnostics). The nucleotides of the forward and reverse primer that are set as bold are complementary to nucleotides 653–670 and 4439–4420 of GenBank acces- sion number X98131, respectively. The primers included an EcoRI and a BamHI recognition site in the forward and reverse primer, respectively (small letters), that allowed the ligation of the PCR product into the multiple cloning site of pJB653, generating pUF1. The recipient strains P. putida KT2440 and P. putida 86-1 Dqor were transformed by electroporation [40]. Clones containing pUF1 were identi- fied by colony blotting and hybridization [47] using the qor probe described above. Growth of recombinant strains and preparation of crude extracts All P. putida pUF1 clones were grown in the presence of 500 lgÆmL )1 ampicillin in mineral salts medium [2] supple- mented with 1 gÆL )1 ammonium sulfate. Induction of qorMSL expression from the Pm promoter of pUF1 was achieved by addition to the medium of the XylS effectors, benzoate and 2-methylbenzoate. For small-scale growth of P. putida KT2440 pUF1 clones, succinate (10 gÆL )1 )and sodium benzoate (8 m M ) were used as sources of carbon. Two 4 L glass fermenters were used to generate biomass for protein purification. Benzoate (8 m M ) was used as the carbon and energy source for growth of P. putida KT2440 pUF1; it was added repeatedly to the cultures. 2-Methyl- benzoate (2 m M ), as an additional XylS effector, was added Table 1. Bacterial strains and plasmids used in this study. Strain/plasmid Genotype and/or relevant properties Reference or source Escherichia coli S17-1 RP4-2 (Tc::Mu) (Km::Tn7) integrated into the chromosome; Tra + , recA, pro, thi, hsdR [44] E. coli XL-1 Blue MRF¢ D(mcrA)183, D(mcrCB-hsdSMR-mrr)173, endA1, supE44, thi-1, rec A1, gyrA96 relA1 lac [F¢ proAB lacI q ZDM15 Tn10 (Tet r )] Stratagene Pseudomonas putida KT2440 r – derivative of P. putida mt-2 [67] P. putida 86 Wild-type strain utilizing quinoline as sole source of carbon and energy [68] P. putida 86-1 Spontaneous Str r mutant of P. putida 86 This work P. putida 86-1 Km-a nptII replacing one copy of qorMSL in P. putida 86-1; nptII in the same orientation with respect to the deleted qor genes; Str r , Kan r ,Qor + This work P. putida 86-1 Km-b nptII replacing one copy of qorMSL in P. putida 86-1; nptII in the opposite orientation with respect to the deleted qor genes; Str r , Kan r ,Qor + This work P. putida 86-1 Dqor Two copies of qorMSL replaced by nptII and aacC1, respectively; nptII is in the same orientation with respect to the deleted qorMSL genes, and aacC1 is in the opposite orientation with respect to the deleted second copy of qorMSL. Str r , Kan r , Gen r ;Qor – This work pUC18 ori colE1 , lacZ, Amp r [42] pSUP202 RP4-Mob + ori colE1 ; Amp r , Cam r , Tet r [44] pBG1 6678 bp segment of P. putida 86 DNA (qorMSL and flanking regions Ôqor upÕ [1898 bp] and Ôqor downÕ [1055 bp]) cloned into SmaI site of pUC18 This work pBG2a derivative of pBG1: 4097 bp XhoI-DraII fragment containing qorMSL replaced by nptII; nptII in the same orientation with respect to the deleted qor genes This work pBG2b derivative of pBG1: 4097 bp XhoI-DraII fragment containing qorMSL replaced by nptII; nptII in the opposite orientation with respect to the deleted qor genes This work pBG3a HindIII fragment of pBG2a containing nptII together with flanking regions Ôqor-upÕ (1534 bp) and Ôqor-downÕ (1047 bp) cloned into the HindIII restriction site of pSUP202 This work pBG3b HindIII fragment of pBG2b containing nptII together with flanking regions Ôqor-upÕ (1534 bp) and Ôqor-downÕ (1047 bp) cloned into the HindIII restriction site of pSUP202 This work pBG4a nptII in pBG3a replaced by aacC1; aacC1 in the same orientation with respect to the deleted qor genes This work pBG4b nptII in pBG3a replaced by aacC1; aacC1 in the opposite orientation with respect to the deleted qor genes This work pJB653 Broad-host-range cloning vector; Pm promoter, xylS for transcriptional regulation; Amp r [52] pUF1 qorMSL (3786 bp PCR amplificate from P. putida 86 DNA) inserted into EcoRI – BamHI sites of pJB653 This work Ó FEBS 2003 Expression of quinoline 2-oxidoreductase genes (Eur. J. Biochem. 