Gene cluster of Arthrobacter ilicis Rü61a involved in the degradation of quinaldine to anthranilate. Characterization and functional expression of the quinaldine 4-oxidase genes qoxLMS

decarboxylase-like protein, an enzyme of the mandelate racemase group, and hypothetical proteins involved in transcriptional regulation, and metabolite transport. (NH 4 ) 2 SO 4 . After washing the column with 85 mM Tris-HCl buffer, pH 7, containing 0.55 M (NH 4 ) 2 SO 4 , and elution with a linear gradient from the washing buffer to 50 mM Tris-HCl, pH 7, the active fractions were pooled and applied to an anion exchange column UNO TM -Q 1 (Biorad, München, Germany) that had been equilibrated in 50 mM Tris-HCl buffer, pH 7. The proteins were eluted with a linear gradient from 0.15 M to 1 M NaCl in the equilibration buffer after washing the column with 0.15 M NaCl in the same buffer. For gel filtration, the enriched Qox was loaded onto the HiLoad 26/60 Superdex 200 prep grade column (Amersham Biosciences, Freiburg, Germany). The column was equilibrated and run at 1 ml/min with 50 mM Tris-HCl, pH 7, containing 0.25 M NaCl. Fractions showing Qox activity were pooled, concentrated by ultrafiltration (membrane cut-off 10 kDa), and stored at -80 °C.


INTRODUCTION
The genetic diversity and flexibility of prokaryotes has led to the evolution of an impressive variety of metabolic pathways to transform or degrade natural as well as numerous xenobiotic compounds. The genes coding for enzymes involved in degradative pathways are often organized as operons and supraoperonic clusters comprising 'pathway modules' (1)(2)(3)(4).
N-heteroaromatic compounds are metabolized and even mineralized by various bacteria (for a review, see 5 and references therein). Quinaldine (2-

methylquinoline) is utilized by Arthrobacter ilicis
Rü61a as a source of carbon, nitrogen, and energy; its degradation proceeds via the "anthranilate pathway" (5,6). The initial step, the hydroxylation of quinaldine in para position to the N-heteroatom, is catalyzed by the inducible enzyme quinaldine 4-oxidase (Qox) 1 . Qox is a molybdo-iron/sulfurflavoprotein with an (LMS) 2 subunit structure and has been classified to belong to the xanthine oxidase family of molybdenum enzymes (7-10; for reviews on molybdenum enzymes, see [11][12][13]. Like many other bacterial molybdenum hydroxylases, e.g., quinoline 2-oxidoreductase (Qor) from Pseudomonas putida 86 (14, 15), isoquinoline 1-oxidoreductase (Ior) from Brevundimonas diminuta 7 (16), CO dehydrogenase from Oligotropha carboxidovorans (17), and aldehyde dehydrogenases from Desulfovibrio gigas and D. desulfuricans (18)(19)(20)(21), Qox contains the molybdopterin cytosine dinucleotide form (MCD) of the molybdenum pyranopterin cofactor (7). X-ray crystal structures of molybdenum hydroxylases have allowed to identify amino acid residues that might possibly be involved in substrate positioning and/or catalytic turnover (17)(18)(19)(20)(21)(22)(23). The catalytic relevance of these residues can be assessed by constructing protein variants carrying amino acid replacements, and their biochemical, spectroscopic and structural characterization. Replacement of a distinct amino acid residue in a protein can be performed by site-directed mutagenesis. However, a prerequisite for such a mutagenesis approach is the availability of a suitable system for the genetic manipulation and for the regulated, functional expression of the genes coding for the enzyme to be investigated. Whereas genes coding for molybdenum hydroxylases containing molybdopterin or the molybdopterin guanine dinucleotide form of the cofactor have been successfully expressed in E. coli (24)(25)(26), attempts to produce MCD-containing enzymes in E. coli clones failed (27, 28, unpublished results of our group). We have recently been working at the construction of expression clones for the 5 MATERIALS AND METHODS Bacterial