PpoC from Aspergillus nidulans is a fusion protein with only one active haem

Article abstract of DOI:10.1042/BJ20091096

In Aspergillus nidulans Ppos [psi (precocious sexual inducer)-producing oxygenases] are required for the production of so-called psi factors, compounds that control the balance between the sexual and asexual life cycle of the fungus. The genome of A. nidulans harbours three different ppo genes: ppoA, ppoB and ppoC. For all three enzymes two different haem-containing domains are predicted: a fatty acid haem peroxidase/ dioxygenase domain in the N-terminal region and a P450 haem-thiolate domain in the C-terminal region. Whereas PpoA was shown to use both haem domains for its bifunctional catalytic activity (linoleic acid 8-dioxygenation and 8-hydroperoxide isomerization), we found that PpoC apparently only harbours a functional haem peroxidase/dioxygenase domain. Consequently, we observed that PpoC catalyses mainly the dioxygenation of linoleic acid (18:2Δ ^9Z,^12Z), yielding 10-HPODE (10-hydroperoxyoctadecadienoic acid). No isomerase activity was detected. Additionally, 10-HPODE was converted at lower rates into 10-KODE (10-keto-octadecadienoic acid) and 10-HODE (10-hydroxyoctadecadienoic acid). In parallel, decomposition of 10-HPODE into 10-ODA (10-octadecynoic acid) and volatile C-8 alcohols that are, among other things, responsible for the characteristic mushroom flavour. Besides these principle differences we also found that PpoA and PpoC can convert 8-HPODE and 10-HPODE into the respective epoxy alcohols: 12,13-epoxy-8-HOME (where HOME is hydroxyoctadecenoic acid) and 12,13-epoxy-10-HOME. By using site-directed mutagenesis we demonstrated that both enzymes share a similar mechanism for the oxidation of 18:2Δ^9Z,^12Z; they both use a conserved tyrosine residue for catalysis and the directed oxygenation at the C-8 and C-10 is most likely controlled by conserved valine/leucine residues in the dioxygenase domain. The Authors Journal compilation

Full text of DOI:10.1042/BJ20091096

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Biochem. J. (2010) 425, 553–565 (Printed in Great Britain) doi:10.1042/BJ20091096
553
PpoC from Aspergillus nidulans is a fusion protein with only one active
haem
1
¨
Florian BRODHUN, Stefan SCHNEIDER, Cornelia GOBEL, Ellen HORNUNG and Ivo FEUSSNER
Department of Plant Biochemistry, Albrecht-von-Haller-Institute for Plant Sciences, Georg-August-University, Justus-von-Liebig-Weg 11, D-37077 Go¨ttingen, Germany
In Aspergillus nidulans Ppos [psi (precocious sexual inducer)-
producing oxygenases] are required for the production of so-
called psi factors, compounds that control the balance between
the sexual and asexual life cycle of the fungus. The genome of
A. nidulans harbours three different ppo genes: ppoA, ppoB and
ppoC. For all three enzymes two different haem-containing do-
mains are predicted: a fatty acid haem peroxidase/dioxygenase
domain in the N-terminal region and a P450 haem-thiolate do-
main in the C-terminal region. Whereas PpoA was shown
to use both haem domains for its bifunctional catalytic
activity (linoleic acid 8-dioxygenation and 8-hydroperoxide
isomerization), we found that PpoC apparently only harbours a
functional haem peroxidase/dioxygenase domain. Consequently,
we observed that PpoC catalyses mainly the dioxygenation
of linoleic acid (18:2ꢀ9Z,12Z), yielding 10-HPODE (10-
hydroperoxyoctadecadienoic acid). No isomerase activity was
detected. Additionally, 10-HPODE was converted at lower rates
into 10-KODE (10-keto-octadecadienoic acid) and 10-HODE
(10-hydroxyoctadecadienoic acid). In parallel, decomposition of
10-HPODE into 10-ODA (10-octadecynoic acid) and volatile
C-8 alcohols that are, among other things, responsible for
the characteristic mushroom flavour. Besides these principle
differences we also found that PpoA and PpoC can convert
8-HPODE and 10-HPODE into the respective epoxy alcohols:
12,13-epoxy-8-HOME (where HOME is hydroxyoctadecenoic
acid) and 12,13-epoxy-10-HOME. By using site-directed
mutagenesis we demonstrated that both enzymes share a similar
mechanism for the oxidation of 18:2ꢀ9Z,12Z; they both use
a conserved tyrosine residue for catalysis and the directed
oxygenation at the C-8 and C-10 is most likely controlled by
conserved valine/leucine residues in the dioxygenase domain.
Key words: Aspergillus nidulans, fatty acid dioxygenase, oxylipin
formation, psi-producing oxygenase (Ppo).
[9,11–13]. Ppo enzymes show a significant sequence identity
with mammalian PGHS (prostaglandin endoperoxide H synthase;
25–29% identity) and fungal 7,8-linoleate diol synthase (40–
45% identity) [10]. In contrast with 7,8-linoleate diol synthase
a search for conserved domains using the NCBI (National
Center for Biotechnology Information) database predicted that
Ppo enzymes contained two different haem domains: a haem
peroxidase/dioxygenase domain in the N-terminal region and a
P450 haem-thiolate domain in the C-terminal region. Indeed,
we have previously shown, on the basis of sequence alignments
and spectroscopic analysis, that PpoA consists of those different
haem domains [14]. Furthermore, we demonstrated that both
domains are essential for the two different catalytic activities
INTRODUCTION
In Aspergillus nidulans (teleomorph Emericella nidulans)
oxidized unsaturated fatty acids, so-called oxylipins, derived
from endogenous fatty acids, have been described to regulate the
developmental life cycle of the fungus [1–7]. In A. nidulans these
oxylipins are collectively called psi (precocious sexual inducer)
factors. Psi factors are a mixture of oxidized 18:1ꢀ9Z, 18:2
ꢀ9Z,12Z
ꢀ9Z,12Z,15Z
ꢀz
and 18:3
(where x:y is a fatty acid containing x car-
bons and y double bonds in position z, counting from the carboxy
group [8]) and are termed psiβ, psiα and psiγ respectively. The
number and the position of the hydroxy groups on the fatty
acid backbone leads to further classification of psi factors: psiB
[OH at C-8, e.g. (8R)-HODE], psiA [OH at C-5 and C-8, e.g.
(5S,8R)-DiHODE] and psiC [OH at C-8 and on the δ-lactone ring]
(where HODE is hydroxyoctadecadienoic acid and DiHODE is
dihydroxyoctadecadienoic acid) [1,2].
The genes encoding enzymes that are involved in the production
of psi factors were first identified by Keller and co-workers
and were termed Ppos (psi-producing oxygenases) [9–11]. The
genome of A. nidulans harbours three different genes possibly
encoding Ppos: ppoA, ppoB and ppoC [9–11]. The same group
showed that deletion of these genes had a significant effect
on: (i) the developmental ratio between asexual conidiospores
and sexual ascospores; (ii) the production of psi factors; and
(iii) the production of secondary metabolites, the mycotoxins
of PpoA; in the N-terminal peroxidase/dioxygenase domain of
ꢀ9Z,12Z
PpoA the dioxygenase reaction takes place and 18:2
is
oxidized to (8R)-HPODE (hydroperoxyoctadecadienoic acid).
This intermediate product is then isomerized by the P450 haem-
thiolate domain of PpoA to 5,8-DiHODE [14] (Figure 1). On the
basis of these results PpoA was assigned as CYP6001A1, a first
member of a new P450 subfamily.
Previously Garscha et al. [15] reported that homogenates of
A. nidulans mutants that are deficient in PpoC failed to form
(10R)-HPODE and (10R)-HODE. From that they concluded
that PpoC is a (10R)-dioxygenase. Recombinant expression
of Aspergillus fumigatus PpoC in insect cells confirmed this
hypothesis [16]. Moreover mutational studies of 7,8-linoleate
Abbreviations used: CmꢀP, 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrroline-N-oxyl; DiHODE, dihydroxyoctadecadienoic acid; HODE, hydroxyocta-
decadienoic acid; HOME, hydroxyoctadecenoic acid; HOTrE, hydroxyoctadecatrienoic acid; HPODE, hydroperoxyoctadecadienoic acid; HPOME,
hydroperoxyoctadecenoic acid; HPOTrE, hydroperoxyocadecatrienoic acid; KODE, keto-octadecadienoic acid; NCBI, National Center for Biotechnology
Information; NIST, National Institute of Standards and Technology; ODA, octadecynoic acid; PGHS, prostaglandin endoperoxide H synthase; Ppo,
psi-producing oxygenase; psi, precocious sexual inducer; RP, reversed-phase; SPME, solid-phase micro-extraction; TMPD, N,N,Nꢀ,Nꢀ-tetramethyl-p-
phenylenediamine.
1
To whom correspondence should be addressed (email ifeussn@uni-goettingen.de).
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F. Brodhun and others
Figure 1 Psi factor formation in A. nidulans and A. fumigatus
Ppo enzymes abstract a hydrogen atom from the C-8 of the backbone of unsaturated C-18 fatty acids and form a carbon-centred radical at this position, a reaction that is presumably accomplished
via a tyrosyl radical mechanism within the N-terminal fatty acid haem peroxidase/dioxygenase domain. In the case of enzymes belonging to PpoA subfamily molecular oxygen attacks the C-8-centred
radical and forms (8R)-HPODE. This intermediate product is either used as a substrate for peroxidase reaction, yielding 8-HODE, or it serves as a substrate for the 8-hydroperoxide isomerase
reaction, yielding mainly 5,8-DiHODE. The isomerase reaction is believed to be accomplished by the C-terminal P450 haem-thiolate domain of PpoA [14]. Thirdly the fatty acid hydroperoxide is
reduced to 8-HODE or transformed to small amounts of 12,13-epoxy-8-HOME respectively. In case of PpoC enzymes the carbon-centred radical migrates to C-10 and molecular oxygen is inserted
at this position, yielding 10-HPODE. This product appears to be unstable and either oxidizes to 10-KODE or decomposes to 10-ODA and volatile compounds, such as 1-octen-3-ol. Furthermore,
10-HPODE can be reduced to 10-HODE or transformed to small amounts of 12,13-epoxy-10-HOME respectively. Ep, epoxy.
diol synthase and PpoC implied a general mechanism for the
Acetonitrile was from Fisher Scientific. Restriction enzymes were
provided by MBI Fermentas.