270) 1569 at a D 550 value of 0.7–1.0. P. putida 86-1 Dqor pUF1 was either grown in benzoate (8 m M , fed repeatedly), or in benzoate (5 m M )plus1H-2-oxoquinoline (2.8 m M )as carbon sources (fed repeatedly). 2-Methylbenzoate was added at a D 550 value of 0.7–1.0. P. putida KT2440 pUF1 as well as P. putida 86-1 Dqor pUF1 cells were harvested by centrifugation (5 500 g,20min)ataD 550 value ‡ 3.0. Crude extracts were prepared by French TM Press treatment at 2.1–2.4 · 10 8 Pa of cell suspensions in 100 m M Tris/HCl buffer (pH 8.5) containing 10 l M phe- nylmethanesulfonylfluoride and 0.05 lLÆml )1 Benzon nuc- lease (Merck, Darmstadt, Germany), subsequent sonification, and removal of debris by centrifugation (48 000 g,45min,4°C). PAGE Non-denaturing PAGE was performed using the high pH discontinuous system according to Hames [48], and 10% and 4% acrylamide (w/v) in the separating and stacking gels, respectively. SDS/PAGE was performed according to the method of Laemmli [49]. Proteins were stained in Coomassie blue R-250 [0.1% (w/v) in 50% (w/v) aqueous trichloroacetic acid], and de-stained in water/methanol/ acetic acid (60 : 30 : 10, v/v/v). Purification of Qor from P. putida 86, P. putida KT2440 pUF1 and P. putida 86-1 D qor pUF1 Qor was purified using ammonium sulfate fractionation (0.8–1.5 M ), hydrophobic interaction chromatography [phe- nyl Sepharose CL-4B (Amersham Pharmacia, Freiburg, Germany) packed into a 15 · 113 mm BioScale MT20 column (Bio-Rad, Mu ¨ nchen, Germany)], and anion exchange chromatography (BioScale DEAE10 column, Bio-Rad) essentially as described by Tshisuaka et al.[2], but omitting the heat precipitation step. Preparation of anti-Qor antisera Polyclonal rabbit Igs were raised against Qor that was purified from wild-type P. putida 86. An initial subcuta- neous injection of Qor protein was followed by boost injections on days 14, 28 and 56, and the sera were collected on day 87 (Eurogentec, Belgium). Western blotting, and immunodetection of Qor protein Proteins separated in SDS/PAGE were transferred onto nitrocellulose membranes (Optitran BA-983 reinforced NC, Schleicher & Schuell, Dassel, Germany) by semidry blotting for 70 min at 0.9 mAÆcm )2 using 25 m M Tris, 190 m M glycine in 20% (v/v) aqueous methanol as continuous blotting buffer [50]. Antisera diluted 1500-fold in blocking solution (Roche Diagnostics), digoxigenin-labelled anti- (rabbit IgG) Igs (diluted 60-fold), and alkaline phospha- tase-labelled anti-digoxigenin Ig (diluted 5000-fold) were used to detect Qor. Colorimetric immunodetection with nitroblue tetrazolium salt and 5-bromo-4-chloro-3-indolyl phosphate was performed as recommended by the supplier (The DIG System User’s Guide for Filter Hybridization, Roche Diagnostics, 1995). Assays for Qor activity and protein content, and determination of the apparent K m and k cat values for quinoline The activity of Qor was determined spectrophotometrically by measuring the quinoline-dependent reduction of the artificial electron acceptor, iodonitrotetrazolium chloride (INT) [2]. One unit was defined as the amount of enzyme that reduces 1 lmol INTÆmin )1 at 25 °C. For activity staining of Qor in PA gels, gels were immersed in the same buffer as used for the spectrophotometric assay, containing substrate and electron acceptor [2]. Protein concentrations were estimated by the method of Bradford as modified by Zor and Selinger [51], using bovine serum albumin as standard protein. The Qor preparations from P. putida