(Received for publication, February 2, 1995; and in revised form, April 13, 1995)
From the
2-Oxo-1,2-dihydroquinoline 8-monooxygenase, which catalyzes the
NADH-dependent oxygenation of 2-oxo-1,2-dihydroquinoline to
8-hydroxy-2-oxo-1,2-dihydroquinoline, is the second enzyme in the
quinoline degradation pathway of Pseudomonas putida 86. This
enzyme system consists of two inducible protein components, which were
purified, characterized, and identified as reductase and oxygenase. The
yellow reductase is a monomeric iron-sulfur flavoprotein (M The soil bacterium Pseudomonas putida 86 utilizes
quinoline as sole source of carbon, nitrogen, and energy. The
occurrence of the metabolites 2-oxo-1,2-dihydroquinoline,
8-hydroxy-2-oxo-1,2-dihydroquinoline, 8-hydroxycoumarin, and
2,3-dihydroxyphenylpropionic acid revealed that the degradation of
quinoline proceeds in this organism via the so called ``coumarin
pathway''(1, 2) . Quinoline 2-oxidoreductase from
this organism, catalyzing the initial nucleophilic attack of quinoline
at C-2, yielding 2-oxo-1,2-dihydroquinoline, is investigated
thoroughly(3, 4) . However, no enzymatic study is
available concerning the further conversions of
2-oxo-1,2-dihydroquinoline in the ``coumarin pathway.'' In
this paper, we present the purification and characterization of
2-oxo-1,2-dihydroquinoline 8-monooxygenase. This novel two-component
enzyme system is compared with other multicomponent enzymes belonging
to the family of non-heme iron oxygenases.
For studies of enzyme induction, the medium
contained (instead of quinoline) 2.8 mM 2-oxo-1,2-dihydroquinoline or glucose (2 g/liter) plus
(NH Large-scale growth of bacteria was
carried out in a 100-liter fermenter with quinoline as substrate.
Bacterial growth and concentrations of quinoline and
2-oxo-1,2-dihydroquinoline were measured spectrophotometrically
according to Bauder et al.(3) . After 9, 23, 32, and
48 h of fermentation, bacteria were fed with 4 mM quinoline.
After total consumption of quinoline and 2-oxo-1,2-dihydroquinoline,
cells were harvested by centrifugation, washed twice with 50 mM Tris-HCl buffer (pH 7.5), and stored at -20 °C.
In Step 2, the supernatant was diluted with the same volume
of standard buffer and loaded for anion-exchange chromatography onto a
DEAE-cellulose DE-52 column (2.5 In Step 3, fractions containing the reductase were
combined and adjusted to 0.8 M
(NH In
Step 4, for a second anion-exchange chromatography, the concentrated
protein solution was applied to a DEAE-Fractogel EMD column (1 In Step 5, for gel
filtration, the protein solution was loaded onto Superose 12 (1.5
In
Step 3`, fractions from DEAE-cellulose DE-52 containing the oxygenase
were pooled and adjusted to 0.6 M
(NH In Step
4`, after concentrating the eluate by ultrafiltration (YM-30 membrane),
it was passed through a Sephacryl S-300 gel filtration column (2.5
In Step 5`, for adsorption chromatography on hydroxyapatite, which
was prepared by the method of Atkinson et al.(6) ,
fractions with oxygenase activity were combined and adjusted to pH 7.2
with 1 M acetate buffer, pH 5.0. Glycerol was included to a
final concentration of 3% (w/v), and 1 M potassium phosphate
buffer, pH 7.2, was added to a final concentration of 10 mM.
The solution was applied to a column of hydroxyapatite (2.5 Since the oxygenase lost 50% specific activity when stored in
standard buffer for 3 days at 4 °C, different stabilizers were
tested: 10% (w/v) glycerol, 10% (v/v) ethanol, 0.2% Triton X-100, 0.1
mM EDTA, 1-10 mM mercaptoethanol, 1 mM glutathione, 0.1-1 mM dithiothreitol, 0.1-1
mM (NH
The molecular weights of
peptides under denaturing conditions were determined using
SDS-polyacrylamide gel electrophoresis(7) .
Acid-labile
sulfur was determined by the formation of methylene blue as described
by Beinert(12) . Bovine milk xanthine oxidase was used as a
standard. Analysis of metals was done with an x-ray fluorescence
spectrometer (System 77 Finnigan Int. Inc., Sunnyvale, CA).
