Genome-to-function characterization of novel fungal P450 monooxygenases oxidizing polycyclic aromatic hydrocarbons (PAHs)

Article abstract of DOI:10.1016/j.bbrc.2010.07.094

Fungi, particularly the white rot basidiomycetes, have an extraordinary capability to degrade and/or mineralize (to CO₂) the recalcitrant fused-ring high molecular weight (≥4 aromatic-rings) polycyclic aromatic hydrocarbons (HMW PAHs). Despite over 30years of research demonstrating involvement of P450 monooxygenation reactions in fungal metabolism of HMW PAHs, specific P450 monooxygenases responsible for oxidation of these compounds are not yet known. Here we report the first comprehensive identification and functional characterization of P450 monooxygenases capable of oxidizing different ring-size PAHs in the model white rot fungus Phanerochaete chrysosporium using a successful genome-to-function strategy. In a genome-wide P450 microarray screen, we identified six PAH-responsive P450 genes (Pc-pah1-Pc-pah6) inducible by PAHs of varying ring size, namely naphthalene, phenanthrene, pyrene, and benzo(a)pyrene (BaP). Using a co-expression strategy, cDNAs of the six Pc-Pah P450s were cloned and expressed in Pichia pastoris in conjunction with the homologous P450 oxidoreductase (Pc-POR). Each of the six recombinant P450 monooxygenases showed PAH-oxidizing activity albeit with varying substrate specificity towards PAHs (3-5 rings). All six P450s oxidized pyrene (4-ring) into two monohydroxylated products. Pc-Pah1 and Pc-Pah3 oxidized BaP (5-ring) to 3-hydroxyBaP whereas Pc-Pah4 and Pc-Pah6 oxidized phenanthrene (3-ring) to 3-, 4-, and 9-phenanthrol. These PAH-oxidizing P450s (493-547 aa) are structurally diverse and novel considering their low overall homology (12-23%) to mammalian counterparts. To our knowledge, this is the first report on specific fungal P450 monooxygenases with catalytic activity toward environmentally persistent and highly toxic HMW PAHs.

Full text of DOI:10.1016/j.bbrc.2010.07.094

Biochemical and Biophysical Research Communications 399 (2010) 492–497
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc
Genome-to-function characterization of novel fungal P450 monooxygenases
oxidizing polycyclic aromatic hydrocarbons (PAHs)
Khajamohiddin Syed, Harshavardhan Doddapaneni 1, Venkataramanan Subramanian 2, Ying Wai Lam,
*
Jagjit S. Yadav
Environmental Genetics and Molecular Toxicology Division, Department of Environmental Health, University of Cincinnati College of Medicine, Cincinnati, OH 45267-0056, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 30 June 2010
Available online 30 July 2010
Fungi, particularly the white rot basidiomycetes, have an extraordinary capability to degrade and/or min-
eralize (to CO2) the recalcitrant fused-ring high molecular weight (P4 aromatic-rings) polycyclic aro-
matic hydrocarbons (HMW PAHs). Despite over 30 years of research demonstrating involvement of
P450 monooxygenation reactions in fungal metabolism of HMW PAHs, specific P450 monooxygenases
responsible for oxidation of these compounds are not yet known. Here we report the first comprehensive
identification and functional characterization of P450 monooxygenases capable of oxidizing different
ring-size PAHs in the model white rot fungus Phanerochaete chrysosporium using a successful genome-
to-function strategy. In a genome-wide P450 microarray screen, we identified six PAH-responsive P450
genes (Pc-pah1–Pc-pah6) inducible by PAHs of varying ring size, namely naphthalene, phenanthrene, pyr-
ene, and benzo(a)pyrene (BaP). Using a co-expression strategy, cDNAs of the six Pc-Pah P450s were
cloned and expressed in Pichia pastoris in conjunction with the homologous P450 oxidoreductase (Pc-
POR). Each of the six recombinant P450 monooxygenases showed PAH-oxidizing activity albeit with
varying substrate specificity towards PAHs (3–5 rings). All six P450s oxidized pyrene (4-ring) into two
monohydroxylated products. Pc-Pah1 and Pc-Pah3 oxidized BaP (5-ring) to 3-hydroxyBaP whereas Pc-
Pah4 and Pc-Pah6 oxidized phenanthrene (3-ring) to 3-, 4-, and 9-phenanthrol. These PAH-oxidizing
P450s (493–547 aa) are structurally diverse and novel considering their low overall homology (12–
23%) to mammalian counterparts. To our knowledge, this is the first report on specific fungal P450 mon-
ooxygenases with catalytic activity toward environmentally persistent and highly toxic HMW PAHs.
