Purification and properties of reductase of the three-component p-cymene methyl hydroxylase from Pseudomonas chlororaphis subsp. aureofaciens

Article abstract of DOI:10.1016/j.procbio.2012.04.021

A novel three-component p-cymene methyl hydroxylase from Pseudomonas chlororaphis subsp. aureofaciens was reported earlier on the basis of genetic characterization and their expression catalyzing methyl group hydroxylation. This enzyme system was inductively synthesized when grown on p-cymene and had an important role in initiating p-cymene metabolism in vivo. In the present study, a NADH-dependent cytochrome c reductase protein has been purified to an electrophoretically homogeneous state and found to be involved in the hydroxylation of methyl group of p-cymene. Molecular mass of the reductase appears to be 38 kDa by SDS/PAGE and 39 kDa by gel filtration apart from one molecule of tightly bound FAD and two atoms each of iron and acid-labile sulfur per molecule of the enzyme. An apparent Km value of the enzyme for NADH is 32 ± 1.2 μM. To the best of our knowledge, this is the first report on the purification of reductase component of p-cymene methyl hydroxylase.

Full text of DOI:10.1016/j.procbio.2012.04.021

Process Biochemistry 47 (2012) 1263–1267
Contents lists available at SciVerse ScienceDirect
Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio
Purification and properties of reductase of the three-component p-cymene
methyl hydroxylase from Pseudomonas chlororaphis subsp. aureofaciens
Tapan K. Duttaa,b,∗, Arindam Duttaa, Joydeep Chakrabortya, Jayita Sarkara, Piyali Pal Chowdhurya,
Irwin C. Gunsalusb,1
a Department of Microbiology, Bose Institute, Kolkata 700054, India
b NHEERL, Gulf Ecology Division, U.S. EPA, Gulf Breeze, FL 32561-5299, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel three-component p-cymene methyl hydroxylase from Pseudomonas chlororaphis subsp. aureofa-
ciens was reported earlier on the basis of genetic characterization and their expression catalyzing methyl
group hydroxylation. This enzyme system was inductively synthesized when grown on p-cymene and
had an important role in initiating p-cymene metabolism in vivo. In the present study, a NADH-dependent
cytochrome c reductase protein has been purified to an electrophoretically homogeneous state and found
to be involved in the hydroxylation of methyl group of p-cymene. Molecular mass of the reductase appears
to be 38 kDa by SDS/PAGE and 39 kDa by gel filtration apart from one molecule of tightly bound FAD and
two atoms each of iron and acid-labile sulfur per molecule of the enzyme. An apparent Km value of the
enzyme for NADH is 32 1.2 ␮M. To the best of our knowledge, this is the first report on the purification
of reductase component of p-cymene methyl hydroxylase.
Received 28 March 2012
Accepted 28 April 2012
Available online 7 May 2012
Keywords:
Methyl hydroxylase
Reductase
p-Cymene
Monooxygenase
Pseudomonas
Purification
© 2012 Elsevier Ltd. All rights reserved.
1. Introduction
group while the other oxygen atom is reduced to water. XylM
shares significant amino acid identity with the integral-membrane
A significant amount of alkyl-substituted aromatics are being
funneled into the environment by a variety of biological and bio-
geochemical processes as well as by industrial activities. These
alkylated aromatic compounds form an important group of envi-
ronmental pollutants or their precursors in the ecosystem [1,2].
Fate of these classes of compounds in the environment is largely
due to microbial activities leading to their assimilation, primarily
initiated via oxidation of side-chain [3–7]. The best known aro-
matic methyl-substituent oxidation pathway is that encoded by
TOL plasmid, pWW0, in Pseudomonas putida mt-2 [6,8,9], involv-
ing oxidation of toluene, m-xylene, and p-xylene to benzoate,
m-toluate, and p-toluate, respectively. The pathway is initiated by
a monooxygenase, which catalyzes the oxidation of toluene (or
m- or p-xylene) to benzyl alcohol. This monooxygenase is a two-
component enzyme consisting of a XylA reductase subunit, which
transfers electrons from NADH through FAD and a [2Fe2S] center
to the membrane-associated XylM hydroxylase subunit. There, one
atom of activated molecular oxygen is inserted into the methyl
non-heme diiron AlkB alkane hydroxylases and steroyl-CoA desat-
urase terminal components [10–12].