Substrate consumption and product formation were monitored by
HPLC (Nucleosil RP18 column, eluent: 30% (v/v) methanol) with
UV-visible spectroscopic detection at 200-400 nm. Substrate
conversion was further confirmed by thin layer chromatography of ethyl
acetate extracts of the acidified assay mixtures (silica gel plates;
solvent system, toluene/dioxane/acetic acid (72:16:1.6)(1) ).
Authentic 8-hydroxy-2-oxo-1,2-dihydroquinoline served as product
reference in both assays. The reaction mixtures contained 1.1 units of
reductase, 12.5 milliunits of oxygenase, 0.05 mM
(NH NADH:acceptor reductase activity was assayed at 25
°C spectrophotometrically as reduction of the artificial electron
acceptors INT (
Extracts of cells, grown on glucose or
succinate, showed neither reductase nor oxygenase activity of
2-oxo-1,2-dihydroquinoline 8-monooxygenase. If cells were grown on
2-oxo-1,2-dihydroquinoline, a specific reductase activity of 1.24
units/mg protein and a specific oxygenase activity of 0.35 units/mg
protein was measured in the cell extracts. Therefore, we propose
that both components of 2-oxo-1,2-dihydroquinoline 8-monooxygenase in P. putida 86 are inducible by the substrate
2-oxo-1,2-dihydroquinoline.
Figure 1:
SDS-polyacrylamide gel
electrophoresis of 2-oxo-1,2-dihydroquinoline 8-monooxygenase. Lane
M, molecular mass standards (M
The preparations of reductase and oxygenase were nearly homogeneous
as shown by SDS-polyacrylamide gel electrophoresis (Fig. 1).
Figure 2:
Absorption spectra of the reductase. The
concentrations were 0.8 mg of reductase/ml of 10 mM potassium
phosphate buffer, pH 7.0. Solid line, reductase as isolated; dashed line, reductase reduced with NADH. Inset, solid line indicates cofactor isolated from the reductase (see
``Experimental Procedures'').
Solutions of purified oxygenase, as isolated in the oxidized state,
had a red-brown color. The UV-visible absorption spectrum showed maxima
at 280, 328, and 460 nm and a shoulder at 545 nm (Fig. 3).
Boiling of the oxygenase resulted in the loss of color. The oxygenase
as isolated was bleached by chemical reduction with dithionite or by
enzymatical reduction with NADH and catalytic amounts of reductase. The
addition of NADH alone did not reduce the oxygenase. This finding
indicated that the reductase, which was reduced directly by NADH,
mediated the electron transfer from NADH to the terminal oxygenase
component.
Figure 3:
Absorption spectra of the oxygenase. The
concentrations were 1 mg (inset, 14 mg) of oxygenase/ml of 50
mM Tris-HCl buffer, pH 7.5. Solid lines, oxygenase as
isolated; dashed line, oxygenase reduced with NADH and
catalytic amounts of reductase; dotted line, oxygenase reduced
with dithionite.
None of the metal cations
Mg In
contrast to the 2-oxo-1,2-dihydroquinoline 8-monooxygenase activity,
the NADH:DCPIP reductase activity was not increased by the addition of
Fe A surprisingly strong activating
effect on 2-oxo-1,2-dihydroquinoline 8-monooxygenase was observed by
the addition of PEG to the enzyme assay. In the absence of PEG, the
assay was nonlinear, initially showing only 14% of activity, which
slowly increased to 38% within 12 min but never reached 100% as
determined in the presence of PEG (Table 2). However, NADH:DCPIP
reductase activity was diminished in the presence of PEG (67% residual
activity).