ꢀ9Z,12Z
directed oxygenation of 18:2
at the C-8 and C-10 by fungal
dioxygenases. It was proposed that conserved leucine/valine
residues in the dioxygenation site of both dioxygenases determine
the oxygenation site [16]. In the same study the formation of
low amounts of the epoxy alcohol 12,13-epoxy-(10R)-HOME
(where HOME is hydroxyoctadecenoic acid) was observed. It
was suggested that, in analogy with the isomerization activity
of the bifunctional 7,8-linoleate diol synthase that transforms
8-HPODE to (7S,8S)-DiHODE, PpoC is also a bifunctional
enzyme with epoxy alcohol synthase activity that converts 10-
HPODE into 12,13-epoxy-(10R)-HOME [16].
In the present study we aimed to compare the biochemical
properties of the two Ppo- enzymes PpoA and PpoC from
A. nidulans, which share a sequence identity of 44%, to elucidate
the basis for differences in the produced compounds. Therefore,
we expressed PpoC in the same expression system as reported for
PpoA [14], purified PpoC and analysed the biochemical properties
of this enzyme.
Cloning and expression of recombinant PpoC in Escherichia coli
A. nidulans ppoC (GenBankTM accession number AY613780.1)
was amplified from fungal cDNA of sexual and vegetative
stages using gene-specific primers containing NdeI and NotI
recognition sites (forward primer, 5ꢀ-CCATATGTTGCGAAGAT-
TTTCTACCTTCAG-3ꢀ; reverse primer, ꢀ 5ꢀ-AGCGGCCGCTC-
AAGCAGAATCAATCTGCTTCTTAG-3 ) via the Phusion® hot-
start high-fidelity DNA polymerase system (Finnzymes). PCR
amplification were performed under the following conditions:
98◦C for 3 min followed by 30 cycles of 98◦C for 30 s, 53◦C for
30 s and 72◦C for 3 min, and the PCR was terminated by 5 min
at 72◦C. The resulting fragment was cloned into pJET2.1/blunt
(Fermantas), yielding the plasmid pJET-PpoC. For expression
in E. coli ppoC was cloned into the pET24a expression vector
(Novagen) using NdeI and NotI, yielding the plasmid pET24a-
PpoC and it was transformed together with the plasmid pGro7
(encoding the chaperone groES–groEL; Takara Bio) into E. coli
BL21 StarTM cells (Invitrogen).
EXPERIMENTAL
Materials
Site-directed mutagenesis
Chemicals were obtained from Sigma–Aldrich and Carl Roth
& Co. Agarose was from Biozym Scientific GmbH. All fatty
acids were purchased from Sigma–Aldrich or Cayman Chemicals.
In vitro mutagenesis was carried out using Phusion® hot-start
high-fidelity DNA polymerase system using the protocol from
the QuickChange® mutagenesis kit (Stratagene). The following
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PpoC is a fusion protein with one active haem
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primers were used: PpoA(V328L) sense (5ꢀ-CAAATATTA-
TCCTAAAAGATTATUCUTUAUCGTACGATTTTGAATATA-
AAC-3ꢀ) and antisense (5ꢀ-GTTTATATTCAAAATCGTACGT-
AGATAATCTTTTAGGATAATATTTG-3ꢀ); PpoA(V328A) sense
(5ꢀ-GTACGCAAATATTATCCTTAAUGUGATTATGCACGAA-
CGATTTTGAATATAAA-3ꢀ) and antisense (5ꢀ-GTTTATATT-
CAAAATCGTTCGTGCATAATCCTTAAGGATAATATTTGC-
GTAC-3ꢀ); PpoC(Y427F) sense (5ꢀ-GAAACCAATGTTCTGTC-
GAATTTAACCTGGCCTTTCGGTGGCACTCTGCTATCAG-
TG-3ꢀ) and antisense (5ꢀ-CACTGATAGCAGAGTGCCACC-
GAAAGGCCAGGTTAAATTCGACAGAACATTGGTTTC-3ꢀ);
PpoC(G1039C) sense (5ꢀ-GGCATCGGTTCTCAAATCTGCCT-
AGGCAAGGACGCCACACTG-3ꢀ) and antisense (5ꢀ-CAGT-
GTGGCGTCCTTGCCTAGGCAGATTTGAGAACCGATG-
CC-3ꢀ); PpoC(L385V) sense (5ꢀ-CATCACCCTGTACGATTAC-
GTACGCACGATCGTTAACCTGAACCGAGTGAACAG-3ꢀ)
and antisense (5ꢀ-CTGTTCACTCGGTTCAGGTTAACGATCG-
performed at a flow rate of 2 ml/min. Fractions containing enzyme
activity were combined and loaded on to a desalting 26/10 column
(GE Healthcare) equilibrated with 50 mM Tris/HCl, pH 8.0. The
desalted protein solution was loaded on to a MonoQ 10/10-
column (GE Healthcare). After loading and washing with 50 mM
Tris/HCl, pH 8.0, the protein was eluted at 3 ml/min with a linear
gradient to 50 mM Tris/HCl, pH 8.0, containing 1 M NaCl within
30 min. Recombinant PpoA and related mutants [PpoA(V328A)
and PpoA(V328L)] were purified as described previously [14].
UV–visible spectroscopy
UV–visible spectroscopy was performed in 50 mM Hepes,
pH 7.4, at room temperature (approx. 24◦C) using a dual-beam
Varian Cary 100 Bio UV/Vis Spectrophotometer.
TGCGTACGTAATCGTACAGGGTGATG-3ꢀ);
and
PpoC-
(L385A) sense (5ꢀ-CATCACCCTGTACGATTACGCACGC-
ACGATCGTTAACCTGAACCGAGTGAACAG-3ꢀ) and anti-
sense (5ꢀ-CTGTTCACTCGGTTCAGGTTAACGATCGTGCG-
TGCGTAATCGTACAGGGTGATG-3ꢀ).
Activity assays
ꢀ9Z,12Z
ꢀ9Z,12Z,15Z
Fatty acids (250 μg of 18:1ꢀ9Z, 18:2
and 18:3
)
were dissolved in 1 ml or 800 μl of 50 mM Tris/HCl, pH 8.0.
The reactions were started by the addition of either 15 μg of
purified PpoC or 200 μl of PpoC bacterial crude extract and
were incubated for 30 min at room temperature. Incubations were
stopped by adding 2 ml of diethyl ether to extract fatty acids and
their derivatives. A second extraction was performed additionally
with 2 ml of diethyl ether and 100 μl of acetic acid. After
evaporating under a nitrogen stream the sample was dissolved in
30 μl of HPLC-eluent (water/acetonitrile/acetic acid; 50:50:0.1,
Expression of recombinant Ppo enzymes in E. coli
E. coli BL21 StarTM cells containing pET24a-PpoC and the
plasmid pGro7 (encoding the chaperone groES–groEL) were
cultivated in 2YT medium [1.6% (w/v) tryptone/1% (w/v) yeast
extract/0.5% (w/v) NaCl] containing 50 μg/ml chloramphenicol,
25 μg/ml kanamycin and 25 μg/ml arabinose to induce the
expression of the chaperone, and 0.02 mM δ-aminolaevulinic
acid. Cells were grown to a D600 of 0.6–0.8. Induction of the
expression of the recombinant protein was performed by adding
IPTG (isopropyl β-D-thiogalactoside) to a final concentration of
0.1 mM and cultures were cultivated for 3 days with shaking at
16◦C. Cells were harvested by centrifugation at 4000 g at 6◦C for
10 min. The pellet was shock-frozen in liquid nitrogen and stored
at −20◦C.
by vol.).
Incubations of 18:2
ꢀ9Z,12Z
and 18O2 (Campro Scientific) with
PpoC were performed in 50 mM Tris/HCl, pH 8.0. The buffer
(900 μl) was degassed and supplied with 18O2 on ice until the
system was saturated. PpoC bacterial crude extract (100 μl) and
ꢀ9Z,12Z
250 μg of 18:2
were added and the mixture was incubated
for 30 min at room temperature. The reaction system was further
enriched with 18O2 during the incubation. The reaction was
terminated by addition of diethyl ether and reaction products were
extracted as described above.
ꢀ9Z,12Z
Incubations of 18:2
with purified PpoA, PpoA(V328A)
and PpoA(V328L) were performed in 50 mM Hepes, pH 7.4, as
described previously [14]. Incubations with 8-HPODE and 10-
HPODE were either performed in 200 μl of PpoC crude bacterial
extract (in 50 mM Tris/HCl, pH 8.0) or with 200 μl of 50 μM
Hepes, pH 7.4, containing 1–5 μM PpoA.