KT2440 pUF1 and from P. putida 86-1 Dqor pUF1 used for the determination of K m app, (quinoline) and k cat app, (quinoline) showed a specific activity of 2 UÆmg )1 and 17 UÆmg )1 , respectively. The kinetic parameters were estimated from Hanes plots. Determination of the metal contents of the Qor proteins, and detection of the nucleotide moiety of the molybdenum cofactor The contents of molybdenum and iron were determined by inductively coupled argon plasma (ICAP) emission spectro- scopy (Thermo Jarrell-Ash Enviro 36 ICAP) by The Chemical Analysis Laboratory of the University of Georgia (Athens, GA, USA). The protein samples used for metal analyses showed specific activities of 15.5 UÆmg )1 ,1.8– 2.3 UÆmg )1 and 18 UÆmg )1 for the Qor proteins from P. putida 86, P. putida KT2440 pUF1 and P. putida 86-1 Dqor pUF1, respectively. For each protein, two independ- ent analyses were performed. For identification of the nucleotide moiety of the molybdenum cofactor, the enzymes were incubated at 95 °C for 10 min in the presence of sulfuric acid (3%, v/v); hydrolysis leads to the release of nucleotides from MCD and FAD. After centrifugation for 10 min at 20 000 g,the supernatant was analyzed by isocratic HPLC on a Lichrospher 100 RP-18 EC column, or a Nucleosil 100– 5C18 column (5 lm particle size, 4 · 250mm)ataflow rate of 1 mLÆmin )1 with 0.2% acetic acid, 0.5% methanol (v/v) with water as the eluent. The compounds were identified by their retention times, as well as the corres- ponding spectra (obtained with a photodiode array detec- tor, Waters 996), and by co-chromatography with authentic reference compounds (CMP, AMP, GMP, FAD). For quantification of CMP, the system was calibrated with external standards. Nucleotides bound loosely to Qor proteins were extracted by boiling the enzymes for 10 min in 20 m M Tris/HCl, pH 7.5, containing 2% SDS (w/v). The extract was separated from the protein by ultra-filtration and analysed by HPLC as described above. Electron paramagnetic resonance (EPR) spectroscopy The Qor samples from P. putida KT2440 pUF1 and from P. putida 86-1 Dqor pUF1 used for the EPR analyses showed a specific activity of 1 UÆmg )1 and 22 UÆmg )1 , 1570 U. Frerichs-Deeken et al. (Eur. J. Biochem. 270) Ó FEBS 2003 respectively. The samples (Qor from P. putida KT2440 pUF1: 12.4 nmol; Qor from P. putida 86-1 Dqor pUF1: 9.6 nmol, in 50 m M Tris/HCl buffer pH 8.5) were reduced in a first step by a tenfold excess of quinoline dissolved in ethanol. For Qor from P. putida KT2440 pUF1, a subsequent reduction with a tenfold molar excess of dithionite (Na 2 S 2 O 4 ) was performed. The samples were transferred into quartz EPR-tubes and frozen in liquid nitrogen within 1 min. EPR spectra at X-band frequencies were recorded on a Bruker ESP 300 spectrometer equipped with a continuous helium flow cryostat (ESR 900, Oxford Instruments) for the temperature range 5–80 K or with a quartz dewar for measurements at liquid nitrogen temper- atures. The magnetic field and the microwave frequency were determined with a NMR gaussmeter and a microwave counter, respectively. The modulation amplitude for spectra recording generally was 0.5 mT. Spectra of Qor from both clones were recorded with identical spectrometer settings. Due to the low spin concentrations, spectra were accumu- lated to achieve a reasonable signal-to-noise ratio. Results and discussion Qor protein from P. putida KT2440 pUF1 Crude extracts of P. putida KT2440 pUF1 clones when grown in the presence of benzoate and/or methylbenzoate contained a prominent protein showing the same electro- phoretic mobility as Qor from wild-type P. putida 86, suggesting that P. putida KT2440 pUF1 synthesized signi- ficant amounts of Qor protein (Fig. 1A). Western blot analysis confirmed the presence of the three subunits of Qor in crude extracts of P. putida