An inducible two-component enzyme system, which catalyzes the
NADH-dependent monooxygenation of 2-oxo-1,2-dihydroquinoline to
8-hydroxy-2-oxo-1,2-dihydroquinoline, was purified from P. putida 86. The enzyme system was termed 2-oxo-1,2-dihydroquinoline
8-monooxygenase with the systematic name 2-oxo-1,2-dihydroquinoline,
NADH:oxygen oxidoreductase (8-hydroxylating). 2-Oxo-1,2-dihydroquinoline 8-monooxygenase revealed a high substrate
specificity toward 2-oxo-1,2-dihydroquinoline, since none of 25 other
compounds tested was converted. However, 8-hydroxyquinoline,
8-hydroxy-2-oxo-1,2-dihydroquinoline, and coumarin caused
substrate-dependent oxygen consumption without being attacked. We
assume that these compounds served as pseudosubstrates, uncoupling
electron transfer from substrate hydroxylation with concomitant
production of H A first
hint that 2-oxo-1,2-dihydroquinoline 8-monooxygenase is a
multicomponent enzyme system was the nonproportional relationship
between enzyme activity and protein concentration. The specific enzyme
activity decreased with protein dilution instead of remaining constant. Since contact of the two soluble enzyme components is a prerequisite
for monooxygenase activity, a delayed binding may explain the nonlinear
time dependence of activity in the absence of PEG (Table 2). The
mechanism of enzyme activation by PEG might be based on strengthening
the component contact due to its hygroscopic property. To profit from
this activating effect, other multicomponent enzyme systems should be
tested for the influence of PEG. Fig. 4presents a model for
the electron transport chain from NADH to the substrate hydroxylating
terminal oxygenase. NADH transmits two electrons simultaneously as
hydride, whereas iron-sulfur centers are restricted to one-electron
reactions. The function of the reductase is to mediate this
two-electron/one-electron transformation by its flavin cofactor. The
oxygenase contains besides its Rieske-type iron-sulfur clusters
additional iron. The necessity of iron for the catalytic function of
the oxygenase component is demonstrated by the fact that metal
chelating agents inhibited 2-oxo-1,2-dihydroquinoline 8-monooxygenase
activity, while NADH:acceptor reductase activity was not affected. This
agrees with the result that added ferrous iron did not increase
NADH:acceptor reductase activity, whereas the complete enzyme reaction
was accelerated. Therefore, we propose that ferrous iron is a weakly
associated cofactor of the oxygenase component, which is abstracted
easily by metal chelating agents or during enzyme purification and
which may be replaced by exogenously added ferrous iron.
Figure 4:
Proposed catalytic mechanism of
2-oxo-1,2-dihydroquinoline 8-monooxygenase.
According
to our data, 2-oxo-1,2-dihydroquinoline 8-monooxygenase belongs to the
group of non-heme iron multicomponent oxygenases, which contain both
iron-sulfur clusters and additional iron in their oxygenase component.
In contrast to flavin-containing single component monooxygenases or
cytochrome P-450 multicomponent monooxygenases, which activate dioxygen
by FAD or heme-bound iron, respectively(24) , non-heme iron
oxygenases are supposed to activate dioxygen by protein-bound
iron(25) . In the case of the oxygenase component of the O-demethylating 4-methoxybenzoate monooxygenase
(putidamonooxin), mononuclear non-heme iron was demonstrated to mediate
the electron transfer from a Rieske-type [2Fe-2S] cluster to
molecular oxygen, thus activating the dioxygen as iron-peroxo complex
for the electrophilic attack of the organic
substrate(26, 27) . This model might be representative
for all known multicomponent non-heme iron oxygenases, which contain
Rieske-type iron-sulfur clusters and mononuclear iron in their terminal
oxygenase component. In Table 3(28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52) ,
2-oxo-1,2-dihydroquinoline 8-monooxygenase is compared with some of
these multicomponent non-heme iron oxygenases. Batie et al.(53) grouped them into the three classes I, II, and III,
based on the number of protein components and on the redox centers
involved in electron transport from NAD(P)H to the terminal oxygenase
component. At least two redox centers, a flavin and a
[2Fe-2S] center, are involved in this electron transfer. A
second [2Fe-2S] center is possible. The redox centers can be
located on one or two protein components, thus constituting two- or
three-component enzyme systems. Class I enzymes are two-component
enzyme systems with a flavin (FMN in class IA, FAD in class IB) and a
plant-type ferredoxin [2Fe-2S] in the reductase. In class II
enzymes, the flavin (always FAD) and the [2Fe-2S] center are
located on separate components. The [2Fe-2S] center is a
plant-type ferredoxin (class IIA) or a Rieske-type center (class IIB).
Class III enzymes contain FAD and plant-type ferredoxin in the
reductase as well as a Rieske-type center in a second component.
According to this classification, 2-oxo-1,2-dihydroquinoline
8-monooxygenase belongs to class IB enzyme systems, as do
2-halobenzoate 1,2-dioxygenase(36, 37) , benzoate
1,2-dioxygenase(38, 39, 40) , and toluate
1,2-dioxygenase(54) . However, the oxygenase components of
these three class IB enzymes are heteromultimers, whereas
2-oxo-1,2-dihydroquinoline 8-monooxygenase is a homomultimer like the
class IA enzymes listed in Table 3.