Ó 2010 Elsevier Inc. All rights reserved.
Keywords:
Phanerochaete chrysosporium
Cytochrome P450 monooxygenase
P450 oxidoreductase
Polycyclic aromatic hydrocarbons
Biodegradation
Pichia pastoris
1. Introduction
genic and/or carcinogenic to living systems [3]. Among the micro-
organisms (bacteria, yeasts, and filamentous fungi) investigated for
Fused-ring polycyclic aromatic hydrocarbons (PAHs) constitute
an important group of highly toxic and significant environmental
chemicals that are generated from different anthropogenic and
industrial processes and accidents such as crude oil spills [1]. The
persistence and genotoxicity of PAHs increase with increasing aro-
matic rings. Consequently, high molecular weight (HMW) PAHs
(P4 benzene rings) are particularly a significant problem both
from the point of view of environmental clean up as well as human
health as these are recalcitrant to biodegradation [2] and are muta-
their ability to break down these hazardous chemicals [1,4,5], only
a few species belonging to actinomycetes and fungi have the ability
to oxidize high molecular weight (HMW) PAHs. In particular, white
rot group of basidiomycete fungi have shown an extraordinary
capability to completely degrade (to CO2) both low molecular
weight (LMW) and HMW PAH compounds [5–7].
Phanerochaete chrysosporium (henceforth abbreviated as PC) is
the most intensively studied model white rot fungus for mecha-
nisms to degrade lignin and a wide range of xenobiotics including
PAHs [6]. Originally, its aromatic ring-oxidizing activity was as-
cribed to the non-specific extracellular peroxidases [8,9] that are
differentially expressed under nutrient-limited (ligninolytic) cul-
ture conditions [10]. Subsequent studies by us and others have
led to increasing evidences on peroxidase-independent degrada-
tion of several aromatics including PAHs under nutrient-sufficient
(non-ligninolytic) culture conditions and involvement of P450
monooxygenation reactions [11–17]. Role of P450 monooxygen-
ation in the rate-limiting initial oxidation of HMW PAHs and
Abbreviations: PAH, polycyclic aromatic hydrocarbon; CYP, cytochrome P450;
POR, cytochrome P450 oxidoreductase; PC, Phanerochaete chrysosporium; PP, Pichia
pastoris.
* Corresponding author. Fax: +1 513 558 4397.
E-mail address: Jagjit.Yadav@uc.edu (J.S. Yadav).
Present address: Carver Center for Genomics, University of Iowa, Iowa City, IA
1
52242, USA.
2
Present address: National Renewable Energy Laboratory, Golden, CO 80401, USA
and Colorado School of Mines, Golden, Co 80401, USA.
0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbrc.2010.07.094

K. Syed et al. / Biochemical and Biophysical Research Communications 399 (2010) 492–497
493
certain LMW PAHs with high ionization potential (>7.35 eV) such
as phenanthrene has also been reported in other basidiomycete
and non-basidiomycete fungi [4,18]. However, despite the past
over 30 years of research on PAH biodegradative fungal species
[1,4,5], the specific P450 monooxygenase enzymes responsible
for this oxidation activity have not been reported. Collectively con-
sidering the above discussion, there is a critical need for identifica-
tion of specific fungal P450 genes and enzymes responsible for the
oxidation of HMW and/or both LMW and HMW PAH compounds,
in order to understand the P450-mediated mechanisms and to
facilitate development of improved biotransformation processes
and applications.