strains, plasmids, and growth conditions. The bacterial strains and plasmids used are listed in Table 1. A. ilicis Rü61a was grown in mineral salts medium (8) at 30 °C with 0.5 ml/l quinaldine. E. coli HB101 (31), which served as host strain for recombinant cosmids, and both E. coli DH5 (32) and E. coli XL1-Blue MRF´ (Stratagene), used for cloning procedures with pUC18, were grown in Luria Bertani (LB) broth (32) at 37 °C. If appropriate, E. coli and P. putida KT2440 cultures contained the following antibiotics: ampicillin (60 g/ml and 500 g/ml for E. coli DH5 and P. putida KT2440 pKP1, respectively), tetracycline (15 g/ml), and kanamycin (50 g/ml). To investigate functional expression of the qox genes in the cosmid clone E. coli HB101 pVK55B/5, it was grown at 37 °C on mineral salts medium containing (per liter) 0.2 g MgSO 4  The medium was supplemented with 15 g/ml of each proline and leucine, and 2 ml/l vitamin solution (33); 0.1 ml/l quinaldine was added when the culture reached an optical density (600 nm) of about 0.8. P. putida KT2440 pKP1 was grown in the mineral salt medium described by Tshisuaka et al. (15) with 8 mM benzoate as carbon and energy source and as XylS effector, and with 1 g/l (NH 4 ) 2 SO 4 . As an additional XylS effector, 2 mM 2-methylbenzoate was added at an optical density (600 nm) of 0.8-1.2.
To generate biomass for protein purification, cells were grown in two 4 l glass fermenters to which benzoate was added repeatedly. At an optical density (600 nm) of about 3, cells were harvested by centrifugation at 14,000 g for 15 min at 4 °C.
For the preparation of electrocompetent cells, E. coli DH5 , E. coli XL1-Blue MRF' and P. putida KT2440 were cultured in TB medium (32). 6 putida KT2440 pVK55B/5 and P. putida KT2440 pVK55/11 were grown in mineral salt medium (8) with 30 mM succinate, 1 g/l (NH 4 ) 2 SO 4 and 1 ml/l vitamin solution (33) at 30 °C. At an optical density (600 nm) of about 0.8, 0.1 ml/l quinaldine was added. Quinaldine conversion was monitored spectrophotometrically in the culture supernatant. Spectra were compared with those of authentic references diluted in the same medium. Qox activity was measured in the cell free extracts, obtained after cell disruption by sonification, and centrifugation at 48,000 g for 40 min at 4 °C.
DNA techniques. Genomic DNA of A. ilicis Rü61a was isolated according to Hopwood et al. (35).
Plasmid and cosmid DNA was isolated with the QIAGEN Plasmid Mini-and Midi Kit, respectively (Qiagen, Hilden, Germany). Gel extraction of DNA fragments for cloning was done with the Nucleo Spin ® Extraction Kit of Macherey-Nagel (Düren, Germany), however, DNA fragments larger than 10 kb were size fractionated in 0.5 % low-melting agarose gels and extracted by agarase treatment. DNA restriction, dephosporylation and ligation, and agarose gel electrophoresis were carried out using standard procedures (32). Electrocompetent cells were generated according to Dower et al. (36) and Iwasaki et al. (37).

Construction of genomic libraries.
In order to generate an enriched gene library for A. ilicis Rü61a, genomic DNA, restricted with SmaI, was separated in agarose gels and vacuum-blotted to nylon membranes (parablot NY plus from Macherey-Nagel, Düren, Germany). Fragments in the size of 4 kb to 5 kb showing positive hybridization signals with the probe "b-DIG" (see below) were extracted from an agarose gel and ligated to the SmaI digested, dephosphorylated vector pUC18 (38). E. coli XL1-Blue MRF´ transformants were screened by colony blotting, and identified by Southern hybridization of SmaI restricted plasmids, using the probe "b-DIG".
For construction of a cosmid library, genomic DNA of A. ilicis Rü61a was partially restricted with HindIII. DNA fragments ranging in size from 15 kb to 25 kb were extracted from a 0.5 % lowmelting agarose gel and ligated to the HindIII digested, dephosphorylated cosmid vector pVK100 (39).