2-Oxo-1,2-dihydroquinoline
8-monooxygenase also bears structural resemblance to other
multicomponent non-heme iron monooxygenases like toluene
4-monooxygenase(55) , phenol hydroxylase(56) , or
xylene monooxygenase(57, 58) , but the oxygenase
components of these enzyme systems apparently do not possess
iron-sulfur centers. Whether 2-oxo-1,2-dihydroquinoline
8-monooxygenase is a true monooxygenase or whether the monohydroxylated
product is due to spontaneous dehydration of an unstable 7,8- or
8,8a-dihydrodiol of 2-oxo-1,2-dihydroquinoline, thus implying
a dioxygenase reaction, is uncertain. Consequently, it remains to be
determined whether 8-hydroxy-2-oxo-1,2-dihydroquinoline is an in
vivo intermediate in the ``coumarin pathway'' of
quinoline degradation by P. putida 86. However, no
intermediate was detected during the enzyme reaction in vitro,
even not under moderate conditions, taking into account the instability
of a putative dihydrodiol intermediate. A 7,8-dihydrodiol was formed
from the substrate analog 2-chloroquinoline by resting cells of P.
putida 86(59) . This activity is now shown to be
independent from 2-oxo-1,2-dihydroquinoline 8-monooxygenase because the
latter enzyme system did not convert 2-chloroquinoline. This paper
demonstrates once more the diversity of hydroxylating enzymes
encountered in quinoline-degrading bacteria. Schwarz et al.(1) described two pathways of quinoline degradation. In
the first step of both pathways, quinoline is converted to
2-oxo-1,2-dihydroquinoline, catalyzed by quinoline 2-oxidoreductase,
which incorporates a hydroxyl group deriving from
water(3, 60) . Whereas the quinoline 2-oxidoreductases
even from distantly related bacteria exhibit far-reaching
similarities(3, 60, 61) ,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
, 38,000), containing flavin adenine
dinucleotide and plant-type ferredoxin [2Fe-2S]. It
transferred electrons from NADH to the oxygenase or to some artificial
electron acceptors. The red-brown oxygenase (M
,
330,000) consists of six identical subunits (M
,
55,000) and was identified as an iron-sulfur protein, possessing about
six Rieske-type [2Fe-2S] clusters and additional iron. It was
reduced by NADH plus catalytic amounts of reductase. For monooxygenase
activity, reductase, oxygenase, NADH, molecular oxygen, and substrate
were required. The activity was considerably enhanced by the addition
of polyethylene glycol and Fe
.
2-Oxo-1,2-dihydroquinoline 8-monooxygenase revealed a high substrate
specificity toward 2-oxo-1,2-dihydroquinoline, since none of 25 other
tested compounds was converted. Based on its physical, chemical, and
catalytic properties, we presume 2-oxo-1,2-dihydroquinoline
8-monooxygenase to belong to the class IB multicomponent non-heme iron
oxygenases.
Materials
DEAE-cellulose DE-52 was obtained from Whatman (Maidstone,
Great Britain); phenyl-Sepharose CL-4B, Sephacryl S-300, Superose 12 HR
10/30, and Superdex 200 prep grade HiLoad 16/60 were from Pharmacia
Biotech (Freiburg, Germany); DEAE-Fractogel EMD was obtained from Merck
(Darmstadt, Germany); HPLC columns were from Bischoff (Leonberg,
Germany); YM-10 and YM-30 ultrafiltration membranes were purchased from
Amicon (Witten, Germany); and PEG(
)6000 and
carrier ampholytes (Servalyt 3-6) were obtained from Serva
(Heidelberg, Germany). Quinoline and 2-oxo-1,2-dihydroquinoline were a
gift from Ruetgerswerke AG (Castrop Rauxel, Germany).
8-Hydroxy-2-oxo-1,2-dihydroquinoline was obtained by
biotransformation.
(
)All other chemicals and
biochemicals were of the highest purity commercially available.
Microorganism and Culture Conditions
P. putida 86 was described by Schwarz et
al.(5) . The strain was grown aerobically at 30 °C on
mineral salts medium containing (g/liter) 4.3 NaHPO
2 H
O, 1.15 KH
PO
, 1.0
NaCl, 0.4 MgSO
7 H
O, 0.05 FeSO
7 H
O, and 0.01 Na
MoO
2 H
O. After sterilization, 4 mM
quinoline was added.
)
SO
(1 g/liter). After 15 h of
growth, cells were washed twice with 50 mM Tris-HCl buffer (pH
7.5) and suspended (adjusting A
to 2.2) in fresh
mineral salts medium containing (i) 2.8 mM
2-oxo-1,2-dihydroquinoline or (ii) 2.8 mM
2-oxo-1,2-dihydroquinoline plus 50 mg/liter chloramphenicol.
Degradation of 2-oxo-1,2-dihydroquinoline was monitored
spectrophotometrically at 323 nm.