cent study [16]. Preparation of fungal microsomes, CO-difference
spectrum analysis, and calculation of total P450 concentration were
performed using established methods [28,29]. Differential gene
expression profiling was performed using a genome-wide custom-
designed 70 mer oligos-based P450 microarray developed in our
previous studies [25,26]. Full-length cDNAs for the identified PAH-
inducible P450 genes (designated Pc-Pah1 through Pc-Pah6) were
cloned using gene-specific RT-PCR as described previously [26].
cDNA sequences are available in the GenBank with accession num-
bers listed in Table 1. Heterologous co-expression of the Pc-Pah
genes along with the homologous Pc-POR [30] in the yeast P. pastoris
(PP) was performed using the pPICZB expression vector system
(Invitrogen). The experimental strategy used for creating the binary
constructs is depicted in Fig. S1. Expression analysis and PAH
compound oxidation in yeast whole cell assays were performed as
detailed in Supplementary information. P450 hydroxylated metab-
olites of the PAH compounds were analyzed by HPLC followed by li-
quid chromatography-electrospray mass spectrometry (LC-ESI/MS)
(Thermo Finnigan) as described in Supplementary information.
Cytochrome P450 monooxygenases are a superfamily of heme-
thiolate proteins that catalyze a broad range of reactions such as
carbon hydroxylation, heteroatom oxygenation, dealkylation, epox-
idation, reduction, dehalogenation [19]. The typical eukaryotic
P450 monooxygenase system contains a P450 monooxygenase
and a P450 oxidoreductase (POR), both of which are normally mem-
brane-associated. This family of proteins has seen a tremendous
growth in the genomic era [[20], http://drnelson.uthsc.edu/Cyto-
chromeP450.html]. However, post-genomic functional character-
ization of orphan P450s in fungal and other genomes has been a
challenging task due to their poor amenability to heterologous
expression in simpler hosts. Whole genome sequencing of PC [21]
has uncovered the presence of a large P450 diversity, comprised
of about 150 P450 genes [22]. While functional genomic studies
on these P450s are emerging [16,23,24], majority remain orphan
with virtually unknown function. Hence, there is a great need to
characterize the role of individual P450s in this model organism.
Our recent studies using the first custom-designed genome-wide
P450 microarray [25,26], have led to a working hypothesis that sub-
strate specific inducibility could be key to identifying the xenobiotic
substrates for orphan P450 enzymes in this organism [16,23,25]. In
this study, we therefore employed a two-stage genome-to-function
strategy, including a genome-wide (microarray-based) transcrip-
tional induction profiling to identify candidate P450 genes respon-
sive to PAHs of varying ring-size followed by co-expression and
catalytic characterization of the identified recombinantly expressed
P450 proteins. These efforts led to identification of a set of six PAH-
responsive P450 monooxygenase genes in the PC genome (desig-
nated Pc-Pah1 through Pc-Pah6). Subsequent co-expression along
with the reductase partner and catalytic analysis revealed their di-
verse PAH substrate specificity particularly towards HMW (4–5
ring) PAHs. Part of the data reported in this manuscript was pre-
sented at the 104th annual general meeting of the American Society
for Microbiology, New Orleans, LA [27].