The terpene p-cymene (also referred to as p-isopropyltoluene),
is an aromatic-terpenoid of biosynthetic origin [13,14] that con-
tains an isopropyl group attached to a benzene ring with a
para-oriented methyl group. Oxidative catabolism of p-cymene had
been studied and the assimilating pathway was already established
[3,15–17]. Oxidation of methyl side-chain is the committed step in
the degradation of p-cymene and is converted to p-cumic alcohol,
p-cumic aldehyde and p-cumate, which is further metabolized to
the intermediates isobutyrate, acetyl CoA and pyruvate. Previous
studies found that P. putida F1 genes involved in the degradation of
p-cymene are located on the cym operon and cymene monoxyge-
nase is a two-component enzyme system [16], although not much
information is known about structural aspect and the mechanistic
details of enzyme involved in p-cymene methyl group hydroxyla-
tion.
The genetic characterization and expression of the three-
component p-cymene monooxygenase involved in p-cymene
methyl hydroxylation in Pseudomonas aureofaciens reclassified as
Pseudomonas chlororaphis subsp. aureofaciens [18] was reported
in our previous communications [19,20]. The present study
reports the purification and properties of the NADH-dependent
cytochrome c reductase enzyme involved in p-cymene methyl
group hydroxylation.
∗
Corresponding author at: Department of Microbiology, Bose Institute, P-1/12
CIT Scheme VIIM, Kolkata 700054, India. Tel.: +91 33 25693241;
fax: +91 33 2355 3886.
E-mail address: tapan@bic.boseinst.ernet.in (T.K. Dutta).
Professor Irwin C. Gunsalus passed away on October 25, 2008.
1
1359-5113/$ – see front matter © 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.procbio.2012.04.021

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T.K. Dutta et al. / Process Biochemistry 47 (2012) 1263–1267
2. Material and methods
2.1. Chemicals
2.4.2. Ammonium sulfate precipitation
Active fractions from DEAE-cellulose column were pooled and
brought to 30% ammonium sulfate saturation by slow addition
of solid ammonium sulfate under gentle stirring at 4 ◦C. During
addition of ammonium sulfate, the pH of the protein solution was
adjusted to 7.0 using 1 N NaOH. After 3 h of stirring, the precipitated
proteins were removed by centrifugation (20,000 × g, 30 min). The
supernatant was then brought to 60% ammonium sulfate satura-
tion and the precipitated proteins were centrifuged as described
above.
NADH, NADPH, horse heart cytochrome c, and Sephadex G-200
were purchased from Sigma Chemical Co., St. Louis, Mo while DEAE-
cellulose (DE 52) anion exchange resin was from Whatman Ltd.,
Kent, England. Phenyl-Sepharose CL-4B for hydrophobic interac-
tion chromatography and Mono Q HR5/5 anion exchange column
(FPLC) were obtained from GE Healthcare Bio-Sciences AB, Upp-
sala, Sweden. Protein standards for gel filtration were obtained
from BioRad, USA while ultrafiltration membranes (PM 10) were
from Amicon, Levington, USA. p-Cymene, p-cumic alcohol, p-cumic
aldehyde and p-cumic acid were purchased from Aldrich Chemical
Co., Milwaukee, Wis. All other chemicals were obtained from com-
mercially available sources and were of analytical reagent grade.
2.4.3. Hydrophobic interaction chromatography
The pellet obtained from 60% ammonium sulfate precipitation
was resuspended in a minimum volume of 50 mM potassium phos-
phate buffer containing 1.7 M ammonium sulfate (pH adjusted to
7.0). Protein was loaded onto a phenyl-Sepharose column (15 ml
bed volume) previously equilibrated with 1.7 M ammonium sul-
fate in 50 mM potassium phosphate buffer as above. The column
was washed with 5 bed volumes of the same buffer. Enzymes were
eluted with 150 ml of linearly decreasing concentration of ammo-
nium sulfate (1.7–0 M) in 50 mM phosphate buffer (pH 7.0) at a flow
rate of 1 ml min−1 in 2 ml fractions.