Cell lysis and protein purification
Harvested cells from 500 ml of culture expressing recombinant
PpoC were resuspended in 50 ml of buffer (50 mM Tris/HCl,
pH 8.0). After addition of lysozyme to a final concentration
of 0.1 mg/ml and PMSF to a final concentration of 0.2 mM,
cells were incubated for 30 min on ice. Cells were disrupted
by sonification (three times, each for a 30 s duration) on ice
using a sonifier cell disruptor B15 (Branson Sonic Power) and
this extract was centrifuged at 54000 g at 4◦C for 20 min to
clear debris, yielding crude bacterial extract. The supernatant
obtained from cell lysis was loaded on to a Source 30Q resin
RP (reversed phase)-HPLC/MS analysis
The HPLC assembly was a Surveyor HPLC system (Thermo
Finnigan) with an EC250/2 100–5 C18 column (2.1 mm ×
250 mm, 5 μm particle size; Macherey-Nagel). The solvent
system for the following gradient was (A) acetonitrile/water/acetic
acid (40:60:0.1, by vol.), and (B) acetonitrile/acetic acid [100:0.1,
by vol.]. The gradient elution profile was as follows: 0–10 min a
flow rate of 0.2 ml/min using 80% (A) and 20% (B); 10–30 min
from 80% (A) and 20% (B) to 100% (B); 30–35 min a flow rate
increase to 0.3 ml/min; 35–40 min 100% (B); 40–44.5 min from
100% (B) to 80% (A) and 20% (B); and 44.5–45 min 80% (A)
and 20% (B) and flow rate decreased to 0.2 ml/min. The mass
spectrometer was an LCQ ion-trap mass spectrometer (Thermo
Finnigan) with electrospray ionization and monitoring of negative
ions. The capillary temperature was 300◦C and the capillary
voltage was 27 kV. For tandem MS analysis, the collision energy
was 1 V.
(a 25 ml XK 16/20 column; GE Healthcare) at a flow rate of
TM
¨
1 ml/min using an AKTAprime system (GE Healthcare). The
column was washed with 50 ml of 50 mM Tris/HCl, pH 8.0
and protein was eluted with a linear gradient of 0–0.3 M NaCl
in 50 mM Tris/HCl, pH 8.0, within 30 min at a flow rate of
2 ml/min. Fractions of 2 ml were collected. Fractions with highest
activity (as judged by a Clark-type O2 electrode; Rank Brothers)
and highest purity (as judged by SDS/PAGE) were combined
and concentrated to a final volume of 2 ml using Vivaspin 20
(100000 molecular-mass cut-off; Sartorius Stedim Biotech). For
gel filtration the concentrated protein extract was loaded on to
a Superdex S200 26/60 pg column (GE Healthcare) equilibrated
with 50 mM Tris/HCl, pH 8.0, containing 1 M NaCl. Elution was
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F. Brodhun and others
Ppo family of A. nidulans, for which functionality of both domains
has been proven [14]. Sequence alignments of the predicted
N-terminal haem peroxidase/dioxygenase domain of A. nidulans
PpoC with those of A. fumigatus PpoC, A. nidulans PpoA,
Gaeumannomyces graminis 7,8-linoleate diol synthase, sheep
PGHS-1 and sheep PGHS-2 (Figure 2A) showed that residues
reported to be essential for the catalytic dioxygenase activity are
conserved in all six sequences. However, sequence alignments
of the predicted C-terminal P450 domain of PpoC with those of
known P450 haem-thiolate enzymes, i.e. the recently described
A. nidulans PpoA [14] and A. fumigatus PpoC [16], showed that
the crucial cysteine residue, which is known as the fifth haem iron
ligand, is neither conserved in PpoC from A. nidulans nor from
A. fumigatus. Instead of a cysteine residue a glycine residue and
a phenylalanine residue respectively, were found at the particular
position of the PpoC sequences (Figure 2B). This may indicate
that the P450 domain is not functional and does not show catalytic
activity (see below).
Analysis of volatile compounds
Volatile compounds were measured from the reaction of 10 ml
of crude bacterial extract incubated with 2.5 mg of fatty
ꢀ9Z,12Z
acid (18:1ꢀ9Z, 18:2
or 18:3ꢀ9Z,12Z,15Z) by SPME (solid-
phase micro-extraction). Incubation of the reaction mixture was
performed in a vial, sealed with a teflon septum and with
an inserted poly(dimethylsiloxane/divinylbenzene)-coated fibre
(Supelco). After 30 min the reaction products were extracted as
described previously [17]. The analysis of the hydroxy fatty acids
was carried out using an Agilent 5973 Network mass-selective
detector connected to an Agilent 6890 gas chromatograph
equipped with a capillary DB-23 column (30 m × 250 μm;
0.25 μm coating thickness). Helium was used as carrier gas
(1 ml/min). The injection temperature was 250◦C, the temperature
gradient was 40◦C for 2 min, 150–200◦C at 15◦C/min and 200◦C
for 3 min. Electron energy of 70 eV, an ion-source temperature of
230◦C and a temperature of 260◦C for the transfer line was used.
Lipid-derived radical trapping by nitroxy radicals and peroxidase
assay
Cloning and functional expression of PpoC
In order to analyse the enzymatic reaction catalysed by PpoC from
A. nidulans, its cDNA was cloned from vegetative and sexual
stages of A. nidulans by using specific primers. Sequencing of the
resulting plasmid confirmed the ORF (open reading frame) was
3933 bp and 1117 amino acids respectively, showing one amino
acid residue exchange (P-215) compared with the published
genome sequence [20]. Functional expression of PpoC was
performed in E. coli BL21 StarTM cells and optimal cultivation
conditions (16◦C for 3 days) were determined. Expression of
PpoC could be enhanced by co-expression of chaperone groES–
groEL and the addition of 0.2 mM δ-aminolaevulinic acid
before induction (aminolaevulinic acid is a precursor of haem
biosynthesis). Protein expression was verified by SDS/PAGE
(Figure 3A).
Lipid-derived radicals generated during the incubation of
ꢀ9Z,12Z
18:2
with PpoC were trapped and analysed as nitroxy
radicals as described previously [14,18]. We used the peroxidase
assay according to Kulmacz [19] to demonstrate peroxidase acti-
vity of PpoC as described previously [14].
Determination of kinetic parameters
Kinetic experiments were performed in 50 mM Tris/HCl, pH 7.9,
at room temperature with a Clark-type O2 electrode. Initial rates
of oxygen consumption were measured for different substrate
concentrations in buffer that was pre-equilibrated against air at
101.325 kPa. The reaction was started by the addition of 200 μl
of PpoC bacterial crude extract to 800 μl of rapidly stirring buffer
ꢀ9Z
containing a defined amount of substrate (sodium salts of 18:1
18:2
,
or 18:3ꢀ9Z,12Z,15Z). Kinetic parameters were determined
ꢀ9Z,12Z
Purification of PpoC
by fitting seven data points to Michaelis–Menten equations by
using the software Origin 6.0.
In order to purify PpoC a protocol was used based on that
described previously for PpoA purification [14]. Extraction
and solubilization of PpoC using crude extract from bacteria
was performed with 50 mM Tris/HCl, pH 8.0. The addition
of detergents and NaCl to the extraction buffer did not
improve protein extraction. For the initial purification step a
Source 30Q resin was chosen to isolate and concentrate PpoC
from the crude cell extract. Fractions showing the highest
activity were combined and purified further using size-exclusion
chromatography. The native molecular mass of PpoC using
gel-filtration chromatography was 480 kDa. This was 4-fold
larger than that determined by SDS/PAGE. Therefore the native
protein is a homotetramer, as has been demonstrated previously
for 7,8-linoleate diol synthase [21] and PpoA [14]. With the
final chromatography step using a MonoQ column most of the
remaining contaminants were removed. After each purification
step the protein fractions showing the highest activity were
combined and subjected to SDS/PAGE (Figure 3A). A protein
band at approx. 120 kDa became subsequently more prominent.
Using this protocol we were able to purify approx. 0.5–2 mg
of protein from 500 ml of cell culture. The activity of the pure
protein was estimated to be 6.5 μmol [O2]/(min × mg) using the
Clark-type O2 electrode. However, we found that the activity of
the purified protein decreased significantly within a few hours.
Neither the addition of 10% (v/v) glycerol nor 5 mM EDTA to
the buffer improved PpoC stability.
Determination of the pH optimum
The pH optimum was determined by measuring initial rates of
oxygen consumption at a defined pH value using a Clark-type
O2 electrode. PpoC bacterial crude extract (200 μl) was added
to 800 μl of buffer containing 100 μM of the sodium salt of
18:2ꢀ9Z,12Z. The following buffers were used for the different pH
ranges: 0.2 M acetate buffer (pH 4.5–5.5), 0.2 M phosphate buffer
(pH 5.5–8.0) and 0.2 M borate buffer (pH 8.0–10.5).
RESULTS
PpoC is a fusion protein of the CYP6001 family
BLAST search analysis of PpoC predicts the presence of two
domains in its sequence. Within the N-terminal region of the
protein a haem peroxidase/dioxygenase domain is predicted,
whereas a P450 haem-thiolate domain is proposed for the
C-terminal part. Therefore PpoC from A. nidulans was assigned
as CYP6001C1 by Dr D.R. Nelson of the P450 Nomenclature
Committee (Department of Molecular Sciences, University of
Tennessee, Memphis, TN, U.S.A.). Similar domain architecture
has also been described recently for PpoA, another member of the
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Figure 2 Partial alignment of the predicted N-terminal haem peroxidase/dioxygenase domain and C-terminal P450 domain from PpoC with sequences from
different fatty acid haem dioxygenases and P450 enzymes
®
(A) Partial amino acid alignment of the predicted haem peroxidase/dioxygenase domain from A. nidulans PpoC (AnPpoC; RefSeq XP_662632) with A. fumigatus PpoC (AfPpoC; GenBank
ABV21633), A. nidulans PpoA (AnPpoA; RefSeq XP_659571), G. graminis 7,8-linoleate diol synthase (GgLDS; Swiss-Prot Q9UUS2.1;), sheep PGHS-1 (Swiss-Prot P05979.2) and sheep PGHS-2
(Swiss-Prot P79208.1). The catalytic tyrosine residue as well as the proximal and distal histidine residues, which are conserved in the six sequences, and have been identified previously [30,41], are
shown on a black background. Additionally, leucine/valine residues that are responsible for direction of oxygenation in A. fumigatus PpoC and G. graminis 7,8-linoleate diol synthase are on a black
background. (*), conserved residues; (:) and (.), similar residues. (B) Partial amino acid alignment of the predicted P450 haem-thiolate domain from A. nidulans PpoC with known P450 enzymes.