3. Results and discussion
3.1. Involvement of P450 enzyme system in biodegradation of HMW
PAH compounds in PC
Addition of the P450 inhibitor piperonyl butoxide (PB) in the
nutrient-rich malt extract (ME) cultures of PC led to a significant
abrogation of the degradation activity towards pyrene and ben-
zo(a)pyrene (Fig. 1). This suggested a key role of P450 monooxy-
genase(s) in initial oxidation of these HMW PAH compounds in
PC, analogous to that reported for the lower PAH phenanthrene
[11]. Comparison between the chemically-killed (CK) control cul-
tures and uninoculated control revealed that unextractable myce-
lial adsorption partially contributed toward the apparent total
disappearance in case of pyrene (ꢀ16.6%) but not in benzo(a)pyr-
ene (BaP) degradation. The PB attenuation effect on biodegradation
was relatively higher for BaP (Fig. 1), implying the involvement of
divergent sets of P450s for the two PAHs. Role of P450s in PAH oxi-
dation was further evidenced by our initial P450 enzyme induction
studies on the whole fungus grown under similar conditions. Re-
sults showed about two fold induction of total P450 enzyme con-
tent (based on typical CO-difference spectrum) in response to
10 ppm pyrene (Table S2). Considering this, we performed a gen-
ome-wide induction profiling for identification of specific PAH-
inducible P450 genes as part of our genome-to-function strategy
to characterize those catalyzing PAH oxidation.
2. Materials and methods
3.2. Microarray-based identification of PAH-responsive P450
monooxygenases (Pc-Pah P450s)
Microbial strains, chemicals, media, and culture conditions are
detailed under Supplementary information. P450 inhibitor studies
and induction of P450s in PC were performed as described in our re-
Genome-wide P450 transcriptional profiling using the P450ome
microarray first developed in our previous studies [25,26] showed
Table 1
Multiple PAH-responsive P450 genes identified using genome-wide P450 microarray analysis on P. chrysosporium.a
P450 ID
CYP name
NCBI Gene Accession number
Protein ID (JGI WGS Ver. 2)
Transcriptional induction (fold-change)b
Naphthalene
Phenanthrene
Pyrene
Benzo(a)pyrene
Pc-pah1
Pc-pah2
Pc-pah3
Pc-pah4
Pc-pah5
Pc-pah6
CYP5136A2
CYP5145A3
CYP5144A7
CYP5136A3
CYP5142A3
CYP5144A5
AY515588
GU270838
AY515589
GU270839
AY515590
AY515591
5852
6327
133311
5001
28946
132481
7.8 4.64
3.28 0.73
2.87 1.24
9.52 2.38
3.24 1.95
1.67 0.65
2.72 0.04
7.34 0.01
1.71 0.11
15.12 7.19
1.32 0.08
7.37 0.26
4.43 0.6
a
PAH treatments and transcriptional profiling were performed using nutrient-rich (malt extract) cultures as described under (Section 2).
The fold-change values are means standard errors obtained by evaluating eight independent spots for each gene and are statistically significant (P 6 0.05 and FDR of
b
0.1). Abbreviations: CYP, Cytochrome P450; JGI, Joint Genome Institute of the US Department of Energy; WGS, Whole genome sequence.

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K. Syed et al. / Biochemical and Biophysical Research Communications 399 (2010) 492–497
120
100
80
60
40
20
0
*
*
Pyr (no-inh)
Pyr-PB
BaP (no-inh)
BaP-PB
Treatment
Fig. 1. Effect of P450 inhibitor piperonyl butoxide (PB) on biodegradation of pyrene and benzo(a)pyrene by P. chrysosporium. Nutrient-rich malt extract broth cultures and
negative controls were prepared as described under (Section 2). Final concentrations of the test PAH and PB in the cultures were 20 ppm and 0.5 mM, respectively. Asterisk
indicates statistically significant (P 6 0.05) effect of the inhibitor. The plotted values represent means standard deviations for three biological replicates. Abbreviations: Pyr,
pyrene; BaP, benzo(a)pyrene; no-inh, no inhibitor.
significant induction (up to ꢀ15-fold) of multiple P450 genes in re-
sponse to the four individual representative PAHs of increasing
ring size (2–5 rings) in nutrient-rich ME cultures (Table 1 and
Fig. S2). A total of six candidate PAH-responsive P450 genes (desig-
nated Pc-Pah1 through Pc-Pah6) were selected, based on a cut off
criterion of P3-fold induction by at least one of the inducer PAHs.