2.2. Organism and culture conditions
Pseudomonas chlororaphis subsp. aureofaciens strain PJC was cul-
tured in PAS medium [21] with p-cymene (1 g l−1) as the sole source
of carbon and energy as described earlier [19,20]. Large quantities of
cells were grown overnight at 30 ◦C under constant shaking (OD660
1.2) in 2.8-liter Fernbach flasks containing 800 ml of the mini-
mal medium (pH 7.0). Cells were harvested by centrifugation and
washed twice with 50 mM potassium phosphate buffer (pH 7.0) and
the cell pellets were frozen in liquid nitrogen and stored at −70 ◦C
until used. Frozen cells (40 g, wet weight) were suspended in 100 ml
of 50 mM potassium phosphate buffer (pH 7.0) and cell suspension
was loaded into pre-cooled French press, Constant Cell Disruption
System, One Shot Model (Constant System Ltd. United Kingdom)
fitted with a 8.0-ml cell and lysed at 30,000 psi for two cycles. Par-
ticulate material was removed by centrifugation at 48,000 × g for
30 min at 4 ◦C. Bacto agar (1.5%), Difco Laboratories, Detroit, MI was
added for solid media.
2.4.4. Mono Q anion-exchange chromatography
The active fractions from phenyl-Sepharose column were
pooled and concentrated on an Amicon concentrator using PM-10
ultrafiltration membrane under nitrogen atmosphere. The con-
centrate (2 ml) was dialyzed against 4-liter of ice-cold 50 mM
potassium phosphate buffer (pH 7.0) with three changes at 4 ◦C.
Dialyzed protein was loaded onto a Mono Q HR5/5 column pre-
equilibrated with 50 mM potassium phosphate buffer (pH 7.0)
designed for fast-performance liquid chromatography (FPLC) sys-
tem, which consisted of a GP250 controller, two P500 pumps, a
UV-1 monitor, a REC-482 recorder and a FRAC200 autosampler (all
from GE Healthcare Bio-Sciences AB, Uppsala, Sweden). The col-
umn was washed with 40 ml of phosphate buffer and enzymes
were eluted with 40 ml of a pre-programmed gradient of NaCl
(0 − 350 mM) in 50 mM potassium phosphate buffer (pH 7.0) at the
rate of 0.5 ml min−1 in 0.5 ml fractions.
2.3. Enzyme assay
The reductase activity was assayed spectrophotometrically by
measuring the reduction of cytochrome c at 550 nm in the pres-
ence of NADH. Reaction was carried out at 25 ◦C in 1 ml of 50 mM
Tris–HCl buffer (pH 8.4) containing 50 ␮M horse heart cytochrome
c and 1 mM NADH. To determine the optimal conditions, dif-
ferent pH values in the range of 7.6–8.6 of Tris–HCl buffer and
different incubation temperatures in the range of 15–50 ◦C were
tested for reductase activity. A molar extinction coefficient of
21,000 M−1 cm−1 [22] for reduced cytochrome c was used to calcu-
late the enzyme activity. Specific activity was defined as units/mg of
enzyme where one unit of the enzyme was the amount catalyzing
the reduction of 1 ␮mol cytochrome c min−1 under the standard
assay conditions.
2.5. Analyses
In vitro transformation of p-cymene to 4-isopropylbenzyl
alcohol (p-cumic alcohol) was analyzed by reverse phase high per-
formance liquid chromatography (HPLC) using a Hewlett-Packard
1090 system equipped with a diode array detector. The reaction
mixture for determining p-cumic alcohol from p-cymene consisted
of 5 mM p-cymene, 2 mM of NADH, 20 ␮g of purified reductase and
saturating amount of membrane fraction (pellet obtained from dis-
rupted cell suspension following centrifugation at 48,000 × g) in
a total volume of 2 ml in 50 mM potassium phosphate buffer (pH
7.0). Following incubation for 2 h at 25 ◦C, the reaction mixture was
acidified to pH 2.0 using concentrated hydrochloric acid, then cen-
trifuged and the supernatant extracted thrice with equal volume of
ethyl acetate. Extract was concentrated and injected onto the HPLC
C18 column system, then eluted with acetonitrile-water (65:35)
for 20 min, and metabolite(s) was detected at 254 nm. Identity of
metabolite was confirmed from comparison of retention time and
UV spectrum with authentic samples.