The predicted CYP450 domains from different Ppo enzymes: A. nidulans PpoC, A. fumigatus PpoC; A. nidulans PpoA [all as given in (A)] and A. fumigatus PpoA (AfPpoA; RefSeq XP_751750) are
aligned with known P450 enzymes. CYP450119, PDB code 1F4T:B; CYP450cin, PDB code 1T2B:B; CYP450130, PDB code 2UUQ:A; CYP450nor, PDB code 1JFB:A; CYP4503A4, PDB code 1TQN:A
and CYP4502C9, PDB code 1R9O:A. Amino acids matching the P450 consensus sequence according to the PROSITE database are shown on a grey background. Additionally the cysteine residue
responsible for the co-ordination of haem iron is shown on a black background. The sequences were aligned using the ClustalW algorithm.
appeared to be weak, a fact that might indicate that haem was
partially lost during the purification procedure.
Spectral analysis and prosthetic groups
The UV–visible absorption spectrum of purified PpoC is shown
in Figure 3(B). The highest absorption maxima were at 280 nm
(probably due to aromatic amino acid site chains; results not
shown) and 403 nm (γ , the Soret band) indicating the presence of
haem. Additionally two weaker maxima at approx. 530 nm and
630 nm were detected. However, the haem absorption spectrum
Identification of products formed by recombinant PpoC
As we found purified PpoC to be highly unstable under our assay
conditions, we decided to investigate most of the biochemical
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Table 1 Oxygenation products of A. nidulans PpoC
Products were identified using RP–HPLC/tandem MS analysis and by detection of characteristic
signals (8-hydroperoxide derivative with m/z 157 (cleavage between C-8 and C-9) and
10-hydroperoxide derivative with m/z 183 (cleavage between C-10 and C-11, as also shown in
Supplementary Figure S2). Volatile products were extracted by SPME and identified by GC/MS
analysis using the NIST database (Figure 6).
Hydroperoxide
products
Hydroperoxide
derived products
Volatile
products
Substrate
ꢀ9Z
18:1
8-HPOME
10-HPOME
8-HPODE
10-HPODE
8-HOME
10-HOME
8-HODE
10-HODE
12,13-Ep-10-HOME
10-ODA
10-KODE
1-octanol, 2-undecenal
ꢀ9Z,12Z
18:2
18:3
1-octen-3-ol
2-octen-1-ol
2-octenal
3-octanone
ꢀ9Z,12Z,15Z
8-HPOTE
8-HOTrE
10-HPOTE
10-HOTrE
2-decenedioic acid
Octadiene isomers
We next examined which products are formed by PpoC
from other fatty acid substrates. We were particularly interested
in products formed from fatty acids that occur endogeno-
usly in A. nidulans, such as other unsaturated C-18 fatty acids.
As expected, 18:1ꢀ9Z and 18:3ꢀ9Z,12Z,15Z were mainly converted into
their corresponding 10-hydroxy/10-hydroperoxy and 8-hydroxy/
ꢀ9Z
8-hydroperoxy derivatives (Table 1). Interestingly, 18:1
was predominantly oxidized at the C-8, yielding 8-HOME/8-
HPOME (HPOME is hydroperoxyoctadecenoic acid), whereas
Figure 3 SDS/PAGE at different stages of purification and UV–visible
absorption of purified PpoC
ꢀ9Z,12Z,15Z
18:3
was mainly oxidized at the C-10, yielding 10-
HOTrE/10-HPOTrE (where HOTrE is hydroxyoctadecatrienoic
acid and HPOTrE is hydroxyoctadecatrienoic acid).
(A)AliquotsaftereachpurificationstepwereanalysedbySDS/PAGE(8%gels).Lane1,crudecell
extract; lane 2, after Source 30Q purification; lane 3, after Sephadex S200 (60/20) purification;
and lane 4, after MonoQ (10/10) purification. MW, molecular mass markers in kDa. (B) A
spectrum of the native enzyme in 50 mM Hepes, pH 7.4, is shown. The UV–visible spectrum
of the purified enzyme showed absorption maxima at 403 nm (γ , Soret band) indicating the
presence of haem. Additionally two weaker maxima at approximately 530 nm and 630 nm are
indicated by arrows.
Additionally, we observed that during incubations of
18:3ꢀ9Z,12Z,15Z with PpoC a polar product was formed that eluted at
3.8 min from RP-HPLC. The corresponding tandem MS spectrum
(m/z 199) of this compound is shown in Supplementary Figure
S1(F) and shows the following signals: m/z 181 (M−–H2O), 155
(M−–44, probably loss of CO2), 139 (M−–60) and 137 (M−–
62). Due to similarity to previously reported tandem MS spectra
of 10-ODA [15] this product was tentatively assigned as (2E)-
decendioic acid.
properties of PpoC by using bacterial crude extract of PpoC-
expressing E. coli cells and using cells expressing the empty
vector as controls.
Bacterial crude extract of PpoC-expressing cells was incubated
Identification of volatile products
ꢀ9Z,12Z
with 18:2
for 30 min on a shaker. After extraction of
ꢀ9Z,12Z
fatty acids and their derivatives we analysed product formation
with RP-HPLC/tandem MS. We detected the formation of
10-HPODE (m/z 311; retention time 22.1 min), 8/10-HODE
(m/z 295; retention time 19.5 min/20.9 min), 10-KODE (where
KODE is keto-octadecadienoic acid; m/z 293; retention time
22.6 min) and 10-ODA (where ODA is octadecynoic acid; m/z
183; retention time 3.8 min) (Figures 4A–4D). Additionally
we found the formation of 12,13-epoxy-10-HOME (m/z 311;
retention time 9.9 min; Figure 4A). The corresponding spectra
of 12,13-epoxy-10-HOME, 8-HODE, 10-HODE, 10-KODE and
10-ODA are shown in Supplementary Figure S1(A–E), available
at http://www.BiochemJ.org/bj/425/bj4250553add.htm, and are
in agreement with previously reported spectra [15,16]. Bacterial
10-ODA, formed during the incubation of PpoC and 18:2
,
has been described as a cleavage product of 10-HPODE [22]. It
is known that this compound is formed from β-scission of the
hydroperoxide, a reaction which is catalysed by metals [22] and
by still unidentified fungal lyases [23]. As additional cleavage
products volatile octenol isomers have also been reported [23–
25]. To elucidate whether similar volatile compounds are also
ꢀ9Z,12Z
formed during the reaction of PpoC with 18:1ꢀ9Z, 18:2
and 18:3ꢀ9Z,12Z,15Z, we analysed the gas phase above the reaction
mixture using SPME and GC/MS analysis. Using the NIST
(National Institute of Standards and Technology) library and
spectra reported previously [26,27], we were able to identify most
of these compounds.
We found that during the reaction of PpoC and 18:1
ꢀ9Z
crude extract of empty vector controls did not metabolize
two
ꢀ9Z,12Z
18:2
to any of these products. Comparing the relative
volatile compounds were formed that could be identified as 1-
octanol and 2-undecenal respectively (Figure 5A and Table 1). The
latter compound has been described as an odorant formed from
8-HPOME [28]. Volatile compounds that were formed during the
were identified as 1-octen-3-ol,
2-octen-1-ol, 3-octenone and 2-octenal respectively (Figure 5B
amounts of different products formed during the incubation, we
found that 10-HODE and 10-HPODE appeared to be the main
ꢀ9Z,12Z
products that are formed from 18:2
by PpoC (Figure 4E),
ꢀ9Z,12Z
whereas 12,13-epoxy-10-HOME and 10-ODA accumulated to
lesser extends.
reaction of PpoC and 18:2
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ꢀ9Z,12Z
Figure 4 RP–HPLC/MS analysis of metabolites formed from 18:2
by recombinant PpoC
ꢀ9Z,12Z
RP–HPLC/MS analysis after 30 min incubation of 18:2
with PpoC in 50 mM Tris/HCl buffer, pH 8.0, and extractive isolation. Shown are: (A) 10-HPODE and 12,13-epoxy-10-HOME (m/z
311; full scan); (B) 8-HODE and 10-HODE (m/z 295; full scan); (C) 10-KODE (m/z 293; full scan); and (D) 10-ODA (m/z 183; full scan). As reported previously [15], 10-HPODE decomposed in the
mass spectrometer into 10-ODA which co-elutes with 10-HPODE at 20.9 min, whereas pre-formed 10-ODA elutes at 3.8. (E) Relative amounts of products formed during the incubation. Results are
+
means S.D. calculated from three different experiments. Ret. time, retention time; Ep, epoxy.
−
and Table 1). Furthermore, we also detected the formation
TMPD (N,N,Nꢀ,Nꢀ-tetramethyl-p-phenylenediamine) as a co-
substrate. When purified PpoC was incubated with 100 μM
TMPD and 100 μM 18:2ꢀ9Z,12Z, we were able to follow the
characteristic increase at 611 nm for several minutes. A typical
progress curve is shown in Supplementary Figure S2 (available
at http://www.BiochemJ.org/bj/425/bj4250553add.htm) and is
indicative for the peroxidase activity of PpoC.
We analysed the pH optimum by measuring the initial rates
of oxygen consumption for the reaction of PpoC crude extract
with 100 μM 18:2ꢀ9Z,12Z. The initial rate of oxygen consumption
reached a maximum between pH 7.4 and 8.0. pH values >8.0 led
to an rapid decrease in enzymatic activity.
of two volatile products during incubations of PpoC with
ꢀ9Z,12Z,15Z
18:3
(Figure 5C). These products could not be reliably
identified by using the NIST library. However, incubation with
ꢀ9Z,12Z,15Z
[13C18]18:3
indicated that these products are octadiene-
isomers (results not shown).
