Functional expression of the quinoline 2-oxidoreductase genesUrsula Frerichs-Deeken1, Birgit Goldenstedt1,2,*, Renate Gahl-Janßen1, Reinhard Kappl3, Ju¨rgen Hu¨ttermann3and Susanne Fetzn
Trang 1Functional expression of the quinoline 2-oxidoreductase genes
Ursula Frerichs-Deeken1, Birgit Goldenstedt1,2,*, Renate Gahl-Janßen1, Reinhard Kappl3,
Ju¨rgen Hu¨ttermann3and Susanne Fetzner1,2,*
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¨t Mu¨nster, Germany;3Fachrichtung 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 putida86-1 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 kcat(quinoline) was about ninefold lower
than that of wild-type Qor The apparent Kmvalues 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 putida86-1 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 + H2Ofi 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 xanthine oxidase family [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 H2O mole-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 phosmonophos-phate group to form molybdenum molybdopterin cytosine dinucleotide (Mo-MCD)
Correspondence to S Fetzner, Institut fu¨r Mikrobiologie,
Westfa¨lische Wilhelms-Universita¨t Mu¨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¨t Mu¨nster, Germany.
(Received 20 November 2002, revised 3 February 2003,
accepted 19 February 2003)
Trang 2The 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 Mg2+-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 putidaKT2440 and in the quinoline degrading strain,
P putida86 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 putida86 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 37C in Luria–Bertani (LB) broth [38] P
put-ida86 was grown in mineral salts medium containing
quinoline as the sole carbon source [2], or in LB broth [38],
at 30C 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) was added to the respective medium
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] and Iwasaki et al [41], respectively
Construction ofP putida 86-1 Dqor
A DNA segment containing the qorMSL genes and flanking regions (qor-up, 1055 bp and qor-down, 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 XhoI and 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 qorgenes, respectively) E coli XL-1 Blue MRF¢ was used
as host strain for pBG2a and pBG2b The nptII inserts together with the flanking regions (qor-up and qor-down) 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 coliS17-1 pBG3a/3b and P putida 86-1 was performed
as described by Masepohl et al [45], except that LB plates 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 XhoI and DraIII
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 Kanrand Genrmutants of P putida 86-1 with a Qor– phenotype DNA isolated from these three P putida 86-1 Dqor (KanrGenr) 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
Trang 3respect to the deleted qorMSL genes, and aacC1 in the
opposite orientation with respect to the deleted second copy
of qorMSL
Expression cloning ofqorMSL 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)1ampicillin in mineral salts medium [2] supple-mented with 1 gÆL)1 ammonium sulfate Induction of qorMSLexpression 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 putidaKT2440 pUF1 clones, succinate (10 gÆL)1) and sodium benzoate (8 mM) were used as sources of carbon Two 4 L glass fermenters were used to generate biomass for protein purification Benzoate (8 mM) 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 ), as an additional XylS effector, was added
Table 1 Bacterial strains and plasmids used in this study.
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
P putida 86-1 Km-a nptII replacing one copy of qorMSL in P putida 86-1; nptII in the same orientation
P putida 86-1 Km-b nptII replacing one copy of qorMSL in P putida 86-1; nptII in the opposite orientation
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 Strr, Kanr, Genr; Q or– This work
qor-up (1534 bp) and qor-down (1047 bp) cloned into the HindIII restriction site
qor-up (1534 bp) and qor-down (1047 bp) cloned into the HindIII restriction site
pBG4b nptII in pBG3a replaced by aacC1; aacC1 in the opposite orientation with respect to the
Trang 4at a D550value of 0.7–1.0 P putida 86-1 Dqor pUF1 was
either grown in benzoate (8 mM, fed repeatedly), or in
benzoate (5 mM) plus 1H-2-oxoquinoline (2.8 mM) as
carbon sources (fed repeatedly) 2-Methylbenzoate was
added at a D550value 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, 20 min) at a D550value‡ 3.0
Crude extracts were prepared by FrenchTM Press
treatment at 2.1–2.4· 108 Pa of cell suspensions in
100 mM Tris/HCl buffer (pH 8.5) containing 10 lM
phe-nylmethanesulfonylfluoride and 0.05 lLÆml)1Benzon
nuc-lease (Merck, Darmstadt, Germany), subsequent
sonification, and removal of debris by centrifugation
(48 000 g, 45 min, 4C)
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 fromP putida 86, P putida
KT2440 pUF1 andP putida 86-1 Dqor pUF1
Qor was purified using ammonium sulfate fractionation
(0.8–1.5M), 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 mM Tris, 190 mM
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 apparentKmandkcatvalues 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 25C 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 Km app, (quinoline) and kcat 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 putida86, 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
95C 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· 250 mm) at a flow rate of 1 mLÆmin)1with 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 mM 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 putida86-1 Dqor pUF1 used for the EPR analyses showed a specific activity of 1 UÆmg)1 and 22 UÆmg)1,
Trang 5respectively The samples (Qor from P putida KT2440
pUF1: 12.4 nmol; Qor from P putida 86-1 Dqor pUF1:
9.6 nmol, in 50 mMTris/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 (Na2S2O4) 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 fromP 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 Dqor
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
the genome of P 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.
Trang 6Conditions of Qor synthesis in wild-typeP putida 86,
P putida 86-1 Dqor 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-ida86 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; oxoOis 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 fromP putida 86-1 Dqor 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 putida86-1 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-ida86-1 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 fromP putida 86,
P putida KT2440 pUF1 and P putida 86-1 Dqor pUF1 The apparent Kmvalues of the Qor proteins for quinoline were similar, whereas the apparent kcatvalue for quinoline
of Qor from strain KT2440 pUF1 was eight- to tenfold lower than that of wild-type Qor and Qor from P put-ida86-1 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.
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) of Q or
in crude extracts after growth on:
Succinate Benzoate Quinoline
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.
Trang 7Metal 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
and 8 g atom of iron per mol of enzyme [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-idaKT2440 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 putidaKT2440 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 fromP putida 86,
P putida KT2440 pUF1 and P putida 86-1 Dqor 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 A280nm/A450nm and A450nm/A550nm 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 Dqor pUF1
by EPR spectroscopy
Mo Reduction of the Qor protein isolated from wild-type
P putida86 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 KT2440 pUF1
(grown on benzoate)
P putida 86–1Dqor pUF1
(grown on benzoate +
1H-2-oxoquinoline)
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.
Trang 8sulfhydryl-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 putidaKT2440 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 slow 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 high-and C) and originating from natural Mo-isotopes with nuclear spin
I¼ 5/2, also small lines of the semiquinone radical form of FAD were observed at g¼ 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 g2-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 g2-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 g1-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
g3-component of FeSII of Qor from P putida KT2440 pUF1 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
g3-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.
Trang 9the 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 putida86-1 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 This work was supported by the Deutsche Forschungsgemeinschaft (Fe 383/4-4), the European Commission within the Xanthine Oxidase Network (Contract No HPRN-CT-1999-00084), and the Fonds der Chemischen Industrie.
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