The cosmids were packaged in vitro into lambda phage particles using a commercial extract (DNA Packaging Kit from Roche, Mannheim, Germany). The preparation was used to infect E. coli HB101, which was selected for tetracycline resistance (Tc r ) and kanamycin sensitivity (Km s ). The clone library was screened by colony blotting and hybridization with a probe described below as "1. Assay for Qox activity, and polyacrylamide gel electrophoresis (PAGE). The activity of Qox was assayed spectrophotometrically by measuring the quinaldine-dependent reduction of the artificial electron acceptor iodonitrotetrazolium chloride (INT) as described by de Beyer and Lingens (7).  Non-denaturing PAGE was performed using the high pH discontinuous system according to Hames (47), preparing resolving gels containing a final acrylamide concentration of 7.5 % (w/v).   in water as eluent. The compounds were identified by their retention times, as well as their UV-spectra (obtained with a photodiode array detector, Waters 996), and by cochromatography with authentic reference compounds (CMP, cytidine, AMP, GMP).

SDS-PAGE
EPR spectroscopy and sample preparation. The samples were filled into EPR quartz tubes (Wilmad) and immediately frozen in liquid nitrogen within one minute. Each sample of Qox protein from P. putida KT2440 pKP1 was first reacted with a 5-10 fold excess of substrate (quinaldine) and measured at 77 K in a nitrogen finger dewar or between 10 K and 65 K in a continuous He-flow ESR900 cryostat (Oxford). In a second step a 60-fold excess of substrate was added to the sample in the quartz tube and measured again. Finally, the sample was exposed to an excess of dithionite (20fold) for complete reduction. The samples were handled under anaerobic conditions. EPR spectra at Xband frequencies (9.5 GHz) were recorded on a Bruker ESP300 spectrometer. 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. For measurements at 65 K and 77 K several spectra at different microwave powers (0.2-10 mW) were recorded to avoid saturation broadening. The spectra below 25 K were recorded at about 10 mW microwave power. To improve the signal/noise ratio X-band spectra were accumulated up to 50 times.
All spectra were base-line corrected.
Nucleotide sequence accession number. The nucleotide sequence of the 23,015 bp insert of pVK55B/5, which includes the genes coding for QoxL, QoxM and QoxS, is deposited in the EMBL Nucleotide Sequence Database under the accession number AJ537472. 2

RESULTS
Degradative capacities of P. putida KT2440 cosmid clones, and synthesis of active quinaldine 4oxidase by P. putida KT2440 cosmid clones and by P. putida KT2440 pKP1.
P. putida KT2440 containing the cosmid pVK100 is able to grow on catechol, but it does not utilize quinaldine or the subsequent intermediates of the anthranilate pathway, namely, 1H-4oxoquinaldine, 1H-3-hydroxy-4-oxoquinaldine, N-acetylanthranilate, and anthranilate. After transformation of P. putida KT2440 with the recombinant cosmids that hybridized with "1.1 DpnI", five of the resulting P. putida KT2440 pVK100 (Tc r , Km s ) clones were able to convert quinaldine to anthranilate cometabolically, suggesting that the genes coding for the enzymes catalyzing the first four steps of the anthranilate pathway are located on the inserts of the cosmids. One of these clones, designated P. putida KT2440 pVK55B/5, was chosen for further investigations. Its cosmid pVK55B/5 harbors the 23 kb insert shown in Fig. 1. P. putida KT2440 pVK55/11, which contains the 10.8 kb region depicted in the lower part of Fig. 1, also shows cometabolic conversion of quinaldine to anthranilate. The specific activity of Qox in cell free extracts of both P. putida KT2440 pVK55B/5 and P. putida KT2440 pVK55/11 was 0.04 U/mg. In contrast, an E. coli HB101 pVK55B/5 clone did not transform quinaldine, and crude extracts did not show Qox activity. This observation is in accordance with other reports on futile attempts to express genes coding for MCD-containing hydroxylases in E. coli (27,28).