As these volatile compounds were also detected during
incubation of 10-hydroperoxy derivatives with empty vector
controls, we suggest that these cleavage reactions were mainly
caused by non-enzymatic reactions, or they may at least be
catalysed independently from PpoC. Similar observations have
been reported previously [15].
Peroxidase activity and pH optimum
Kinetic properties
In order to determine whether the high amounts of 10-HODE
(Figure 4E) can be formed by PpoC within an enzymatic
peroxidase reduction, we performed a peroxidase assay using
Kinetic parameters were determined by measuring the initial rates
of oxygen consumption for the reaction with different substrate
concentrations using the Clark-type O2 electrode. The reaction
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Figure 5 GC/MS analysis of volatile compounds of PpoC and different fatty acid substrates
Volatile compounds formed during the incubation of PpoC with (A) 18:1ꢀ9Z , (B) 18:2ꢀ9Z,12Z and (C) 18:3ꢀ9Z,12Z,15Z . The products were identified with SPME, GC/MS and by using the NIST library and
ꢀ9Z
ꢀ9Z,12Z
spectra reported previously [26,27]. Incubations of PpoC with 18:1 led to the production of 1-octanol and 2-undecenal, whereas during incubations with 18:2
1-octen-3-ol, 2-octen-1-ol,
ꢀ9Z,12Z,15Z
2-octenal and 3-octenon were predominantly formed. 18:3
was converted by PpoC into two volatile products which could not be identified using the NIST library. By experiments with
ꢀ9Z,12Z,15Z
[
13C]-labelled 18:3
these products were tentatively assigned as octadiene isomers.
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Figure 6 Conversion of 8-HPODE and 10-HPODE into epoxy alcohols by PpoC
The results from RP-HPLC/MS analysis after incubation of (A and C) 10-HPODE or (B and D) 8-HPODE with (A and B) empty vector controls or (C and D) recombinant PpoC bacterial crude extract
after 40 min incubation at room temperature. Ret. time, retention time; Ep, epoxy.
Table 2 Kinetic parameters for different substrates
and detected the formation of the corresponding 12,13-epoxy-
8-HOME. The individual RP-HPLC/MS chromatograms of
both experiments are shown in Figure 6. The corresponding
tandem MS spectra of both epoxy alcohol derivatives, which
are in agreement with those published previously [16], are
shown in Supplementary Figure S3(A) and S3(B) (available at
http://www.BiochemJ.org/bj/425/bj4250553add.htm). It should
be noted, however, that we also detected small amounts of both
epoxy alcohols when using cell lysates of empty vector controls
(Figures 6A and 6B). As the detected amounts in control assays
were much lower compared with incubations with PpoC, we
conclude that the major part of the detected epoxy alcohols are
formed by an enzymatic reaction as proposed previously in [16],
but by the peroxidase/dioxygenase domain of Ppo enzymes, rather
than by the isomerase domain.
Kinetic analysis was performed with a Clark-type O2 electrode. Initial time-dependent rates of
oxygen consumption were measured with different substrate concentrations and Km values were
determined by fitting seven data points to the Michaelis–Menten equation using the software
Origin6.0. Topreventmycelformationthesodiumsaltsofeachfattyacidwereusedasasubstrate
+
for the kinetic analysis. Results are means S.D. calculated from three different experiments.
−
Substrate
Km (μM)
ꢀ9Z
+
18:1
18:2
18:3
13.1 1.7
−
ꢀ9Z,12Z
+
3.6 0.97
−
ꢀ9Z,12Z,15Z
+
7.9 1.6
−
was performed with PpoC crude extract; thus we were able to
determine kinetic parameters that are independent of the enzyme
To investigate the origin of both oxygen atoms in the epoxy
ꢀ9Z
ꢀ9Z,12Z
concentration, such as the Km. We used sodium salts of 18:1
,
alcohol derivatives, we incubated 18:2
with bacterial crude
18:2ꢀ9Z,12Z and 18:3ꢀ9Z,12Z,15Z as substrates and measured the initial
rate of oxygen consumption for the different concentrations in
triplicate. The reaction was performed at pH 7.9 (pH optimum)
and was started by the addition of substrate to the oxygen-
saturated stirring solution. The Km values are summarized in
Table 2 and suggest that 18:2ꢀ9Z,12Z may be the preferred substrate.
lysate under an atmosphere enriched in 18O2. The MS spectrum
of the epoxy alcohol product is shown in Figure 7 and displays
two major signals: m/z 311 corresponding to [16O]-labelled 12,13-
epoxy-10-HOME and m/z 315 for [18O]-labelled 12,13-epoxy-10-
HOME. As no abundant signal with m/z 313 could be detected,
we conclude that the epoxy and the hydroxy group contain either
two atoms of 16O or two atoms of 18O. This indicates that both
oxygen atoms of the epoxy alcohol group are derived from the
same substrate molecule, the fatty acid hydroperoxide.
Investigating the epoxy alcohol synthase activity of Ppo enzymes
We next examined a potential epoxy alcohol synthase activity
of the related PpoA enzyme (purified to homogeneity). Therefore
we incubated 10-HPODE with different concentrations of
PpoA. In Figure 8 the relative amounts of 12,13-epoxy-10-
HOME, 10-ODA and 10-HPODE are plotted against PpoA
concentrations. We observed a linear correlation between the
PpoA concentrations used and product formation or substrate
consumption respectively. Thus we suggest PpoA exhibits
We detected the formation of 12,13-epoxy-10-HOME (approx.
3% of all products formed) when PpoC was incubated with
18:2ꢀ9Z,12Z. It has been suggested that this product is formed
from 10-HPODE by A. fumigatus PpoC [16]. To clarify
the enzymatic formation of epoxy alcohols by PpoC, we
incubated 10-HPODE with PpoC-containing bacterial crude
extract. Clearly, formation of 12,13-epoxy-10-HOME could
be detected. Next, we also incubated 8-HPODE with PpoC
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ꢀ9Z,12Z
Figure 7 MS analysis of 12,13-epoxy-10-HOME formed from 18:2
PpoC under 18O2
by
ꢀ9Z,12Z
RP-HPLC/MS analysis of the 12,13-epoxy-10-HOME obtained after incubation of 18:2
with recombinant PpoC bacterial crude extract under an enriched 18O2 atmosphere.
Figure 9 Relative amounts of 8-HODE formed by wild-type PpoC and PpoA
compared with their corresponding mutants
(A) Relative amounts of 8-HODE formed by recombinant wild-type PpoC (PpoC_WT), PpoC with
the L385A substitution (PpoC_L385A) and PpoC with the L385V substitution (PpoC_L385V)
bacterial crude extract in 50mM Tris/HCl, pH 8.0. (B) Relative amounts of 8-HODE formed
by purified wild-type PpoA (PpoA_WT), PpoA with the V328A substitution (PpoC_V328A)
and PpoA with theV328L substitution (PpoC_V328L) in 50 mM Hepes, pH 7.4. Results are
Figure 8 Conversion of 10-HPODE into 12,13-epoxy-10-HOME and 10-ODA
as a function of PpoA concentration
+
means S.D. calculated from three different experiments.
−
The reaction system contained 500 μM 10-HPODE and the indicated concentration of purified
PpoA in 50 μM Hepes, pH 7.4. The reaction was terminated after 40 min. Ep, epoxy.
PpoC (Figure 9A) we observed that replacement of Leu385 with
the smaller valine residue enhanced the formation of 8-HODE by
2–3-fold compared with the wild-type enzyme. Reducing the size
to a smaller residue, alanine, enhanced the formation of 8-HODE
by 3–4-fold as judged by RP-HPLC/MS analysis.
epoxy alcohol synthase activity with 10-HPODE as a substrate,
paralleling the results obtained with PpoC (see above). As
reported previously 8-HPODE is predominantly converted into
5,8-DiHODE by the 8-hydroperoxide isomerase P450 domain of
PpoA [14]. The tandem MS spectrum of this product is shown in
Supplementary Figure S3(C). Additionally, we detected traces of
12,13-epoxy-8-HOME formed by PpoA from 8-HPODE.
We previously reported that we were unable to detect formation
ꢀ9Z,12Z
of any 10-HODE/10-HPODE during incubations of 18:2
and PpoA by RP-HPLC. Using an improved RP-HPLC/MS
approach we now observed the formation of 10-HODE/10-
HPODE and especially found that approx. 30% of the formed
hydroxides were 10-HODE (Figure 9B). The replacement of
Val328 in PpoA with an alanine residue reduced the formation
of 8-HODE and increased the production of 10-HODE, whereas
replacement with leucine only led to a modest increase in 8-HODE
formation compared with the wild-type enzyme (Figure 9B).
Site-directed mutagenesis of determinants that control site of
dioxygenation
Val385 is a conserved residue in the peroxidase/dioxygenase
domain of PpoC, PpoA (Val328), 7,8-linoleate diol synthase
(Val330) and PGHS (Val349 in sheep PGHS-1). In the latter,
Val349 is known to be important for substrate positioning and
the dioxygenase reaction [29]. To compare the oxygenation
mechanisms of A. nidulans PpoA and PpoC, we mutated the
corresponding amino acids in both enzymes; Leu385 of PpoC was
replaced with either valine or alanine, whereas Val328 of PpoA
was replaced with either alanine or leucine. Crude bacterial extract
from wild-type or mutated PpoC and purified wild-type or mutated
PpoA was incubated with 18:2ꢀ9Z,12Z. The relative amounts of
hydroxy products formed were determined by RP-HPLC/MS and
the results are visualized as column diagrams (Figure 9). For
Site-directed mutagenesis of the catalytic tyrosine residue
As shown in Figure 1(A) Tyr427 of A. nidulans PpoC is conserved
in the enzymes analysed. Previous mutagenesis studies have
indicated that this tyrosine residue is important for catalytic
activity [14,29–31] presumably by generating a tyrosyl radical
[32,33]. Replacement of PpoC Tyr427 with phenylalanine (Y427F)
led to an inactive enzyme that showed no detectable dioxygenase
activity when incubated with 18:2ꢀ9Z,12Z. The expression
of the PpoC(Y427F) mutant was confirmed by SDS/PAGE
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PpoC is a fusion protein with one active haem
563
analysis (see Supplementary Figure 4 available at available at
http://www.BiochemJ.org/bj/425/bj4250553add.htm).