KT2440 pUF1 clones (Fig. 1B). Qor from P. putida KT2440 pUF1 was enriched 91-fold with a yield of 43% (Table 2). Whereas the specific activity of Qor purified to electrophoretic homogeneity from wild-type P. putida 86 usually varied between 19 and 23 UÆmg )1 , the specific activity of Qor preparations purified from P. putida KT2440 pUF1 was only 0.8–2.7 UÆmg )1 . P. putida 86-1 D qor As the wild-type strain P. putida 86 is known to be able to synthesize Mo–MCD, a deletion mutant lacking the genes that code for Qor might be a suitable host for the expression cloning of genes coding for Mo–MCD-containing molyb- denum hydroxylases. By replacing two copies of qorMSL in thegenomeofP. putida 86-1 by nptII and aacC1, the mutant P. putida 86-1 Dqor was obtained. It had lost the ability to grow on quinoline, and it did not synthesize Qor protein. However, it was able to utilize 1H-2-oxoquinoline, i.e., the product of the Qor-catalyzed reaction, with a growth rate comparable to that of wild-type P. putida 86. This indicates that the mutations did not affect any subsequent step of the quinoline degradation pathway. Fig. 1. Synthesis of Qor protein by P. putida KT2440 pUF1. (A) Non- denaturing PAGE. Lane 1, crude extract of P. putida KT2440; lane 2, crude extract of P. putida KT2440 pJB653; lanes 3–5, crude extracts of different P. putida KT2440 pUF1 clones; lane 6, Qor purified from wild-type P. putida 86; lane 7, crude extract of P. putida 86 grown in mineral salts medium containing quinoline as sole carbon source; lane 8, crude extract of P. putida 86 grown in LB broth. (B) Immuno- detection of Qor subunits in Western blot of crude extracts separated by SDS/PAGE. Lane 1, crude extract of P. putida KT2440; lane 2, crude extract of P. putida KT2440 pJB653; lanes 3–5, crude extracts of different P. putida KT2440 pUF1 clones; lane 6, Qor purified from wild-type P. putida 86. Ó FEBS 2003 Expression of quinoline 2-oxidoreductase genes (Eur. J. Biochem. 270) 1571 Conditions of Qor synthesis in wild-type P. putida 86, P. putida 86-1 D qor pUF1 and P. putida 86 pJB653 Qor of the wild-type strain P. putida 86 has been described as an inducible enzyme [2]. In crude extracts of P. putida 86 cells grown on quinoline, the specific activity of Qor was about 0.2 UÆmg )1 of protein, whereas the specific Qor activity in crude extracts of succinate- or benzoate-grown cells was below 0.001 UÆmg )1 (Table 3). Succinate-grown cells of P. putida 86-1 Dqor pUF1 did not contain any detectable Qor activity (Table 3), as expression of the qorMSL genes inserted into the multiple cloning site of pJB653 from the Pm promoter is controlled by the plasmid-encoded XylS protein, that is activated by benzoate effectors [52]. The presence of the expression vector pJB653 in P. put- ida 86 did not significantly influence the specific Qor activities in extracts of succinate-grown cells, and quino- line-grown cells (Table 3). However, in benzoate-grown cells of P. putida 86 pJB653, the specific Qor activity was more than 110-fold higher than in benzoate-grown cells of the wild-type strain P. putida 86. As benzoate as such is not an inducer of Qor synthesis in P. putida 86, the effect of benzoate in P. putida 86 pJB653 probably is mediated by the plasmid-encoded XylS protein. The family of AraC/ XylS proteins comprises positive transcriptional regulators that are characterized by significant amino acid sequence homology extending over a 100-residue stretch constituting the DNA binding domain [53–56]. In P. putida 86, a putative xylS homologue designated oxoS has been previ- ously identified upstream of the oxoO gene that codes for a protein involved in the quinoline degradation pathway; oxoO is localized about 7 kb upstream of the qorMSL genes [57]. We may speculate that the degradation pathway is regulated by the XylS-type