The expression plasmid pKP1 has been constructed from the broad host range expression vector pJB653 (41)  Purification of Qox from P. putida KT2440 pKP1. Qox was purified in a four step procedure to near electrophoretic homogeneity (Fig. 2B). The results of the enzyme purification are listed in Table 2.
The enzyme was purified 85-fold from the crude extract with a yield of 7 %. Qox from P. putida KT2440 pKP1 consists of three subunits with molecular mass of about 20 kDa, 35 kDa, and 80 kDa, as observed for the wild-type enzyme (8).
HPLC analysis of the non-protein part of the enzyme after acidic hydrolysis indicated the presence of CMP and AMP with retention times of 4.6 min and 7.9 min, respectively. GMP or free cytidine were not detected in the sample. While the CMP is released from the molybdopterin cytosine dinucleotide cofactor, AMP derives from the FAD cofactor.
The apparent K m values of Qox purified from the expression clone were very similar to those of the wild-type Qox; its k cat values were even higher ( Table 3).
Analysis of redox-active centers in Qox from P. putida KT2440 pKP1 by EPR spectroscopy. EPR spectroscopy is capable to selectively monitor the paramagnetic states of the various redox centers present in the Qox enzymes. In this way typical fingerprint spectra of redox centers (e.g., the rapid or very rapid species of the Mo(V)-cofactor) can be compared between the enzyme preparations to yield information on the presence and integrity of these centers. Here the comparison focuses on Qox of the wild-type specimen A. ilicis Rü61a and the enzyme produced by P. putida KT2440 pKP1. The spectra obtained at 65 K after addition of substrate are shown in Fig. 4. For Qox isolated from P. putida  The transcriptional order of the genes coding for the subunits of Qox is 5´-qoxL-M-S-3´.
Among the heterotrimeric molybdoenzymes no conservation in gene arrangement is obvious. While in many cases the genes coding for the three subunits of these enzymes are transcribed in the order 5´medium-small-large-3´ (28,(52)(53)(54), other enzymes are known whose genes are arranged in the order 5´-large-small-medium-3´ (55) or even with a gap between the gene for the large subunit and the genes for the medium and the small subunit (54). These divergencies may lead to the assumption that there is no ancestral common transcriptional unit for these enzymes.
The qoxL gene is 2388 bp in length, coding for a protein of 795 amino acids (aa). A potential ribosome-binding site (AAGGAGA) is located fourteen nucleotides upstream of the start codon ATG.
146 nucleotides upstream of the qoxL start codon a putative -35 region was detected (TTGACG) which, however, is not followed by a recognizable -10 region in the usual distance of [16][17][18][19] nucleotides (56). QoxL exhibits the well conserved motifs assumed to be involved in binding the pyranopterin cofactor (MoCoI-MoCoV) (19, 57) (Fig. 6A) (58), were identified in QoxM, indicating that it harbors the FAD cofactor (Fig. 6B). The motif TIGG is described to create a pocket for the adenosine and to contact the pyrophosphate moiety of the FAD molecule in XDH from R. capsulatus (23). In XDHARc, T206 of the amino-terminal motif (first motif in Fig. 6B) as well as the double glycine of this motif also interact with the pyrophosphate (23). In QoxM, Q33 corresponds to this residue; the medium-sized subunits of Qor from P. putida 86 (28)  aa, shows the two cysteine rich motifs strictly conserved in the prokaryotic molybdenum hydroxylases, which probably bind the two different [2Fe2S] clusters (60). The N-terminal motif following the sequence 40 CX 4 CGXCX 11 C 60 is homologous to the plant-type ferredoxin signature pattern; the second motif 117 CGXCX 31 CXC 154 has a binding motif that is typical for molybdenum hydroxylases. While the latter motif is presumed to bind the FeSI center, which is "proximal" to the molybdenum cofactor, the first motif may bind the "distal" FeSII center (57).