N-terminal region of the enzymes and a cytochrome P450 region
in the C-terminal part. Recently, we showed that the bifunctional
activity of PpoA is indeed related to the presence of both
domains: within the peroxidase/dioxygenase domain of PpoA
the (8R)-dioxygenase reaction takes place, whereas in the P450
domain the (8R)-hydroperoxide isomerization is accomplished
[14]. Although PpoC is predicted to be a fusion protein with
a similar C-terminal P450 domain, we found on the basis of
sequence alignments, that the crucial cysteine residue is not
conserved in its sequence (Figure 2B). In P450 enzymes the
corresponding residue acts as the fifth haem iron ligand and
mutation of this cysteine residue leads to an inactive enzyme
[37]. Therefore it appeared doubtful that this domain is functional
in PpoC and has catalytic activity. To analyse the influence of this
changed domain structure we isolated PpoC cDNA, expressed the
enzyme in E. coli and investigated its biochemical properties.
Our results indicate that both fusion proteins, PpoA and PpoC,
have many properties in common, but there are also essential
differences that are most likely caused by the differences in
their C-terminal P450 domain. PpoA and PpoC resemble 7,8-
linoleate diol synthase tetrameric haem proteins that catalyse the
Site-directed mutagenesis of the predicted P450 haem-thiolate
domain
As shown in Figure 2(B) we found that the predicted P450 domain
of PpoC does not contain the conserved cysteine residue that
is known to be the fifth haem iron ligand in P450 enzymes
[34]. Substitution of this cysteine residue (Cys1006) in PpoA for
an alanine residue led to an enzyme that showed dioxygenase
activity, but no detectable isomerase activity, presumably through
distortion of the P450 haem moiety [14]. As PpoC contains a
glycine residue instead of a cysteine residue at this particular
position, we hypothesized that replacement of Gly1039 with a
cysteine residue may reconstitute the haem-binding moiety and
thereby lead to an enzyme with isomerase activity, as it is known
for PpoA. However, we did not detect formation of any DiHODE
ꢀ9Z,12Z
derivative when PpoC(G1039C) was incubated with 18:2
.
Moreover, the enzyme showed the same catalytic activity as the
wild-type enzyme. Moreover, we also incubated PpoC(G1039C)
with 8-HPODE and upon this incubation with 8-HPODE no
DiHODE was detectable.
oxidation of 18:2ꢀ9Z,12Z. Whereas PpoA predominantly oxidizes
ꢀ9Z,12Z
18:2
at the C-8, yielding 8-HPODE, PpoC mainly performs
oxygenation at the C-10 forming 10-HPODE. These dioxygenase
reactions are both catalysed within the peroxidase/dioxygenase
domain of the fusion proteins (Figure 9). Sequence alignments
showed that PpoA and PpoC contain the conserved tyrosine
residue within their haem peroxidase/dioxygenase domain (Tyr374
in PpoA and Tyr427 in PpoC) that is also found in 7,8-
linoleate diol synthase (Tyr376), sheep PGHS-1 (Tyr385) and sheep
PGHS-2 (Tyr370). Previous studies showed that dioxygenase
activity was reduced or even abolished in these enzymes
when this tyrosine residue was mutated [14,29,30]. As also
reported previously for PpoA, we now provide evidence that
replacement of this tyrosine residue with a phenylalanine
residue also abolished the dioxygenase activity of PpoC. Thus
Radical spin-trapping adducts
In order to investigate the formation of carbon-centred fatty acid
radicals during the reaction of PpoC and 18:2
ꢀ9Z,12Z
we used the
method of Koshiishi and co-workers to scavenge the hypothetical
radical with the radical scavenger CmꢀP (3-carbamoyl-2,2,5,5-
tetramethyl-3-pyrroline-N-oxyl) [35,36]. Recently, we adapted
this approach for the PpoA/linoleate system and found the
formation of two radical spin-trapping adducts, which we
tentatively assigned as the trapped C-8 and C-10 radical adducts
[14]; this finding that now proves to be consistent with our
observation that PpoA is also able to form 10-HODE/10-HPODE
(Figure 9B). To test the existence of radical intermediates in
the PpoC/linoleate system we performed the trapping reaction
with CmꢀP using PpoC and 18:2ꢀ9Z,12Z. Linoleate allyl radical
CmꢀP adducts of m/z 463 were identified using RP-HPLC/MS.
Two distinct peaks were be detected: a major peak eluting at
28.8 min and a smaller one at 30.8 min [14]. We suggest from this
result that the major peak corresponds to the trapped C-8 radical
adduct, whereas the smaller peak may correspond to the C-10
radical adduct, in analogy with the PpoA/linoleate system. The
MS spectra of both trapping adducts of the PpoC/linoleate system
showed a characteristic fragment with m/z 185; this fragment
arose from the reduced form of CmꢀP through heterolysis of
the C–O–N bond [36]. We also detected trapping adducts with
m/z 479 that may correspond to nitroxyl radical–lipid epoxy allyl
adducts [36].
we conclude that PpoC also uses a tyrosyl radical for the
ꢀ9Z,12Z
abstraction of the hydrogen from the C-8 of 18:2
and
forms a carbon-centred radical at this position. The unpaired
electron is delocalized and can migrate from the C-8 to the
C-10 by shifting the C-9–C-10 double bond to C-8–C-9. The
formation of carbon-centred substrate radicals at the C-8 and
C-10 was also supported by experiments in which we trapped
these radicals using the radical scavenger CmꢀP as reported
before for other enzymes [14,36].
The results thus far led us to ask what the amino acid
determinants that directed the oxygenation at C-8 or C-10
respectively were. Recently, Garscha et al. [16] reported the
recombinant expression of a PpoC from A. fumigatus that
catalyses a similar reaction to PpoC. By replacing Leu384 and
Val388 with larger and smaller amino acids they could show
that these residues act as determinants that direct oxygenation
ꢀ9Z,12Z
either at C-8 or C-10 of 18:2
[16]. As similar substitutions
of the equivalent residue in G. graminis 7,8-linoleate diol
synthase (Val330) showed a related effect, they developed a
general model for the oxygenation by fungal 8-dioxygenases
DISCUSSION
The present study aimed to characterize A. nidulans PpoC, a
putative fatty acid dioxygenase, and to compare it with A. nidulans
PpoA, another member of the A. nidulans Ppo-family. The Ppo
enzyme family in A. nidulans consists of three dioxygenase
proteins, PpoA, PpoB and PpoC. Genetic evidence indicated Ppo
products were involved in the regulation of the developmental
life cycle of A. nidulans [13]. Interestingly, an NCBI-conserved-
domain search predicted that all three protein sequences seem
to be fusion proteins because of the presence of two conserved
and 10-dioxygenases respectively. This model proposed that
ꢀ9Z,12Z
Leu384 shields the C-8 of 18:2
in the active site of (10R)-
dioxygenase or PpoC [16]. To gain further insights into the
oxygenation mechanisms, we also mutated the related Val328 (in
PpoA) and Leu385 (in PpoC) in the Ppo enzymes from A. nidulans.
This experiment enabled, for the first time, the direct comparison
of two dioxygenases present in the same species, with highly
similar amino acid sequences, but different product specificities.
Replacement of PpoC Leu385 with an alanine residue or a valine
domains:
a haem peroxidase/dioxygenase domain in the
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564
F. Brodhun and others
residue gave similar effects as reported previously [16] that may
also indicate that Leu385 shields the C-8 of 18:2ꢀ9Z,12Z. However,
the replacement of Val328 in PpoA with a leucine residue or
an alanine residue conversely indicated that this residue might
shield C-10 rather than C-8. This effect may be explained by a
different positioning, or even by a different orientation, of the
fatty acid in the active site of both enzymes, leading to the
conclusion that our results, in contrast with those of Garscha
et al. [16], indicate that there either is no general model for
oxygenation by these fungal dioxygenases or that the role of the
mutated amino acids is as yet unclear. Clearly a crystal structure
is needed to further address this question. A further explanation
may be that the isomerase activity of recombinant 7,8-linoleate
diol synthase is very weak and, therefore, 8-HODE/8-HPODE can
accumulate. This accumulation might influence the equilibrium in
a different way compared with PpoA, where almost all 8-HPODE
is immediately converted into 5,8-DiHODE.
This was further confirmed by incubations of PpoA with 10-
HPODE (Figure 8). In contrast with 8-HPODE this substrate was
not converted into a dihydroxy fatty acid but rather transformed
into 12,13-epoxy-10-HOME. The finding that the amount of
12,13-epoxy-10-HOME showed a linear dependency on the
concentration of the PpoA used in the experiment additionally
indicated an enzymatic reaction. However, as we also found
production of epoxy alcohols in negative controls, in minor
amounts, we suggest that epoxy alcohol derivatives can also be
formed non-enzymatically from 10-HPODE and 8-HPODE under
the used assay conditions.
As the isomerase activity of the bifunctional PpoA is located
in the P450 domain and PpoC seems to contain an inactive P450
domain, due to the lack of a highly conserved cysteine residue,
we tried to obtain a bifunctional PpoC by mutation analysis.