Protein concentration in cell extract was measured by the
method of Bradford [23], with bovine serum albumin as the stan-
dard.
Enzyme purity and relative mobility (Mr) of polypeptides were
determined by sodium dodecyl sulfate-polyacrylamide gel elec-
trophoresis (SDS-PAGE) [24] with 10% gels and low-molecular
weight standard proteins (Mrs 14,400–94,000). Proteins were
2.4. Enzyme purification
All purification steps were performed at 4 ◦C.
2.4.1. Anion-exchange chromatography on DEAE-cellulose
A 100 ml volume of crude extract obtained from 40 g of P. chloro-
raphis subsp. aureofaciens cells was applied to a DEAE-cellulose
DE-52 anion-exchange column (bed volume 100 ml) previously
equilibrated with 50 mM potassium phosphate buffer (pH 7.0).
After the column was washed with 500 ml of same buffer, the
enzymes were eluted by a linear gradient of 0 − 0.5 M of NaCl in
500 ml of 50 mM potassium phosphate buffer (pH 7.0) at a flow
rate of 4 ml min−1. The elutant was collected in 5 ml fractions.

T.K. Dutta et al. / Process Biochemistry 47 (2012) 1263–1267
1265
stained with Coomassie blue R250. The relative mobility of native
protein was estimated by gel filtration [25] from a Sephadex G-
200 column (1.0 cm × 90 cm) that had been calibrated with a set
of molecular weight standards namely ␥-globulin, 158 kDa; bovine
serum albumin, 66 kDa; ovalbumin, 45 kDa; carbonic anhydrase,
29 kDa and lysozyme, 14.3 kDa. The void volume of the column was
determined by using blue dextran 2000 (Sigma, USA). Protein was
eluted by 50 mM potassium phosphate buffer (pH 7.0) at a flow rate
of 0.5 ml min−1. The elution profile was continuously monitored by
measuring absorbance at 280 nm.
were microwave frequency 9.05 GHz, microwave power 10 mW,
modulation amplitude 5 G, and temperature 20 K.
3. Results and discussion
3.1. Purification of NADH-dependent cytochrome c reductase
The reductase component of p-cymene methyl hydroxylase was
purified from Pseudomonas chlororaphis subsp. aureofaciens that
had been cultivated aerobically in the presence of p-cymene as
sole source of carbon and energy. By disrupting the cultivated cells
with a high pressure homogenizer device, NADH-dependent reduc-
tase activity was easily released into the soluble fraction. However,
purification of the terminal oxygenase component has so far been
unsuccessful due to the membrane bound nature of the protein [12]
and its inactivation during solubilization. Therefore, the reductase
enzyme is considered to be a membrane-extrinsic protein that pos-
sibly combines with the surface of the cytoplasmic membrane by
hydrophobic interactions. Moreover, the in vitro formation of p-
cumic alcohol from p-cymene by the purified reductase protein in
presence of an otherwise inactive crude membrane fraction con-
taining terminal oxygenase component and NADH was observed
during HPLC analysis of the organic extract of the reaction mix-
ture, supporting the role of the reductase as an electron transport
component in p-cymene hydroxylation (data not shown). In the
standard assay conditions, the transformation of p-cymene was
found to be 1.7 nmol min−1 ␮g−1 of purified reductase. However,
parallel control with membrane fraction from non-induced culture
did not exhibit p-cymene hydroxylation activity. The results of the
purification procedure of the reductase are given in Table 1. Based
on the elution profiles of proteins obtained from various column
chromatographic processes, it had been observed that the crude cell
extract contained only one protein capable of reducing cytochrome
c. These processes led to a 92-fold purification of the reductase with
6% recovery of total activity originally present in the crude extract.
2.6. Reaction stoichiometry and kinetic parameters
The stoichiometry of the reductase catalyzing transfer of elec-
trons from NADH to cytochrome c was determined from 1 ml of
50 mM Tris–HCl buffer (pH 8.4) containing 200 ␮M cytochrome c
and 50 ␮M NADH and 2.0 ␮g of reductase, incubated at 25 ◦C, with
the formation of reduced cytochrome c monitored spectropho-
tometrically at 550 nm. The change in A550 which could not be
modified by further addition of reductase enzyme or oxidized
cytochrome c represented the quantity of cytochrome c reduced
by 50 ␮M NADH. Kinetic parameters were determined by using
purified enzyme and monitoring the reduction of cytochrome c as
described above. The kinetic values for NADH were obtained with
a constant cytochrome c concentration of 50 ␮M and varying con-
centrations of NADH from 10 to 250 ␮M in the presence of 2.0 ␮g of
reductase. Each individual reaction was done in triplicate. Apparent
Km and Vmax values were obtained using a computer-aided direct fit
to the Michaelis-Menten equation (Sigmaplot 10, Enzyme module;
Systat Software, San Jose, CA, USA).