transcriptional activator OxoS, that might bind quinoline as an effector. In P. putida 86 pJB653, the plasmid-encoded XylS protein when activa- ted by its effector benzoate might recognize the putative DNA binding site of OxoS and activate transcription of the catabolic gene cluster. Qor protein from P. putida 86-1 D qor pUF1 Immunodetection of the subunits of Qor in Western blots confirmed that the deletion mutant P. putida 86-1 Dqor containing the expression vector pJB653 did not synthesize Qor protein, whereas P. putida 86-1 Dqor pUF1 grown on benzoate or on a mixture of benzoate and 1H-2-oxoqui- noline formed Qor (not shown). From a 4 L fermenter of P. putida 86-1 Dqor pUF1 fed repeatedly with benzoate and 1H-2-oxoquinoline as carbon sources, between 16 and 18 g of wet biomass were obtained after cultivation for 24–28 h. Table 4 summarizes the enrichment of Qor from P. put- ida 86-1 Dqor pUF1. The protein preparations showed specific activities of 20–23 UÆmg )1 , that is comparable to the activity of wild-type Qor. Kinetic properties of the Qor proteins from P. putida 86, P. putida KT2440 pUF1 and P. putida 86-1 D qor pUF1 The apparent K m values of the Qor proteins for quinoline were similar, whereas the apparent k cat value for quinoline of Qor from strain KT2440 pUF1 was eight- to tenfold lower than that of wild-type Qor and Qor from P. put- ida 86-1 Dqor pUF1 (Table 5). Table 2. Purification of Qor protein from P. putida KT2440 pUF1. Starting material was 34 g of wet biomass. In crude extracts, quinoline- independent INT reduction mediated by unspecific reductases of strain KT2440 impedes accurate measurement of quinoline-dependent INT reduction catalyzed by Qor. ppt, precipitation. Fraction Activity (Units) Protein (mg) Specific activity (UÆmg )1 ) Purification (-fold) Yield (%) Crude extract 85.3 3696 0.023 1 100 Ammonium sulfate ppt 87.4 516 0.17 7 102 Phenyl Sepharose CL-4B 71.9 76 0.95 41 84 BioScale DEAE10 36.5 17.4 2.10 91 43 Table 3. Activity of Qor in crude extracts of wild-type P. putida 86, P. putida 86 pJB653 and P. putida 86-1 Dqor pUF1 grown on different carbon sources. Strain Specific activity (UÆmg )1 )ofQor in crude extracts after growth on: Succinate Benzoate Quinoline P. putida 86 < 0.001 < 0.001 0.21 P. putida 86 pJB653 0.001 0.11 0.18 P. putida 86-1 Dqor pUF1 0 0.10 – a a As benzoate is necessary as an XylS effector for expression of qorMSL from pUF1, P putida 86-1 Dqor pUF1 is not able to grow on quinoline as a sole source of carbon. Table 4. Purification of Qor protein from P. putida 86-1 Dqor pUF1. Starting material was 27 g of wet biomass. ppt, precipitation. Fraction Activity (Units) Protein (mg) Specific activity (UÆmg )1 ) Purification (-fold) Yield (%) Crude extract 312 3045 0.10 1 100 Ammonium sulfate ppt 271 416 0.65 6.4 87 Phenyl Sepharose CL-4B 299 78 3.83 37.5 96 BioScale DEAE10 206 9.3 22.1 216.7 66 1572 U. Frerichs-Deeken et al. (Eur. J. Biochem. 270) Ó FEBS 2003 Metal content of the Qor proteins and analysis of nucleotides released from the Qor proteins from P. putida 86, P. putida KT2440 pUF1 and P. putida 86-1 Dqor pUF1 Native Qor is expected to contain 2 g atom of molybdenum and8gatomofironpermolofenzyme[1,2].However, with the analytical method performed (direct analysis without preceding digestion), only 0.8 g atom of molyb- denum and 5.5 g atom of iron were detected per mol of wild-type Qor. The iron content of Qor from P. put- ida KT2440 pUF1 corresponded to that of wild-type Qor, however, its molybdenum content was tenfold lower (Table 5); this could explain the decrease in activity. The molybdenum cofactor of wild-type Qor has previ- ously been identified as Mo-MCD [14]. Treatment of Qor proteins with sulfuric acid and subsequent analysis of the preparation by reverse-phase HPLC showed the presence of CMP and AMP (from FAD). GMP