To reconstitute the P450 haem-binding moiety and to obtain a
functional 8-hydroperoxide isomerase domain we replaced Gly1039
ꢀ9Z,12Z
We investigated further the downstream products of the
with a cysteine residue and incubated the enzyme with 18:2
dioxygenase reaction of PpoC. The 10-HPODE that was
and 8-HPODE. No functional 8-hydroperoxide isomerase activity
was retrieved. This might indicate that several more residues in
the PpoC sequence are important for correct binding of haem and
a correct folding of the P450 domain. However, for conclusive
identification of further essential amino acids which constitute
a functional P450 motif a crystal structure will be required. A
domain-switching approach may also be suitable to address this
question in the future, but the lack of information about the crystal
structure of the enzyme is also hindering further investigations in
that direction.
ꢀ9Z,12Z
formed during incubations of PpoC and 18:2
was mainly
converted into 10-HODE, which was formed by an enzymatic
peroxidase reaction of PpoC within the C-terminal haem
peroxidase/dioxygenase domain. Another product of this domain
was 10-KODE. However, 10-HPODE appeared to be unstable on
its own and 10-ODA and volatile compounds such as 2-octen-1-ol,
2-octenal, 3-octanone and 1-octen-3-ol were formed, most likely
by chemical fragmentation from this PpoC product. The latter
ꢀ9Z,12Z
compound has also been reported to be formed from 18:2
by homogenates of Lentinus decadetes [38], Psalliota bispora
[23–25] and Tricholoma matsutake [39]. Although the reaction
was discovered 25 years ago, the enzyme involved in formation
of these compounds, known for the mushroom flavour, was never
identified from either of these sources. Interestingly, at first an
independent metabolic reaction leading to formation of the same
volatiles 2-octen-1-ol and 1-octen-3-ol was described for the moss
Physcomitrella patens [27]. Here a bifunctional lipoxygenase uses
the C-20 fatty acid arachidonic acid and the C-8 alcohols are
formed via the (12S)-hydroperoxide of this fatty acid [40].
As a further product 12,13-epoxy-10-HOME was formed
during the reaction of PpoC and 18:2ꢀ9Z,12Z. This product has
also been reported to be formed by PpoC from A. fumigatus [16].
It was suggested that this enzyme has epoxy alcohol synthase
activity and specifically converts 10-HPODE into 12,13-epoxy-
10-HOME in analogy with the isomerization reaction that is
performed with 8-HPODE by 7,8-linoleate diol synthase [16].
Indeed, by incubating purified 10-HPODE with PpoC bacterial
crude extract we confirmed the enzymatic conversion of this
substrate into 12,13-epoxy-10-HOME. Thus we showed for the
first time that 10-HPODE is in fact the precursor of 12,13-epoxy-
10-HOME. Additionally, we found that PpoC can also convert
8-HPODE into 12,13-epoxy-8-HOME but not into 5,8-DiHODE.
This finding underlines that a functional P450 domain, as in case
of PpoA, is essential for an isomerization reaction but not for
epoxy alcohol synthase activity.
Taken together, our results show that PpoA and PpoC are
fusion proteins that employ similar mechanisms to catalyse the
formation of 8- and 10-hydroperoxy fatty acids. In this context
the leucine/valine residues that have been reported recently for
A. fumigatus PpoC and G. graminis 7,8-linoleate diol synthase
ꢀ9Z,12Z
seem also to determine the position of oxygenation of 18:2
,
i.e. C-8 and C-10, of dioxygenases in A. nidulans, but a general
mechanism as to how these amino acid residues influence the
site of oxygenation remains unclear. PpoC and PpoA both have
an epoxy alcohol synthase side activity and can convert 8-
HPODE and 10-HPODE into the corresponding epoxy alcohols.
However, this is employed by the peroxidase/dioxygenase domain
of these enzymes. This is supported further by the fact that PpoA
converts 8-HPODE mainly into 5,8-DiHODE, whereas PpoC
cannot perform a similar reaction, probably due to a degenerated
P450 haem-thiolate domain.
AUTHOR CONTRIBUTION
Florian Brodhun and Ivo Feussner designed the research. Florian Brodhun, Stefan
Schneider,CorneliaGo¨belandEllenHornungperformedtheexperiments.FlorianBrodhun,
Stefan Schneider, Cornelia Go¨bel, Ellen Hornung and Ivo Feussner analysed the data.
Florian Brodhun, Cornelia Go¨bel, Ellen Hornung and Ivo Feussner wrote the manuscript.
ACKNOWLEDGEMENTS
As 7,8-linoleate diol synthase displays its bifunctionality
ꢀ9Z,12Z
We thank the following colleagues at Georg-August-University, Go¨ttingen, Germany:
Sabine Freitag and Theres Riemekasten for expert technical assistance; Nambirajan
Govindarajan for initial cloning experiments; and Dr I. Heilmann for critical reading of the
manuscript prior to submission. Assignment of PpoC and comments on the PpoC P450
sequence by Dr D.R. Nelson (Department of Molecular Sciences University of Tennessee,
Memphis, TN, U.S.A.) are also gratefully acknowledged.
by conversion of 18:2
into 8-HPODE, followed by
the isomerization yielding 7,8-DiHODE, we next investigated
whether purified PpoA also harboured a similar epoxy alcohol
synthase side activity, besides the described dioxygenase and
isomerase activity. Incubations of purified PpoA and 8-HPODE
led mainly to the formation of 5,8-DiHODE by the 8-
hydroperoxide isomerase P450 domain as reported. As we also
detected low amounts (< 1% of total reaction products) of 12,13-
epoxy-8-HOME during these incubations we concluded that, like
PpoC, PpoA also harbours an epoxy alcohol synthase side activity.
FUNDING
This work was supported by the German Research Foundation in the framework of the
International Research Training Group 1422.
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PpoC is a fusion protein with one active haem
565
22 Labeque, R. and Marnett, L. J. (1987) 10-Hydroperoxy-8,12-octadecadienoic acid. A
REFERENCES
diagnostic probe of alkoxyl radical generation in metal–hydroperoxide reactions. J. Am.
Chem. Soc. 109, 2828–2829
1
Mazur, P., Meyers, H. V., Nakanishi, K., El-Zayat, A. A. E. and Champe, S. P. (1990)
23 Wurzenberger, M. and Grosch, W. (1984) The formation of 1-octen-3-ol from the
10-hydroperoxide isomer of linoleic acid by a hydroperoxide lyase in mushrooms
(Psalliota bispora). Biochem. Biophys. Acta 794, 25–30
Structural elucidation of sporogenic fatty acid metabolites from Aspergillus nidulans.
Tetrahedron Lett. 31, 3837–3840
Mazur, P., Nakanishi, K., Elzayat, A. A. E. and Champe, S. P. (1991) Structure and
synthesis of sporogenic Psi factors from Aspergillus nidulans. J. Chem. Soc. Chem.
Commun. 20, 1486–1487
2
24 Wurzenberger, M. and Grosch, W. (1984) Origin of the oxygen in the products of the
enzymatic cleavage reaction of linoleic acid to 1-octen-3-ol and 10-oxo-trans-8-decenoic
acid in mushrooms (Psalliota bispora). Biochem. Biophys. Acta 794, 18–24
25 Wurzenberger, M. and Grosch, W. (1984) Stereochemistry of the cleavage of the
10-hydroperoxide isomer of linoleic acid to 1-octen-3-ol by a hydroperoxide lyase from
mushrooms (Psalliota bispora). Biochem. Biophys. Acta 795, 163–165
26 Stumpe, M., Bode, J., Go¨bel, C., Wichard, T., Schaaf, A., Frank, W., Frank, M., Reski, R.,
Pohnert, G. and Feussner, I. (2006) Biosynthesis of C9-aldehydes in the moss
Physcomitrella patens. Biochim. Biophys. Acta 1761, 301–312
27 Wichard, T., Go¨bel, C., Feussner, I. and Pohnert, G. (2005) Unprecedented
lipoxygenase/hydroperoxide lyase pathways in the moss Physcomitrella patens. Angew.
Chem. Int. Ed. Engl. 44, 158–161
28 Grosch, W. (1987) Reactions of hydroperoxides-products of low molecular weight. In
Autoxidation of Unsaturated Lipids (Chan, H. W.-S., ed.), pp. 95–139, Academic Press,
London
3
4
5
6
7
8
9
Champe, S. P. and el-Zayat, A. A. (1989) Isolation of a sexual sporulation hormone from
Aspergillus nidulans. J. Bacteriol. 171, 3982–3988
Champe, S. P., Rao, P. and Chang, A. (1987) An endogenous inducer of sexual
development in Aspergillus nidulans. J. Gen. Microbiol. 133, 1383–1387
Calvo, A. M., Gardner, H. W. and Keller, N. P. (2001) Genetic connection between fatty acid
metabolism and sporulation in Aspergillus nidulans. J. Biol. Chem. 276, 25766–25774
Calvo, A. M., Wilson, R. A., Bok, J. W. and Keller, N. P. (2002) Relationship between
secondary metabolism and fungal development. Microbiol. Mol. Biol. Rev. 66, 447–459
Tsitsigiannis, D. I. and Keller, N. P. (2007) Oxylipins as developmental and host-fungal
communication signals. Trends Microbiol. 15, 109–118
Andreou, A., Brodhun, F. and Feussner, I. (2009) Biosynthesis of oxylipins in
non-mammals. Prog. Lipid Res. 48, 148–170
Tsitsigiannis, D. I., Zarnowski, R. and Keller, N. P. (2004) The lipid body protein, PpoA,
coordinates sexual and asexual sporulation in Aspergillus nidulans. J. Biol. Chem. 279,
11344–11353
29 Thuresson, E. D., Lakkides, K. M., Rieke, C. J., Sun, Y., Wingerd, B. A., Micielli, R.,
Mulichak, A. M., Malkowski, M. G., Garavito, R. M. and Smith, W. L. (2001)
Prostaglandin endoperoxide H synthase-1: the functions of cyclooxygenase active site
residues in the binding, positioning, and oxygenation of arachidonic acid. J. Biol. Chem.