Subsequent qRT-PCR analysis on selected genes and treatments
verified the microarray results. For instance, qRT-PCR based fold-
change values for Pc-Pah2 were 2.45 0.03 (naphthalene),
4.21 0.44 (phenanthrene), and 2.47 0.11 (benzo(a)pyrene) as
against 2.72 0.04, 3.28 0.73, and 1.71 0.11, respectively in
microarray analysis. The house-keeping gene GAPDH remained un-
changed. Of the six PAH-responsive genes, a set of four genes each
was inducible by naphthalene (Pc-Pah2, Pc-Pah3, Pc-Pah5 and
Pc-Pah6) and phenanthrene (Pc-Pah1 through 4) whereas a set of
two genes each was inducible by pyrene (Pc-Pah1 and Pc-pah4),
and BaP (Pc-pah2 and Pc-pah3) (Table 1). The two pyrene-inducible
genes (Pc-Pah1 and Pc-Pah4) were also inducible in the presence of
the lower PAH phenanthrene. The two BaP-inducible genes (Pc-
Pah2 and Pc-Pah3) were also inducible by the lower PAHs naphtha-
lene and phenanthrene. Taken together, a Pc-Pah gene inducible by
the higher PAH (4- or 5- ring) was also inducible by a lower PAH
(2- and/or 3- ring).
significance considering the expression difficulties encountered in
our previous efforts on independent expression of Pc-Pah P450s
without the homologous POR. SDS–PAGE analysis of the six micro-
somal preparations showed a clear band coinciding with Pc-POR
when compared to the control (Fig. 2A). Western blot analysis
using anti-His antibody showed full-length expression of all six
Pc-Pah P450 proteins (Fig. 2B). Active nature of the expressed
recombinant P450 proteins was evident from their characteristic
CO-difference spectra (Fig. 2C). The observed maximum absorption
peaks were at 448 nm (Pc-Pah4), 450 nm (Pc-Pah1 and Pc-Pah3),
451 nm (Pc-Pah2), and 452 nm (Pc-Pah5 and Pc-Pah6). P450 and
POR expression levels varied for the six Pc-Pah clones (Fig. 2D),
with Pc-Pah5 and 6 showing the highest P450 concentration.
Pc-Pah6 had the highest co-expressing POR activity, an observation
consistent with the corresponding band intensity in the SDS–PAGE
gel (Fig. 2A).
3.4. Catalytic analysis of the recombinant fungal P450 enzymes
3.4.1. Substrate specificity
Whole-cell yeast biocatalysts are considered ideal for screening
of hydrophobic compounds such as PAHs as the use of cells allows
ready uptake as well as alleviates the need for isolation of micro-
somes and in vitro reconstitution of the P450 assay [31]. In whole
cell assays, the six recombinant Pc-Pah enzymes showed catalytic
activity toward PAHs, albeit with varying activity and substrate
specificity (Fig. 3). No activity was observed in the empty vector
control yeast. While all six Pc-Pah P450s showed pyrene oxidation,
quantitative differences in the activity were observed among the
individual enzymes; Pc-Pah5 and Pc-Pah4 showed relatively high-
er pyrene oxidation activity as compared to the other four Pc-Pah
P450s. In comparison, a tighter substrate specificity was observed
for phenanthrene as only Pc-Pah4 (ꢀ65%) and Pc-Pah6 (ꢀ14%)
could oxidize this compound (Fig. 3). This is significant considering
that phenanthrene is not a substrate for lignin peroxidases because
of its high ionization potential (>7.35 eV). Anthracene, another 3-
ring PAH, was, however, not detectably oxidized by any of the six
Pc-Pah P450s in this study. Only Pc-Pah1 and Pc-Pah3 showed
activity towards BaP, with the former showing relatively higher
levels of oxidation (32.6% vs. 14.3%). The co-expressed POR levels
(Fig. 2D) did not seem to quantitatively correlate either with the
relative P450 expression levels or with the PAH oxidation activity
(Fig. 3) of the individual Pc-Pah P450s. Common specificity of Pc-
Pah4 and Pc-Pah6 towards phenanthrene, albeit to a varying ex-
tent, may be because of their evolution from a common progenitor
Interestingly, five of the six PAH-responsive P450 genes iden-
tified here also showed upregulation under defined high nitrogen
(HN) culture conditions in our earlier study [26], further con-
firming their optimal expressibility under non-ligninolytic condi-
tions. We also observed inducibility of two of the genes (Pc-Pah4
and Pc-Pah5) in response to alkylphenols [16], indicating their
broader substrate range against environmental xenobiotics.