was not present in any Qor extract, indicating that the host strains did not incorporate Mo-MGD, or free GMP, into the cofactor binding domain of the Qor protein. Similar amounts of CMP were released from the three Qor proteins (Table 5). However, especially in the nearly inactive Qor protein from P. putida KT2440 pUF1, it may be possible that the nucleotide is occupying the CMP binding site of the Qor protein, without being part of an MCD cofactor. To detect loosely bound CMP, nucleotides were extracted from the proteins by boiling in aqueous SDS. This method led to the release of about 0.4 mol of CMP per mol of enzyme, however, approximately the same amounts of CMP were released from the different Qor enzymes. Thus, the low activity observed for the Qor protein from P. putida KT2440 pUF1 seems to be correlated to a deficiency in the metal, not to a deficiency in the organic part of the molybdenum cofactor. However, we cannot exclude that the pyranopterin part of the cofactor is somehow defective in Qor from strain KT2440 pUF1. UV/Visual spectra of the Qor proteins from P. putida 86, P. putida KT2440 pUF1 and P. putida 86-1 D qor pUF1 The UV/Visual spectra of Qor purified from P. putida KT2440 pUF1 and of wild-type Qor were very similar, except for the absorption around 305 nm, that was signi- ficantly decreased in Qor from P. putida KT2440 pUF1. This decrease might reflect a deficiency in the pyranopterin cofactor. The ratios A 280nm /A 450nm and A 450nm /A 550nm of 4.5–5 and 2.8–3, respectively, were identical in both proteins, indicating the presence of the full set of iron–sulfur clusters and stoichiometric amounts of FAD. The UV/Visual spectrum of Qor from P. putida 86-1 Dqor pUF1 was typi- cal for a molybdo-iron/sulfur flavoprotein; it lacked the marked decrease at 305 nm observed in the Qor protein from P. putida KT2440 pUF1 (Fig. 2). Analysis of redox-active centers in Qor from P. putida KT2440 pUF1 and P. putida 86-1 D qor pUF1 by EPR spectroscopy Mo. Reduction of the Qor protein isolated from wild-type P. putida 86 with its substrate quinoline led to the forma- tion of the Mo(V)-rapid species that is readily observable at 77 K; the Mo(V) rapid species is indicative of the monooxo-monosulfido-type molybdenum center [2] and is thought to represent a complex of substrate with enzyme [5]. The typical, almost axial, spectrum in Fig. 3A shows the splitting of the H-D-exchangeable proton attributed to the Table 5. Metal content, amount of CMP released by hydrolysis with sulfuric acid and kinetic parameters of the Qor proteins. Source of Qor Metal content (g atom per mol of enzyme) a CMP released by hydrolysis (mol per mol of enzyme) b Kinetic parameters Mo Fe CMP K m app (quinoline) (m M ) k cat app (quinoline) (s )1 ) P. putida 86 (grown on quinoline) 0.8 5.5 1.2 0.18 c 74 P. putida KT2440 pUF1 (grown on benzoate) 0.08 5.4 1.3 0.12 8.7 P. putida 86–1Dqor pUF1 (grown on benzoate + 1H-2-oxoquinoline) 0.5 3 1.2 0.12 85.4 a Average of two determinations; b average of three experiments; c [1]. Fig. 2. UV/Visual spectra of Qor proteins. Solid line, Qor purified from wild-type P. putida 86; dotted line, Qor purified from P. putida KT2440 pUF1; dashed line, Qor from P. putida 86-1 Dqor pUF1. The increased absorption at 280 nm of the latter is due to contaminating colourless proteins. Ó FEBS 2003 Expression of quinoline 2-oxidoreductase genes (Eur. J. Biochem. 