Sequence analysis of the qoxLMS flanking regions. Within the 23 kb region, fifteen putative ORFs were identified upstream of the qoxLMS gene cluster, and one ORF downstream of qoxS ( Fig. 1; Table   4). The hypothetical protein encoded by the nearly complete ORF 1, which starts 574 nucleotides downstream of qoxS but lacks some C-terminal residues, is related to chaperone like proteins homoprotocatechuate (90). The ORF 15 protein indeed exhibits a high degree of similarity to known HHDD isomerases/OPET decarboxylases (see Table 4).
ORF 16 codes for a putative protein composed of 434 aa with a M r of 47,172 that shows significant similarity to members of the enolase superfamily. These enzymes catalyze diverse overall reactions which however are initiated by a common step, i.e., abstraction of the -proton of a carboxylic acid to form an enolic intermediate (91,92). The members of the enolase superfamily were whereas the second carboxylate oxygen is stabilized through a strong hydrogen bond to E317. In MLE, these functions probably are taken over by the residues K169, K167, and E327 (91). Sequence alignment with the ORF 16 protein revealed K220, K218, and E382 as homologous residues. As enolases are devoid of the KXK motif of MR and MLR (91), it is unlikely that the ORF16 protein belongs to the enolase subgroup. In the reverse MR reaction, H297 is the (R)-specific catalytic base that abstracts the -proton from (R)-mandelate, assisted by D270 (91,93). These two residues are not conserved in MLE, but they actually were found in the ORF 16 protein in positions H351 and D324.
Based on the pattern of conserved residues, we suggest that the gene product of ORF 16 belongs to the mandelate racemase subgroup of enzymes. This subgroup apart from MR includes (D)-galactonate 21 dehydratase, (D)-glucarate dehydratase, (L)-rhamnoate dehydratase, and some reading frames with unassigned functions (91,93).

DISCUSSION
The qoxLMS genes from Arthrobacter ilicis Rü61a were expressed in a Pseudomonas host, yielding catalytically competent Qox protein. Active quinaldine 4-oxidase was produced by cosmid clones containing the whole 23 kb insert, or a 10.8 kb fragment (Fig. 1), and by P. putida KT2440 pKP1, where expression of the qox genes is regulated by the Pm promotor which in turn is induced by benzoate-activated XylS protein. The specific activity for Qox in the crude extract of P. putida KT2440 pKP1 was as high as that found in crude extracts of the wild-type strain A. ilicis Rü61a. The specific activity of Qox purified from P. putida KT2440 pKP1 even exceeded that determined by Stephan et al. (8) for preparations of wild-type Qox. The biochemical and spectroscopic properties of the Qox protein purified from P. putida KT2440 pKP1 were similar to those of the wild-type enzyme.
EPR spectroscopy revealed identical spectral patterns of the FAD-radical signal and the catalytically competent very rapid species in both enzymes (Fig. 4), indicating that the environment of the centers and particularly the mode of substrate/product binding at the site of the Mo(V)-cofactor are very similar. The sole appearance of the FeSII signals at lower temperatures in the enzyme of P. putida KT2440 pKP1 are in accordance with the findings of redox and rapid freeze experiments of Qox from A. ilicis Rü61a for which the redox potential of FeSII was 180 mV higher than that of FeSI.
Consequently, the FeSII signal was observed first under single turnover conditions in kinetic EPR experiments (10). Hence, it is concluded that the difference of the redox potentials of both FeS centers in Qox from P. putida KT2440 pKP1 should be similar. The axial type FeSI signal is not found for other proteins of the xanthine oxidase family. Its presence in Qox from the expression clone points to a conserved structural environment of this cluster which also has been shown to be proximal to the molybdenum cofactor (10,57,94). The spectral signature of the FeSII center of Qox of P. putida KT2440 pKP1 is similar to that of A. ilicis Rü61a but shows some slight differences in g-factor and line shape. These changes seem to be related to the simultaneous presence of substrate and dithionite reduced clusters. Since this cluster presumably is located close to the surface of the domain it is more by guest on March 24, 2020 http://www.jbc.org/ Downloaded from 22 susceptible to solvent effects and generally shows a relaxation behaviour different from FeSI cluster (10,11,57,95). On the whole, our results suggest that the Qox protein produced by the expression clone is identical to the wild-type enzyme, implying that the host strain P. putida KT2440 is able to provide all the accessory functions that are required for the assembly of this complex enzyme. Qox is in fact the first MCD-containing enzyme to be synthesized in a catalytically fully competent form by a heterologous host. There is no overexpression of the qoxLMS genes in P. putida KT2440 pKP1, however overexpression was not our primary goal, but we intended to construct a system that allows the genetic manipulation of the qox genes by mutagenesis approaches, and the production of protein variants of Qox.