3.3. Recombinant co-expression of Pc-Pah P450s and Pc-POR in Pichia
pastoris
Bioinformatic analyses on the six Pc-Pah genes and their corre-
sponding cDNAs, cloned and sequenced in this study for recombi-
nant expression, revealed variable structural features such as 8 to
12 introns with variable distribution (Fig. S3) and genome-wide
localization. Interestingly, two of the genes Pc-Pah3 and Pc-Pah6,
are part of a tandem gene cluster of 12 P450 members in the PC
genome. Pc-Pah5 is also on the same genome scaffold and is sepa-
rated from the upstream 4 tandem P450s cluster by the interven-
ing 3 non-P450 genes.
The co-expression strategy enabled a successful active expres-
sion of the individual Pc-Pah P450 proteins. This strategy assumes

K. Syed et al. / Biochemical and Biophysical Research Communications 399 (2010) 492–497
495
nant yeast cultures (Table S3) confirming their role in the observed
PAH catalysis. PB did not exert any growth inhibition, confirming
the enzymatic basis of the observed activity inhibition. Comparison
of the P450 inhibitor effect on the whole fungus versus the recom-
binant Pc-Pah P450 yeast clones showed complete inhibition of
BaP-oxidizing P450 activity in both the biological systems. In con-
trast, only partial inhibition of activity was observed for pyrene in
the whole fungus (unlike the yeast clones), indicating the likely
presence of additional pyrene-oxidizing P450s (non-responsive to
PB) in the PC genome. This is not unexpected considering the ob-
served general amenability of pyrene to oxidation by all six
P450s in this study.
MWM
1
2
3
4
5
6
C
A
116.3
97.4
66.2
55.4
B
3.4.2. Catalytic specificity
Initial HPLC screening on the PAH oxidation reaction extracts
for the individual reactive P450s showed variable number of
metabolite peaks for individual PAHs. No such peaks were ob-
served in the empty vector control yeast. Subsequent LC-ESI/MS
analysis (Fig. S4) confirmed the observed HPLC patterns of the
metabolite peaks and allowed identification of the oxidation
metabolites by accurate mass measurement on the whole reaction
extracts using authentic standards (Table S4). Phenanthrene reac-
tion extracts for the Pc-Pah4 and Pc-Pah6 clones showed three
peaks (I, II, III) with same measured monoisotopic mass (m/z) but
different retention time (Rt). The Rt values of the three peaks ex-
actly matched with those of the authentic standards (Table S4).
Based on the Rt and m/z comparisons, the peaks were thus identi-
fied as 3-phenanthrol (peak I), 4-phenanthrol (peak III), and 9-phe-
nanthrol (peak II). Among the three metabolites, 9-phenanthrol
was the most abundant followed by 4- and 3-phenanthrols, respec-
tively. In contrast, pyrene reaction extracts for each of the 6 Pc-Pah
P450s showed two peaks exhibiting the same m/z but different Rt.
The second peak (II) showed Rt coinciding with that of the authen-
tic standard 1-hydroxypyrene. In contrast with phenanthrene and
pyrene, a single peak was observed in the benzo(a)pyrene reaction
extracts from Pc-Pah1 and Pc-Pah3. As the Rt and m/z values of the
peak were the same as those of the authentic standard, this peak
was identified as 3-hydroxybenzo(a)pyrene (Table S4).