270) 1573 sulfhydryl-group of the one electron reduced complex [2,24]. When the Qor protein isolated from P. putida KT2440 pUF1 (specific activity: 1 UÆmg )1 ) was reacted with quino- line, a rapid-type EPR-signal of rather small intensity was detected (Fig. 3B). In contrast, the catalytically competent Qor protein purified from P. putida 86-1 Dqor pUF1 produced the rapid EPR-signal in considerably higher amounts (Fig. 3C). As both Qor samples were treated and recorded under identical experimental conditions, the relative quantities of the Mo(V)-rapid species could be estimated from the EPR intensities. This comparison showed that the amount of Mo(V)-species formed in Qor from P. putida KT2440 pUF1 was approximately 25-fold lower than in Qor from P. putida 86-1 Dqor pUF1. In accordance with the results of the metal analyses, the very low intensity of the Mo(V) rapid EPR signal suggested that most of the Qor molecules are deficient in molybdenum. This is in line with the finding that only very weak EPR- signals of reduced FeS-clusters were detected after addition of substrate (almost no electron transfer from quinoline via Mo to FeS), but clearly are formed by direct reduction of the FeS-clusters with dithionite (see below). Thus, although P. putida KT2440 pUF1 presumably is able to catalyse the synthesis and insertion of a cytidine dinucleotide cofactor into recombinant Qor as suggested by the release of CMP after hydrolysis of the enzyme, it appears that the assembly of intact Mo-MCD is a bottleneck in strain KT2440 pUF1, leading to the incorporation of a defective, molybdenum deficient cofactor into the maturing Qor protein. The rapid EPR-signal of Qor from P. putida 86-1 Dqor pUF1 (Fig. 3C) shows some minor differences as compared to the signal of the wild-type protein. The distortions marked by arrows in trace C are caused by signals of the resting species which are associated with inactive Mo(V)-centers formed during the preparation process in varying amounts [24]. The finding that in each of the three Qor enzymes the majority of the Mo(V) species was represented by the rapid type EPR signal indicates that the molybdenum centers are predominantly in the correct monooxo-monosulfido form. The ÔslowÕ type signal, associated with the inactive desulfo (¼ dioxo) form [2], could not be identified in the spectral patterns indicating that this species is, if at all, present only in negligible amounts. Besides the low intense resonances visible at the high- and low-field side of the rapid EPR-signal (traces A and C) and originating from natural Mo-isotopes with nuclear spin I ¼ 5/2, also small lines of the semiquinone radical form of FAD were observed at g ¼ 2.004. Fe/S. When the temperature was lowered to about 20 K the characteristic rhombic EPR-patterns of two [2Fe)2S] clusters, FeSI and FeSII, became visible. Their assignment is given in Fig. 4A for the wild-type Qor reduced with quinoline. In this case, the g 2 -component of FeSII is superimposed by the intense and saturation broadened Mo(V)-signal. An identical spectrum of FeS-clusters and Mo(V) contribution was found for Qor from P. putida 86-1 Dqor pUF1 reduced with quinoline as shown in Fig. 4C. In contrast, for Qor from P. putida KT2440 pUF1 only extremely weak signals of the FeS-centers (not shown) were present after reduction with substrate. When this sample was subsequently reduced with a tenfold excess of dithio- nite, the signals of both FeS-centers appeared in appreciable intensity as indicated in Fig. 4B. The absence of Mo(V)- signals reveals the g 2 -component of FeSII. It is noted that the g-factors of the FeSI and II signals of the Qor proteins from the wild-type strain and from P. putida 86-1 Dqor pUF1 are identical, whereas the g 1 -components for Qor from P. putida KT2440 pUF1 are shifted slightly to lower g-factors. Such spectral differences depending on the mode of reduction have been reported for wild-type Qor [24]. In general, the differences in g-factor of the FeS-signals are less than 0.003 compared to the corresponding signals of wild-type Qor [24]. An exception is found for the g 3 -component ofFeSII of Qor from P. putida KT2440pUF1 that is shifted to a lower g-factor of 1.858 as compared to 1.871 for the wild-type Qor (Fig. 