Assembly of the Fe/S protein (QoxS) and the flavoprotein (QoxM) is thought to involve ubiquitously conserved pathways. However, biosynthesis of the MCD form of the molybdenum pyranopterin cofactor and its insertion into QoxL requires not only proteins involved in molybdenum uptake and MPT biosynthesis, but also a tailoring enzyme forming MCD from MPT, and maybe even a specific chaperone for MCD insertion (for reviews on molybdate uptake and biosynthesis of the molybdenum cofactor, see: 96,97). Sequence comparisons of known genes of the moa, moe and mod operons of E. coli with the P. putida KT2440 genome revealed corresponding sequences with significant similarities, suggesting that strain KT2440 is able to synthesize the MPT cofactor. The successful expression of fully active Qox and the release of CMP upon acidic hydrolysis of the enzyme indicated that P. putida KT2440 is also able to provide the MCD cofactor, and moreover, to insert it into the maturing Qox protein.
It is remarkable to note that the gene product of ORF 1 (Fig. 1), which due to its similarity to the XdhC protein (61) is thought to be involved in cofactor insertion during Qox assembly in the wildtype strain, is not required for formation of functional Qox by the P. putida expression clone.
On the basis of X-ray crystal structure analyses of aldehyde oxidoreductases from D. gigas repressor, its DNA binding site is located between the tetR gene and the vicinal gene tetA that is transcribed in the opposite direction. The tetA gene, which codes for an energy-dependent tetracycline/Mg 2+ antiporter, is the target of the TetR mediated regulation (105). Such an arrangement has been found for other tetR-like genes and their targets. However, we have not been able to identify possible DNA binding sites for the regulator between ORF 8 and ORF 9, or in front of qoxL or ORF 2.
Nevertheless, involvement of the putative ORF 8 protein in regulation of transcription of these genes can not be excluded, since the DNA sequences recognized by the HTH-motifs appear to be very diverse among different TetR like regulators.
Conclusions. Identification, sequencing, cloning, and functional, heterologous expression of the qoxLMS gene cluster has been achieved in this work. The available expression system will allow the genetic manipulation of the qox genes by site-directed mutagenesis. Of special interest is the investigation of the functional role of the glutamate residue E736, which corresonds to the glutamate residues strictly conserved in enzymes of the xanthine dehydrogenase family and which has been predicted to be involved in the catalytic mechanism. Residues thought to be involved in substrate positioning are also important targets for mutagenesis studies; their alteration might give us an idea about the molecular basis of substrate specificity and regioselectivity of hydroxylation.
The identification of a number of putative genes that might be functionally related to quinaldine oxidation may open up further investigations on the anthranilate pathway and its regulation.

ACKNOWLEDGMENTS
We thank Renate Gahl-Janßen for excellent technical assistance and Sonja Sielker for the kinetic data    The sample in B was exposed to a ca. 10-fold excess of substrate; in A,C to a 60-fold excess. (vr = very rapid species, r = rapid species)

Fig. 5:
EPR spectra of Qox from P. putida KT2440 pKP1 reduced with a 10-fold excess of substrate (A) and fully reduced by subsequent addition of dithionite (B) in comparison to the spectrum of Qox from A.
ilicis Rü61a (C). Spectrum A was recorded at 15 K, B and C at 25 K. The assignment of EPR lines to the two 2Fe2S-centers I and II is indicated.