Taken together, catalytic activity of the Pc-Pah P450s towards
varying ring-size PAHs revealed their functional diversity albeit
with some redundancy. While all Pc-Pah P450s catalyzed the oxi-
dation of pyrene, specificity was observed in respect of phenan-
threne and BaP (Fig. 4). It is interesting that the multifunctional
Pc-Pah P450s (those catalyzing oxidation of more than one PAH)
showed a pattern of ring-size specificity. For instance, the P450s
showed activity either with phenanthrene and pyrene (3 and 4 ring
PAH) or pyrene and BaP (4 and 5 ring PAH) but none of them oxi-
dized both phenanthrene and BaP (3 and 5 ring PAH) (Fig. 4). While
the substrate responsiveness (induction) profiling strategy proved
critical in successful identification of relevant PAH-oxidizing P450s
(Table 1), the induction patterns did not show absolute correlation
with the respective substrate specificities (Fig. 3).
Pc-Pah1
Pc-Pah2
Pc-Pah3
Pc-Pah4
Pc-Pah5
Pc-Pah6
PP
0.004
0.003
0.002
0.001
0
C
400
410
420
430
440
450
460
470
480
490
500
-0.001
-0.002
-0.003
-0.004
-0.005
Wavelength (nm)
D
Pc-Pah P450 (pmol per mg) POR activity (nmol per min per mg)
300
250
200
150
100
50
0
Pah1
Pah2
Pah3
Pah4
Pah5
Pah6
C
Pc-Pah P450
Fig. 2. Co-expression and activity characterization of Pc-Pah P450 monooxygenases
and the homologous P450 reductase partner (Pc-POR) in P. pastoris. The expressed
enzymes were assessed in the yeast microsomes prepared as described under
(Section 2) (A) SDS–PAGE analysis of the microsomal protein preparations from
recombinant P450-POR yeast clones Pc-Pah1 through Pc-Pah6 (lanes 1–6) and
negative control yeast (lane c). Proteins were separated on 7% SDS–PAGE and
detected by staining with SYPRO Ruby (BioRad). The protein band coinciding with
Pc-POR (estimated 81.6 kDa) is shown by an arrow. (B) Western blot analysis of Pc-
Pah proteins using anti-his antibody. (C) Characteristic CO-difference spectra for the
individual Pc-Pah proteins (Pc-Pah 1 through 6) and negative control. (D) P450
enzyme concentrations and POR activity levels of the individual Pc-Pah clones
versus the negative control (C). P450 concentration values are based on the CO-
difference spectra shown in panel C. POR activity values are the means for three
biological replicates (the SD values varied between 0.07 and 0.23).
Formation of oxidized products in the single P450 enzyme-
based oxidation reactions confirmed that Pc-Pah P450s are in-
volved in the initial oxidation step of the PAH biodegradation
process. P450-mediated oxidation of the PAH ring in eukaryotes
occurs via formation of an epoxide which in turn is converted into
hydroxylated products by non-enzymatic isomerization and/or by
the action of epoxide hydrolase [4]. The relative abundance of the
three phenanthrols was consistent with the regiospecificity profile
observed for the whole fungus [1]; 9-phenanthrol was found to be
the most abundant whereas 3-phenanthrol the least. In this con-
text, it is interesting that recent whole fungus-based studies using
nutrient-limited (defined low nitrogen) cultures showed the for-
mation of a single monohydroxylated product, 9-phenanthrol
[17]. In previous PC studies [11], it was unclear whether a single
gene as has been hypothesized for members of the same P450
genomic cluster in our previous genome-wide bioinformatic study
on P450ome of this organism [22].
The 450 inhibitor PB almost completely abrogated the observed
PAH-oxidizing activity of all six Pc-Pah enzymes in the recombi-

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K. Syed et al. / Biochemical and Biophysical Research Communications 399 (2010) 492–497
70
60
50
40
30
20
10
0
Pc-Pah1
Pc-Pah2
Pc-Pah3
Pc-Pah4
Pc-Pah5
Pc-Pah6
Pc-Pah P450
Phenanthrene Pyrene Benzo(a)pyrene
Fig. 3. Relative PAH-oxidizing activities of the six recombinant Pc-Pah P450 enzymes towards varying ring-size PAH compounds in whole cell assays. Each culture was spiked
with a test PAH and the oxidation activity was assessed at 36 h of incubation. The values represent means standard deviations for three biological replicates and are
statistically significant (P 6 0.05).