Enzyme Purification
All purification steps were carried out at 4 °C, except
for fast protein liquid chromatography (DEAE-Fractogel EMD and Superose
12), which was performed at room temperature. All pH values given refer
to room temperature.Purification of the Reductase
In Step 1, frozen
cells (40 g, wet weight) were thawed in 40 ml of standard buffer (50
mM Tris-HCl, pH 7.3) and disrupted 5 min in time intervals of
0.5 s in a sonifier (model 450, Branson Inc., Danbury, CT) with maximal
power. The suspension was centrifuged at 48,000 g for
40 min.
39 cm) equilibrated in
standard buffer. The column was washed with the same buffer, and bound
proteins were desorbed with a linear gradient (800 ml) of KCl
(0-0.6 M) in standard buffer at a flow rate of 0.6
ml/min. This purification step largely separated the reductase from the
oxygenase.
)
SO
. For hydrophobic interaction
chromatography, the solution was applied to a column of
phenyl-Sepharose (2.5
5 cm) equilibrated in 50 mM Tris-HCl buffer, pH 7.5, containing 0.8 M (NH
)
SO
and 1 mM dithiothreitol. The column was rinsed with 200 ml of the same
buffer, and proteins were eluted with a linear gradient (300 ml) of
0.8-0 M (NH
)
SO
,
50-10 mM Tris-HCl (pH 7.5), 0-10% (v/v) ethanol,
containing 1 mM dithiothreitol. The flow rate was 3 ml/min.
The combined fractions with reductase activity were rinsed with 50
mM Tris-HCl buffer, pH 7.8, containing 1 mM dithiothreitol, by ultrafiltration with a YM-10 membrane.
14 cm) equilibrated in 50 mM Tris-HCl buffer, pH 7.8,
containing 1 mM dithiothreitol. The column was rinsed with 3
ml of the same buffer, and a linear gradient (35 ml) of 0-0.5 M (NH
)
SO
was applied at a
flow rate of 1 ml/min. Active fractions were pooled and concentrated to
0.5 ml by ultrafiltration with a YM-10 membrane.
28 cm), which was equilibrated in 100 mM Tris-HCl
buffer, pH 7.8, containing 1 mM dithiothreitol. Proteins were
separated at a flow rate of 0.5 ml/min. Fractions containing the
reductase were pooled and stored at -80 °C.
Purification of the Oxygenase
Preparation
of crude extract (Step 1`) and chromatography on DEAE-cellulose DE-52
(Step 2`) were performed as described above for the reductase, except
that a crude extract from 20 g (wet weight) of cells was used.)
SO
and 3% (w/v) glycerol. The
solution was loaded on a column of phenyl-Sepharose (2.5
5 cm),
equilibrated in 50 mM Tris-HCl buffer, pH 7.5, containing 0.6 M (NH
)
SO
, 3% (w/v)
glycerol, and 0.1 mM 2-oxo-1,2-dihydroquinoline. The column
was washed with the same buffer, and the oxygenase was eluted by
omitting (NH
)
SO
from the buffer. To
avoid severe losses of oxygenase activity, fast performance of all
steps demanding high (NH
)
SO
concentrations was substantial (flow rate, 10 ml/min).
92 cm; flow rate, 0.5 ml/min) using standard buffer containing
50 mM KCl and 0.1 mM 2-oxo-1,2-dihydroquinoline.
5
cm), equilibrated in 10 mM potassium phosphate buffer, pH 7.2,
containing 3% (w/v) glycerol and 0.1 mM
2-oxo-1,2-dihydroquinoline. The column was washed with the same buffer.
Proteins were eluted with a linear gradient (300 ml) of 10-300
mM potassium phosphate buffer, pH 7.2 (3% (w/v) glycerol and
0.1 mM 2-oxo-1,2-dihydroquinoline), at a flow rate of 0.8
ml/min. For reproducible results, hydroxyapatite was used only once.
Fractions containing the oxygenase were pooled, concentrated by
ultrafiltration with a YM-30 membrane, and stored at -80 °C.
)
Fe(SO
)
,
and 0.1 mM 2-oxo-1,2-dihydroquinoline. Further attempts to
preserve enzyme activity were made by using other buffers (pH
6-8.5) or purification procedures such as precipitation with
(NH
)
SO
, PEG, or ethanol,
chromatography on Superdex 200, chelating Sepharose, calcium tartrate
or Mono-Q (anion exchange), and preparative native gel electrophoresis.
Gel Electrophoresis
Progress in enzyme purification was monitored by
SDS-polyacrylamide gel electrophoresis with 10% separating and 4%
stacking gels(7) . The 1-mm slab gels were stained in 0.2%
(w/v) Coomassie Blue R-250 in water/methanol/acetic acid (40:50:10).