4B). The change of the g 3 -factor of FeSII may indicate that the electronic structure was influenced probably by an unknown alteration of the immediate environment of this FeS-cluster. For completeness, it should be mentioned here that a weak signal of yet unknown origin is observed in all reduced Qor samples. Although it is located close to the g-factor of Fig. 3. EPR spectra of the rapid species in Qor from wild-type P. putida (A), P. putida KT2440 pUF1 (B) and P. putida 86-1 Dqor pUF1 (C) formed after reduction with substrate quinoline. Spectra were recorded at 77 K at a microwave power of 2 mW. Trace B is multiplied by a factor of six to show the small signals of the rapid species in this sample. The arrows indicate the position of contribution of the resting species particularly to spectrum C. 1574 U. Frerichs-Deeken et al. (Eur. J. Biochem. 270) Ó FEBS 2003 the FAD radical signal its saturation and temperature behaviour points to a metal centered species. The EPR analyses showed that, apart from some small contribution of nonfunctional species (resting), the EPR- signals of wild-type Qor and of Qor from P. putida 86-1 Dqor pUF1 are virtually superimposable, indicating identi- cal cofactor composition and arrangement. Conclusions Assembly of [2Fe)2S] clusters as well as flavin and Mo-MPT biosynthesis [58] are thought to involve ubiquitously conserved pathways, but additional reactions that modify Mo-MPT appear to be restricted to certain organisms. In E. coli, for example, all known molybdenum enzymes contain MGD as the organic part of the molybdenum cofactor, and attempts to express genes encoding Mo-MCD- containing enzymes in E. coli failed [23,29] (K. Parschat & S. Fetzner, unpublished results). In this work, we tested whether expression of the qorMSL genes from P. putida 86 in P. putida KT2440 and in a qorMSL deletion mutant of P. putida 86-1 results in the formation of catalytically active enzyme. The expression clone P. putida 86-1 Dqor pUF1 synthes- ized catalytically competent Qor protein that in its kinetic and spectroscopic properties seemed identical to wild-type Qor. This clone did not allow overproduction of Qor, however, as about 6–8 mg of Qor protein can be purified from 10 g of wild-type P. putida 86 biomass, protein production was not the primary goal of this work. This expression system will allow the genetic manipulation of the qor genes by mutagenic approaches, and the synthesis of enzyme variants, that after purification by the established protocol, will be available for further biochemical and spectroscopic characterization. The mutant P. putida 86-1 Dqor may also be a suitable recipient for the expression cloning of genes coding for other Mo-MCD-containing hydroxylases. Bacterial strains synthesizing molybdenum hydroxylases, or isolated molybdenum hydroxylases catalyzing regio- specific hydroxylation reactions, are useful biocatalysts for industrial processes to manufacture hydroxy-substi- tuted N-heteroaromatic compounds [59–63]. Enzyme engineering may be used to improve the stability or catalytic efficiency of the enzymes, or to alter their substrate specificity [64–66]. Most of the molybdenum hydroxylases catalyzing the hydroxylation of N-hetero- aromatic compounds contain the Mo-MCD cofactor [5,8,9]. Thus, a system enabling the genetic manipulation and regulated expression of genes coding for Mo-MCD- containing hydroxylases might also be of biotechnological importance. Acknowledgements We thank S. Valla, Norwegian University of Science and Technology, Trondheim, Norway, for kindly providing pJB653 and M. Sohni, Oldenburg, for selecting the streptomycin resistant mutant of P. put- ida 86. We thank W. Wackernagel, Oldenburg, and the late W. Klipp, Bochum, for the generous gift of plasmids and strains. 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