OH
3-hydroxybenzo(a)pyrene was identified as the sole oxidation
product [15].
Taken together, matching regiospecificity of the recombinant
Pc-Pah P450s and the parent whole fungus (PC) towards oxidation
of different PAHs and gene inducibility under in vivo conditions
confirm the authenticity and physiological relevance of the identi-
fied P450s in PC. Considering that the white rot fungi possess a
3-phenanthrol
HO
Pc-Pah4 &
Pc-pah6
comprehensive enzymatic machinery to completely degrade (to
CO2) PAHs, the P450 monooxygenases may link up with the PAH
mineralization pathway components under appropriate condi-
tions; such common pathway components (non-ligninolytic and
4-phenanthrol
ligninolytic) for cycling of PAH metabolites have been indicated
in previous studies [18,32]. Hence, the presence of P450s in white
rot fungi assumes greater significance than in other biological sys-
tems that have a standalone P450 enzyme system for PAH metab-
olism such as in non-basidiomycete fungi [4] and animals [19].
Interestingly, there was a partial overlap in catalytic regiospecific-
ity [33,34] between the fungal Pc-Pah P450 enzymes and mamma-
lian PAH-oxidizing P450s (human/rat/mouse CYP1A1, 1A2, and
1B1), possibly because of low overall protein level homology
(12–23%) (Table S5). This phenomenon holds promise for future
basic structure-activity relationship studies to generate improved
versatile biocatalysts for detoxification and biotransformation
applications.
Phenanthrene
OH
9-phenanthrol
OH
Pc-Pah1to
Pc-pah6
1-hydroxypyrene
Pyrene
4. Conclusions
Pc-Pah1&
Pc-pah3
We report a successful genome-to-function approach to func-
tionally characterize orphan P450s in the P. chrysosporium genome,
a strategy that could be extendable to other genomes (prokaryotes
and other eukaryotes such as fungi, insects, plants, and animals)
with orphan P450s. To our knowledge, this report constitutes the
first genome-wide comprehensive identification and functional
characterization of fungal P450 monooxygenases catalyzing oxida-
tion of varying ring-size PAH compounds, namely phenanthrene,
pyrene and BaP. This study opens up avenues for investigating sim-
ilar PAH-oxidizing P450 enzymes in other fungal systems and for
exploiting these structurally and catalytically diverse P450 en-
zymes for future basic and applied research on P450-mediated
mechanisms and processes in bioremediation and biotechnological
applications.
OH
Benzo(a)pyrene
3-hydroxybenzo(a)pyrene
Fig. 4. PAH oxygenation reactions catalyzed by the individual recombinant Pc-Pah
enzymes (Pc-Pah1 to Pc-Pah6) of P. chrysosporium.
P450 or multiple P450s are involved in the formation of multiple
phenanthrols via formation of different epoxides (9, 10-oxide
versus 3, 4-oxide). In this context, our results showed involve-
ment of a single P450 in the formation of all three phenanthrols,
suggesting that individual P450 (Pc-Pah4 or Pc-Pah6) can oxidize
phenanthrene at both positions (9,10 or 3,4). Each of the six Pc-
Pah P450s oxidized pyrene into two mono hydroxylated products,
of which one was identified as 1-hydroxypyrene. BaP was oxi-
dized to 3-hydroxybenzo(a)pyrene by Pc-Pah1 and Pc-Pah3. This
is consistent with the whole fungus study on PC wherein
Acknowledgments
The work was supported by the NIH’s National Institute of Envi-
ronmental Health Science (NIEHS) grants R01ES10210 and
R01ES015543 to JSY.

K. Syed et al. / Biochemical and Biophysical Research Communications 399 (2010) 492–497
497
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Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bbrc.2010.07.094.
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