Analytical isoelectric focusing was performed in rehydrated gels as
recommended by Pharmacia(8) .Molecular Weight Estimation
The native molecular weight of the reductase was determined
by gel filtration through Superose 12 in 50 mM Tris-HCl
buffer, pH 7.8, containing 150 mM NaCl. The native molecular
weight of the oxygenase was estimated by gel filtration on Superdex 200
in 100 mM Tris-HCl buffer, pH 7.5.Absorption Spectra
Absorption spectra were measured in cells of 1-cm path length
at 25 °C with a Uvicon 930 spectrometer (Kontron Instruments,
Neufahrn, Germany).Flavin, Acid-labile Sulfur, and Metal Determinations
The flavin cofactor was extracted from the reductase by two
methods. The component in 10 mM potassium phosphate buffer (pH
7.0) was boiled for 4 min or treated with trichloroacetic acid as
described by Siegel(9) . After removal of protein by
centrifugation and ultrafiltration, the yellow filtrate was examined by
UV-visible spectroscopy (, 11,300 M
cm
(10) ) and
by reversed phase HPLC on a Lichrospher RP18 column according to
Nielsen et al.(11) . 0.2 M formic acid, 0.1 M ammonia/methanol (3:1) was used as eluent.
Protein Determination
Protein concentrations were determined according to Bradford (13) with bovine serum albumin as standard.Enzyme Assays
The activity of 2-oxo-1,2-dihydroquinoline 8-monooxygenase
was measured at 25 °C spectrophotometrically at 365 nm as NADH
consumption (standard assay, , 3,400 M
cm
(14) ) or
polarographically as oxygen uptake with a Clark-type oxygen electrode
(YSI4004, Yellow Springs Instrument Co., Yellow Springs, OH). The
reaction was optimized for temperature, pH, and buffer as well as for
the concentrations of buffer, PEG, Fe
, NADH, and
2-oxo-1,2-dihydroquinoline. The 21 mM Tris-HCl buffer (pH 7.5)
contained 7% PEG, 0.05 mM
(NH
)
Fe(SO
)
, 0.25 mM NADH, and suitable amounts of reductase and oxygenase components.
The reaction was started by the addition of 0.2 mM 2-oxo-1,2-dihydroquinoline. All estimations were corrected for
NADH or oxygen consumption recorded in the absence of substrate. 1 unit
of 2-oxo-1,2-dihydroquinoline 8-monooxygenase activity was defined as
the amount of enzyme that consumed 1 µmol of NADH or 1 µmol of
oxygen per min. The enzyme activity was not proportional to the crude
extract concentration in the assay mixture. To achieve a proportional
relationship, the activity of the reductase was measured in the
presence of an excess amount of the oxygenase component and vice
versa. These components to be added had been enriched by the
purification steps 1 and 2. They were stored at -80 °C until
use.
)
Fe(SO
)
2 mM NADH, and 2 mM 2-oxo-1,2-dihydroquinoline in 50 mM Tris-HCl buffer (pH 7.5). Transformation of other putative
substrates, causing oxygen consumption, was monitored likewise. Enzyme
activity under anaerobic conditions (achieved by repeated degassing and
flushing with nitrogen) was examined by thin layer chromatography as
described above.
, 19,300 M
cm
(15) ), DCPIP (
,
21,000 M
cm
(16) ), cytochrome c (
, 21,000 M
cm
(17) ), and potassium
hexacyanoferrate III (ferricyanide) (
, 1,020 M
cm
(18) ). The
reaction mixture contained 27 mM Tris-HCl buffer (pH 7.5),
0.05 mM electron acceptor, and 0.25 mM NADH. The
reaction was started by the addition of 5-20 µl of protein
solution. 1 unit of NADH:acceptor reductase activity was defined as the
amount of enzyme that reduced 1 µmol of electron acceptor per min.
Inhibitors
The effects of several putative inhibitors on NADH:DCPIP
reductase activity and on 2-oxo-1,2-dihydroquinoline 8-monooxygenase
activity (standard assay) were examined in the absence of
Fe. Assay mixtures were preincubated 2 min with each
compound (0.5 mM). The protein concentrations were 14
µg/ml in the NADH:DCPIP reductase assay and 760 µg/ml in the
standard assay.