276, 10347–10357
10 Tsitsigiannis, D. I., Bok, J. W., Andes, D., Nielsen, K. F., Frisvad, J. C. and Keller, N. P.
(2005) Aspergillus cyclooxygenase-like enzymes are associated with prostaglandin
production and virulence. Infect. Immun. 73, 4548–4559
30 Garscha, U. and Oliw, E. H. (2008) Critical amino acids for the 8(R)-dioxygenase activity
of linoleate diol synthase. A comparison with cyclooxygenases. FEBS Lett. 582,
3547–3551
11 Tsitsigiannis, D. I., Kowieski, T. M., Zarnowski, R. and Keller, N. P. (2005) Three putative
oxylipin biosynthetic genes integrate sexual and asexual development in Aspergillus
nidulans. Microbiolology 151, 1809–1821
31 Koeduka, T., Matsui, K., Akakabe, Y. and Kajiwara, T. (2002) Catalytic properties of rice
α-oxygenase. A comparison with mammalian prostaglandin H synthases. J. Biol. Chem.
277, 22648–22655
12 Tsitsigiannis, D. I. and Keller, N. P. (2006) Oxylipins act as determinants of natural
product biosynthesis and seed colonization in Aspergillus nidulans. Mol. Microbiol. 59,
882–892
32 Su, C., Sahlin, M. and Oliw, E. H. (1998) A protein radical and ferryl intermediates are
generated by linoleate diol synthase, a ferric hemeprotein with dioxygenase and
hydroperoxide isomerase activities. J. Biol. Chem. 273, 20744–20751
33 Smith, W. L., DeWitt, D. L. and Garavito, R. M. (2000) Cyclooxygenases: structural,
cellular, and molecular biology. Annu. Rev. Biochem. 69, 145–182
34 Denisov, I. G., Makris, T. M., Sligar, S. G. and Schlichting, I. (2005) Structure and
chemistry of cytochrome P450. Chem. Rev. 105, 2253–2278
35 Takajo, T., Tsuchida, K., Murahashi, T., Ueno, K. and Koshiishi, I. (2007) Intramolecular
rearrangement of linolenate peroxyl radicals in lipoxygenase reactions at lower oxygen
content. J. Lipid Res. 48, 855–862
13 Tsitsigiannis, D. I., Kowieski, T. M., Zarnowski, R. and Keller, N. P. (2004) Endogenous
lipogenic regulators of spore balance in Aspergillus nidulans. Eukaryotic Cell 3,
1398–1411
14 Brodhun, F., Go¨bel, C., Hornung, E. and Feussner, I. (2009) Identification of PpoA from
Aspergillus nidulans as a fusion protein of a fatty acid heme dioxygenase/peroxidase and
a cytochrome P450. J. Biol. Chem. 284, 11792–11805
15 Garscha, U., Jerneren, F., Chung, D., Keller, N. P., Hamberg, M. and Oliw, E. H. (2007)
Identification of dioxygenases required for Aspergillus development: studies of products,
stereochemistry, and the reaction mechanism. J. Biol. Chem. 282, 34707–34718
16 Garscha, U. and Oliw, E. H. (2009) Leucine/valine residues direct oxygenation of linoleic
Acid by (10R)- and (8R)-dioxygenases: expression and site-directed mutagenesis of
(10R)-dioxygenase with epoxyalcohol synthase activity. J. Biol. Chem. 284,
13755–13765
36 Koshiishi, I., Tsuchida, K., Takajo, T. and Komatsu, M. (2005) Quantification of lipid alkyl
radicals trapped with nitroxyl radical via HPLC with postcolumn thermal decomposition.
J. Lipid Res. 46, 2506–2513
37 Yoshioka, S., Takahashi, S., Hori, H., Ishimori, K. and Morishima, I. (2001) Proximal
cysteine residue is essential for the enzymatic activities of cytochrome P450cam. Eur. J.
Biochem. 268, 252–259
17 Pohnert, G. (2000) Wound-activated chemical defense in unicellular planktonic algae.
Angew. Chem. Int. Ed. Engl. 39, 4352–4354
18 Koshiishi, I., Tsuchida, K., Takajo, T. and Komatsu, M. (2006) Radical scavenger can
scavenge lipid allyl radicals complexed with lipoxygenase at lower oxygen content.
Biochem. J. 395, 303–309
19 Kulmacz, R. J. (1987) Prostaglandin-G2 levels during reaction of prostaglandin-h
synthase with arachidonic-acid. Prostaglandins 34, 225–240
20 Galagan, J. E., Calvo, S. E., Cuomo, C., Ma, L. J., Wortman, J. R., Batzoglou, S., Lee,
S. I., Basturkmen, M., Spevak, C. C. and Clutterbuck, J. et al. (2005) Sequencing of
Aspergillus nidulans and comparative analysis with A. fumigatus and A. oryzae. Nature
438, 1105–1115
38 Matsui, K., Sasahara, S., Akakabe, Y. and Kajiwara, T. (2003) Linoleic acid
10-hydroperoxide as an intermediate during formation of 1-octen-3-ol from linoleic acid
in Lentinus decadetes. Biosci. Biotechnol. Biochem. 67, 2280–2282
39 Akakabe, Y., Matsui, K. and Kajiwara, T. (2005) Stereochemical correlation between
10-hydroperoxyoctadecadienoic acid and 1-octen-3-ol in Lentinula edodes and
Tricholoma matsutake mushrooms. Biosci. Biotechnol. Biochem. 69, 1539–1544
40 Senger, T., Wichard, T., Kunze, S., Go¨bel, C., Lerchl, J., Pohnert, G. and Feussner, I.
(2005) A multifunctional lipoxygenase with fatty acid hydroperoxide cleaving activity from
the moss Physcomitrella patens. J. Biol. Chem. 280, 7588–7596
21 Su, C. and Oliw, E. H. (1996) Purification and characterization of linoleate 8-dioxygenase
from the fungus Gaeumannomyces graminis as a novel hemoprotein. J. Biol. Chem. 271,
14112–14118
41 Picot, D., Loll, P. J. and Garavito, R. M. (1994) The X-ray crystal structure of the
membrane protein prostaglandin H2 synthase-1. Nature 367, 243–249
Received 17 July 2009/30 September 2009; accepted 30 October 2009
Published as BJ Immediate Publication 30 October 2009, doi:10.1042/BJ20091096
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Biochem. J. (2010) 425, 553–565 (Printed in Great Britain) doi:10.1042/BJ20091096
SUPPLEMENTARY ONLINE DATA
PpoC from Aspergillus nidulans is a fusion protein with only one active
haem
1
¨
Florian BRODHUN, Stefan SCHNEIDER, Cornelia GOBEL, Ellen HORNUNG and Ivo FEUSSNER
Department of Plant Biochemistry, Albrecht-von-Haller-Institute for Plant Sciences, Georg-August-University, Justus-von-Liebig-Weg 11, D-37077 Go¨ttingen, Germany
ꢀ9Z,12Z
Figure S1 Tandem MS analysis of products formed from 18:2
by PpoC
ꢀ9Z,12Z
Tandem MS spectra of (A) 12,13-epoxy-10-HOME, (B) 8-HODE, (C) 10-HODE, (D) 10-KODE and (E) 10-ODA are shown, which were formed by PpoC bacterial crude extract and 18:2
.
Additionally, in (F) the tandem MS spectrum of 2-decendioic acid is shown, which was obtained after incubations of PpoC with 18:3ꢀ9Z,12Z,15Z . Above each tandem MS spectrum the characteristic
fragmentation patterns are indicated, as reported previously [1,2].
1
To whom correspondence should be addressed (email ifeussn@uni-goettingen.de).
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F. Brodhun and others
Figure S2 Peroxidase activity assay of recombinant and purified PpoC
ꢀ9Z,12Z
For the peroxidase activity assay purified PpoC was incubated with 100 μM 18:2
and
100 μM TMPD. The increase in the absorption at 611 nm, due to oxidized TMPD, is visualized
as a function of time.
Figure S3 Tandem MS analysis of products formed from 10-HPODE and 8-HPODE by PpoC and PpoA
(A) Tandem MS spectrum of 12,13-epoxy-10-HOME that was either formed from incubation of 10-HPODE with purified PpoA or PpoC bacterial crude extract. (B) Tandem MS spectrum of
12,13-epoxy-8-HOME that was formed by PpoC bacterial crude extract incubated with 8-HPODE. (C) PpoA formed from 8-HPODE predominantly formed 5,8-DiHODE as reported previously [3].
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PpoC is a fusion protein with one active haem
Figure S4 SDS/PAGE of expressed recombinant PpoC mutants
A SDS/PAGE analysis of different PpoC mutants is shown. Lane 1, empty vector control; lane 2:
PpoC with the L385A substitution; lane 3: PpoC with the Y427F substitution; lane 4: PpoC with
the G1039C substitution. MW, molecular-mass markers in kDa.
REFERENCES
1
2
3
Garscha, U., Jerneren, F., Chung, D., Keller, N. P., Hamberg, M. and Oliw, E. H. (2007)
Identification of dioxygenases required for Aspergillus development: studies of products,
stereochemistry, and the reaction mechanism. J. Biol. Chem. 282, 34707–34718
Garscha, U. and Oliw, E. H. (2009) Leucine/valine residues direct oxygenation of linoleic
acid by (10R)- and (8R)-dioxygenases: expression and site-directed mutagenesis of
(10R)-dioxygenase with epoxyalcohol synthase activity. J. Biol. Chem. 284, 13755–13765
Brodhun, F., Go¨bel, C., Hornung, E. and Feussner, I. (2009) Identification of PpoA from
Aspergillus nidulans as a fusion protein of a fatty acid heme dioxygenase/peroxidase and a
cytochrome P450. J. Biol. Chem. 284, 11792–11805
Received 17 July 2009/30 September 2009; accepted 30 October 2009
Published as BJ Immediate Publication 30 October 2009, doi:10.1042/BJ20091096
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