2.7. Flavin determination
Flavin was extracted by heating at 95 ◦C for 2 min from the
purified reductase at 1 mg/ml in 50 mM potassium phosphate
buffer (pH 7.0). Precipitated protein was removed by centrifugation
(10,000 × g, 5 min) and the supernatant was lyophilized and dis-
solved in 10 ␮l of 50 mM potassium phosphate buffer (pH 7.0). The
flavin coenzyme was identified by reverse phase chromatography
on a C18 column as described above, eluted with acetonitrile-
KH2PO4 (50 mM, pH 3.5), (10:90) and detected at 254 nm. Identity
of flavin component was confirmed from comparison of retention
time and UV spectrum with that of standard flavins (FAD and FMN).
3.2. Properties of NADH dependent cytochrome c reductase
Polyacrylamide gel electrophoresis (PAGE) of the purified reduc-
tase enzyme preparation in the presence and absence of sodium
dodecyl sulfate (SDS) revealed one Coomassie Brilliant Blue-
positive band on the gel. The enzyme was composed of only one
kind of polypeptide, which had an estimated relative mobility
of ∼38,000 from SDS-PAGE determination (Fig. 1A, Lane 2). The
molecular mass of the reductase was determined to be 39 kDa on
a calibrated Sephadex G-200 gel filtration column, as compared to
38.433 kDa calculated from the amino acid sequence of the protein
[19], suggesting that the purified reductase was a monomer of the
38 kDa polypeptide.
Solution of the enzyme was brownish yellow in color and gave
absorption maxima at 460 nm, a wavelength slightly longer than
the absorbance maximum of free FAD (448 nm, Fig. 1B). The spec-
trum bore similarity to NADH:acceptor reductase components of
methane and xylene monooxygenases [29,30]. The flavin coenzyme
isolated from the reductase protein had a spectrum corresponding
to that of free FAD (Fig. 1B). The flavin exhibited the same mobility
and UV spectrum as FAD as compared with that of FMN on reverse
phase chromatography. The concentration of FAD was determined
at 1.07 mol/mol of enzyme, indicating that each molecule of NADH-
cytochrome c reductase contains one FAD molecule. Furthermore,
cloning and sequencing of the reductase gene (cymA) and the
deduced amino acid sequence showed the typical FAD binding
motif as well as the N-terminal cysteine residues finger print for
the presence of chloroplast type [2Fe–2S] redox center [19]. These
observations suggest the protein is the reductase component of
2.8. Iron and labile sulfide determination
The iron content of the NADH-dependent cytochrome c reduc-
tase was determined by using the o-phenanthroline method [26]
using the purified protein, which was previously deproteinized
with trichloroacetic acid (5% final concentration) to release iron
component of the iron-sulfur cluster. The acid-labile sulfide con-
tent of the reductase was determined by the method of Brumby
et al. [27] modified in the manner suggested by Suhara et al. [28],
while prior to the addition of N,N-dimethyl-p-phenylenediamine
and ferric chloride, the purified enzyme was incubated with the
alkaline-zinc reagent for 2 h to release acid labile sulphide compo-
nent.
2.9. EPR spectroscopy
Electron paramagnetic resonance (EPR) spectrum of the purified
reductase was recorded using a Varian E-112 X-band spectrometer
equipped with an Air Products (Allentown, PA) variable tempera-
ture cryostat and a Varian TE102 mode cavity. The conditions used

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T.K. Dutta et al. / Process Biochemistry 47 (2012) 1263–1267
Table 1
Purification of reductase.