Inducibility of 2-Oxo-1,2-dihydroquinoline
8-Monooxygenase
Cells of P. putida 86, grown on
glucose as sole source of carbon and energy, did not metabolize
2-oxo-1,2-dihydroquinoline in the presence of chloramphenicol, an
inhibitor of protein synthesis. Without chloramphenicol, these cells
degraded 2-oxo-1,2-dihydroquinoline after a lag phase of 2 h. Cells,
grown on 2-oxo-1,2-dihydroquinoline, converted this substrate
immediately when added anew without being affected by the addition of
chloramphenicol. Thus, the degradation of 2-oxo-1,2-dihydroquinoline
was dependent on protein biosynthesis induced by
2-oxo-1,2-dihydroquinoline.Purification of Reductase and Oxygenase of
2-Oxo-1,2-dihydroquinoline 8-Monooxygenase
The results of a
typical purification procedure are summarized in Table 1. The
reductase was purified 108-fold in a yield of 3%. The oxygenase
activity was enriched only 2.4-fold. However, as judged by
SDS-polyacrylamide gel electrophoresis, a high enrichment of the
oxygenase protein in relation to other cell proteins was evident (Fig. 1). A rough estimate by comparing the gels of the
oxygenase and reductase suggests that the increase in specific activity
should have been around 100-fold. We assume that the oxygenase lost
activity during the purification procedure despite stabilizing it with
glycerol and substrate. Other tested stabilizers or purification
procedures were even less effective in preserving enzyme activity.
):
phosphorylase b (94,000), bovine serum albumin (67,000),
ovalbumin (43,000), carbonic anhydrase (30,000), soybean trypsin
inhibitor (20,100), and lactalbumin (14,400). Lanes 1-5,
reductase after purification steps 1-5; lanes
1`-5`, oxygenase after purification steps 1`-5` (see
``Experimental Procedures'').
Molecular Weights and Subunit
Composition
The native molecular weight of purified
reductase was determined to 39,000 by gel filtration. Its molecular
weight determined under denaturing and reducing conditions by
SDS-polyacrylamide gel electrophoresis was 37,000. Thus, we propose the
reductase to be a monomer. Gel filtration of the oxygenase resulted in
a native molecular weight of 330,000. Since SDS-polyacrylamide gel
electrophoresis of the oxygenase revealed only one protein band
corresponding to a molecular weight of 55,000, we suggest that the
oxygenase is composed of six identical subunits.Isoelectric Focusing
The isoelectric
point of the purified reductase was 4.6. The oxygenase, which was
nearly homogeneous as judged by SDS-polyacrylamide gel electrophoresis,
separated in isoelectric focusing as a cluster of bands within the
range of pH 4.9-5.4. As observed by other
authors(19, 20) , the formation of multiple bands
might be an artifact due to complex formation of proteins with carrier
ampholytes.Absorption Spectra
In the oxidized state,
the reductase was yellow in color. The UV-visible absorption spectrum
showed maxima at 271 and 456 nm, a broad double peak at 337 and 381 nm,
and shoulders at about 427 and 550 nm (Fig. 2). The reductase
was reduced and bleached by the addition of NADH. After boiling and
centrifuging a reductase solution, the supernatant had an absorption
spectrum (Fig. 2, inset) typical for a flavin.
Flavin and Iron-Sulfur Contents
Analysis
by HPLC revealed an amount of 0.7 mol of FAD/mol of reductase.
Spectrophotometrical determination of the FAD content resulted in a
ratio of 0.9 mol of FAD/mol of reductase. Average contents of 1.7
± 0.3 g atom iron and 1.6 ± 0.15 g atom acid-labile
sulfur were determined per mol of reductase. 1 mol of oxygenase
contained 13.1 ± 1.8 g atom iron and 9.1 ± 0.9 g atom
acid-labile sulfur. The absorption spectra ( Fig. 2and Fig. 3) indicate that the iron is not bound to a heme-like
structure but is part of iron-sulfur clusters. Electron paramagnetic
resonance studies revealed that a plant-type ferredoxin
[2Fe-2S] cluster with g = 1.95 is present
in the reductase component, whereas the oxygenase component harbors
Rieske-type [2Fe-2S] clusters (g
=
1.89).
(
)We propose that the reductase contains
one FAD and one plant-type ferredoxin [2Fe-2S] cluster and
that the oxygenase includes six Rieske-type [2Fe-2S] clusters (i.e. one [2Fe-2S] per subunit) and additional iron.
Enzyme Reaction
Reductase, oxygenase,
NADH, molecular oxygen, and substrate were required for
2-oxo-1,2-dihydroquinoline 8-monooxygenase activity. Polarographic
determinations of O uptake with limiting concentrations of
either 2-oxo-1,2-dihydroquinoline or NADH revealed a 1:1:1
stoichiometry for 2-oxo-1,2-dihydroquinoline:O
:NADH. From
the highest specific activities under conditions of the standard assay,
turnover numbers of 1235 min
(reductase) and 320
min
(oxygenase) were calculated. No catalytic
activity was detectable under anaerobic conditions. The pH optimum of
the enzyme activity was pH 7.5. The optimal temperature was in the
range of 25-30 °C.
Electron Acceptors
The reductase
component of 2-oxo-1,2-dihydroquinoline 8-monooxygenase showed
NADH:acceptor reductase activity not only toward the oxygenase
component of this enzyme system but also toward some artificial
electron acceptors. Reductase with a specific activity of 40 units/mg,
measured in the presence of the oxygenase component (standard assay),
showed specific activities of 53, 89, 404, and 501 units/mg with INT,
DCPIP, cytochrome c, and ferricyanide, respectively. The
oxygenase showed no oxidoreductase activity with these acceptors. In
the DCPIP reductase assay, the reductase would reach only 1.3% activity
if NADH was replaced by NADPH.Cofactor Requirements and Activation
The
addition of Fe and FAD led to an increase in
2-oxo-1,2-dihydroquinoline 8-monooxygenase activity (Table 2). We
suggest that these additives might supplement cofactors lost during
purification. FMN did not replace FAD, and NADPH was less effective as
electron donor than NADH (Table 2). A preincubation of the enzyme
components with Fe
and FAD (30 min, 4 °C) did not
further enhance activity.
, Ca
, Fe
,
Co
, and Ni
(50 µM each) substituted Fe
in its enzyme-activating
effect. However, the addition of 50 µM Mn
, Zn
, or Cu
resulted in 14, 56, or 100% loss of activity, respectively,
compared to the activity without any supplementary metal salt.
or FAD. The NADH:DCPIP reductase was inhibited by
Zn
and Cu
(33 and 100% inhibition),
but it was not affected by Mn
.
2-Oxo-1,2-dihydroquinoline (0.2 mM) did not influence the
NADH:DCPIP reductase activity.
Inhibitors
Substances that modify
sulfhydryl groups (p-hydroxymercuribenzoate, N-ethylmaleimide, and iodoacetate, 0.5 mM each)
affected both NADH:DCPIP reductase activity (98, 66, and 43%
inhibition) and the activity of the complete monooxygenase system (97,
30, and 23% inhibition). Metal chelating agents (EDTA,
4,5-dihydroxy-1,3-benzene disulfonic acid, and 1,10-phenanthroline, 0.5
mM each) did not influence the NADH:DCPIP reductase activity,
but the complete enzyme system was inhibited (12, 14, and 95%).Substrate Specificity
8-Hydroxyquinoline,
8-hydroxy-2-oxo-1,2-dihydroquinoline, and coumarin (0.1 mM each) caused substrate-dependent oxygen consumption without being
converted. No oxygen consumption was observed with quinoline,
2-/8-monochloroquinoline, 2-/8-monomethylquinoline, quinoline
2-/8-monocarboxylic acid, 4-/5-/6-/7-monohydroxyquinoline,
2,4-/2,6-dihydroxyquinoline, isoquinoline,
1-oxo-1,2-dihydroisoquinoline, pyridine, acridine, indole, carbazole,
hypoxanthine, 2-hydroxynaphthalene, and anthraquinone (0.1 mM each).
O
. This effect has been reported
for some other oxygenases, e.g. orcinol
hydroxylase(21) , salicylate hydroxylase(22) , or
4-methoxybenzoate monooxygenase(23) . Since exogenously added
H
O
decayed spontaneously under our test
conditions, even in absence of any protein, H
O
consequently was undetectable in the enzyme assay.
(
)the oxygenases catalyzing the next degradation step
attack the same substrate (2-oxo-1,2-dihydroquinoline) at different
positions. The 5,6-dioxygenating enzyme of Comamonas
testosteroni
consequently totally differs from the
enzyme system of P. putida 86, hydroxylating at C-8 (this
paper). The capabilities of enzymes, performing nucleophilic as well as
electrophilic regio- (and stereo-)selective hydroxylations should give
a stimulus to their industrial use for biotransformations(62) .
, average value of the
g-tensor, g
= 1/3 (g + g + g); HPLC, high
pressure liquid chromatography; INT,
2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium
chloride.
We are grateful to Dr. A. Riedel
(Universitt Regensburg, Germany) for performing
electron paramagnetic resonance spectroscopy. We thank Prof. Dr.
Schreiber for recording and interpreting x-ray fluorescence spectra and
K. Kapassakalis for technical assistance during fermentation.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.