Purification step
Protein (mg)
Activitya (U)
Sp act (U/mg)
Recovery (%)
Purification fold
Cell-free extract
3200
228
42.6
6.8
1612
616
408
268
101
0.5
2.7
9.6
39.4
45.9
100
38
25
17
6
1
5
19
79
92
DEAE-cellulose DE-52
Ammonium sulfate fractionation
Phenyl sepharose
Mono Q
2.2
a
Enzyme activity was determined by the NADH-dependent cytochrome c reductase assay.
Fig. 1. (a) SDS-PAGE analysis of the NADH dependent cytochrome c reductase. Lane 1, molecular weight markers (kDa) containing phosphorylase b (94), bovine serum
albumin (67), ovalbumin (43), carbonic anhydrase (30), trypsin inhibitor (20.1), and ␣-lactalbumin (14.4); and lane 2, purified reductase, 0.5 ␮g. (b) Absorption spectra of
NADH dependent cytochrome c reductase in 50 mM potassium phosphate buffer (pH 7.0). Inset, cofactor isolated from the reductase. mAU, milli-absorbance unit.
p-cymene methyl hydroxylase, an iron–sulfur flavoprotein, con-
taining 1 molecule each of FAD and [2Fe–2S] center/molecule of
enzyme.
hydroxylation of a methyl side-chain of p-cymene similar to that
reported in P. putida F1. However, genetic studies have suggested
that this monooxygenase consists of a novel three-component sys-
tem, where products of the cymA, cymB and cymM genes function
as an electron-transfer protein, enhancer/activator protein and a
The stoichiometry of the NADH-cytochrome c reductase activ-
ity was determined and the oxidation of 50 nmol NADH produced
104 nmol of reduced cytochrome c. The activity of the enzyme
varied with pH with maximum activity between pH 7.6 and 8.6
(optimum pH 8.4) while the optimum temperature was found to be
25 ◦C. The reductase was inactivated when exposed to higher tem-
peratures, incubation at 30 ◦C, 37 ◦C and 50 ◦C for 10 min reduced
its activity by 42%, 66% and 98% respectively. The reductase could
be stored at −70 ◦C in 50 mM potassium phosphate buffer (pH 7.0)
containing 60% glycerol (v/v) for a month without major loss of
activity.
The activity of the purified enzyme could not be stimulated by
the addition of FAD indicating tight association with the enzyme
molecule which was not lost during the purification process.
The content of iron and acid-labile sulfide was determined to
be in ratios of 1.92 0.14 and 1.86 0.11 atoms molecule−1 of
the NADH-dependent cytochrome c reductase enzyme, respec-
tively, confirming the presence of one [2Fe–2S] center per enzyme
molecule. Moreover, the EPR spectrum of the dithionite-reduced
purified reductase revealed g values at 2.04, 1.95, and 1.89 (Fig. 2),
which agrees very well with those of typical chloroplast type
ferredoxins [31]. The NADH oxidation catalyzed by the reductase
displayed Michaelis–Menten kinetics, with apparent Km and Vmax
values for NADH of 32 1.2 ␮M and 1412 38 ␮mol min−1 mg−1
of protein respectively. With the purified enzyme, NADPH could
not replace the cofactor requirement for cytochrome c reductase
activity.
Fig. 2. EPR spectrum of the NADH dependent cytochrome c reductase. A 200-␮l
volume of the reductase (40 ␮M) was anaerobically reduced with a slight excess of
sodium dithionite.
The p-cymene methyl hydroxylase (monooxygenase) sys-
tem encoded in P. chlororaphis subsp. aureofaciens catalyses the

T.K. Dutta et al. / Process Biochemistry 47 (2012) 1263–1267
1267
terminal hydroxylase, respectively. The reductase component
CymA from P. chlororaphis subsp. aureofaciens showed 87% iden-
tity in 349 amino acid residues with the corresponding reductase
component CymAb from P. putida F1. In this study, the electron-
transfer component of p-cymene monooxygenase, the product of
cymA, was purified to homogeneity followed by the characteriza-
tion of the properties of this reductase enzyme. Understanding the
mechanistic details of this complex protein including that of the
role of the possible enhancer/activator protein will be the subject
of further investigations.
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Acknowledgments
This manuscript is dedicated to the memory of I. C. Gunsalus
(Gunny). This work was supported in part by a National Research
Council Research Associate Award, USA (T.K.D.) and Bose Institute,
Kolkata, India.
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