The selective oxidation of substituted aromatic hydrocarbons and the observation of uncoupling via redox cycling during naphthalene oxidation by the CYP101B1 system

Article abstract of DOI:10.1039/c7cy00088j

The cytochrome P450 monooxygenase enzyme CYP101B1, from Novosphingobium aromaticivorans DSM12444, efficiently and selectively oxidised a range of naphthalene and biphenyl derivatives. Methyl substituted naphthalenes were better substrates than ethylnaphthalenes and naphthalene itself. The highest product formation activity for a singly substituted alkylnaphthalene was obtained with 2-methylnaphthalene. The oxidation of alkylnaphthalenes was regioselective for the benzylic methyl or methine C-H bonds. The products from 1- and 2-ethylnaphthalene oxidation were highly enantioselective with a single stereoisomer being generated in significant excess. The disubstituted substrate, 2,7-dimethylnaphthalene, had a higher product formation activity than either 1- and 2-methylnaphthalene. Methyl substituted biphenyls were also better substrates than biphenyl and had similar biocatalytic parameters to 1-methylnaphthalene. CYP101B1 catalysed oxidation of 2- and 3-methylbiphenyl was selective for attack at the methyl C-H bonds. The exception was the turnover of 4-methylbiphenyl which generated 4′-(4-methylphenyl)phenol as the major product (70%) with 4-biphenylmethanol making up the remainder. The drug molecule diclofenac was also regioselectively oxidised to 4′-hydroxydiclofenac by CYP101B1. The activity of the CYP101B1 system with naphthalene was more complex and the rate of NADH oxidation increased over time but very little product, 1-naphthol, was generated. Addition of samples of 1-naphthol and 2-naphthol and low concentrations of 1,4-naphthoquinone induced rapid NADH oxidation activity in the in vitro turnovers in both the presence and absence of the cytochrome P450 enzyme. Hydrogen peroxide was generated in these reactions in absence of the P450 enzymes demonstrating that the ferredoxin and ferredoxin reductase in combination with quinones from naphthol oxidation and oxygen can undergo redox cycling giving rise to a form of uncoupling of the reducing equivalents.

Full text of DOI:10.1039/c7cy00088j

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The selective oxidation of substituted aromatic
hydrocarbons and the observation of uncoupling
via redox cycling during naphthalene oxidation by
the CYP101B1 system†
Cite this: DOI: 10.1039/c7cy00088j
*
Emma A. Hall, Md Raihan Sarkar and Stephen G. Bell
The cytochrome P450 monooxygenase enzyme CYP101B1, from Novosphingobium aromaticivorans
DSM12444, efficiently and selectively oxidised a range of naphthalene and biphenyl derivatives. Methyl
substituted naphthalenes were better substrates than ethylnaphthalenes and naphthalene itself. The highest
product formation activity for
a
singly substituted alkylnaphthalene was obtained with
2-methylnaphthalene. The oxidation of alkylnaphthalenes was regioselective for the benzylic methyl or
methine C–H bonds. The products from 1- and 2-ethylnaphthalene oxidation were highly enantioselective
with a single stereoisomer being generated in significant excess. The disubstituted substrate, 2,7-
dimethylnaphthalene, had a higher product formation activity than either 1- and 2-methylnaphthalene.
Methyl substituted biphenyls were also better substrates than biphenyl and had similar biocatalytic parame-
ters to 1-methylnaphthalene. CYP101B1 catalysed oxidation of 2- and 3-methylbiphenyl was selective for
attack at the methyl C–H bonds. The exception was the turnover of 4-methylbiphenyl which generated 4′-
(4-methylphenyl)phenol as the major product (70%) with 4-biphenylmethanol making up the remainder.
The drug molecule diclofenac was also regioselectively oxidised to 4′-hydroxydiclofenac by CYP101B1. The
activity of the CYP101B1 system with naphthalene was more complex and the rate of NADH oxidation in-
creased over time but very little product, 1-naphthol, was generated. Addition of samples of 1-naphthol
and 2-naphthol and low concentrations of 1,4-naphthoquinone induced rapid NADH oxidation activity in
the in vitro turnovers in both the presence and absence of the cytochrome P450 enzyme. Hydrogen per-
oxide was generated in these reactions in absence of the P450 enzymes demonstrating that the ferredoxin
and ferredoxin reductase in combination with quinones from naphthol oxidation and oxygen can undergo
redox cycling giving rise to a form of uncoupling of the reducing equivalents.
Received 16th January 2017,
Accepted 9th March 2017
DOI: 10.1039/c7cy00088j
rsc.li/catalysis
ity and leakage due to oxygen reduction competing with slow
inter-protein electron transfer can reduce the effectiveness of
Introduction
There is great interest in applying cytochrome P450 (CYP) en-
zymes as biocatalysts for the regio- and stereo-selective inser-
tion of an oxygen atom into chemically inert carbon–hydrogen
bonds.1–7 This monooxygenase activity requires two electrons
that are usually derived from NAD(P)H and these are deliv-
ered one at a time, as required, to the CYP enzymes by
electron transfer proteins.8 Electron transfer is often the rate
determining step in CYP catalysis and the activities of many
CYP enzymes are compromised when alternative electron
transfer systems are used.9–11 In addition uncoupling reac-
tions which can arise from poor enzyme/substrate compatibil-
the enzyme. Both result in oxygen reduction without product
formation.12–14 The identification and application of highly
active monooxygenase systems greatly facilitates biocatalytic
C–H bond oxidation of hydrocarbons and aromatics.15–17
Therefore the isolation and characterisation of new complete
systems which are capable of performing challenging reac-
tions, such as the selective oxidation of aromatic molecules,
is of paramount importance in the field of fine chemical
synthesis.
Many bacterial and fungal monooxygenase enzymes have
been investigated as potential biocatalysts for the oxidation
aromatic molecules.18–20 The fungal peroxygenases from
Agrocybe aegerita and Coprinellus radians were able to oxidise
a broad range of polyaromatic hydrocarbons.21 Certain mem-
bers of the self-sufficient CYP102 family of P450 mono-
oxygenases, which are highly active for fatty acid substrates,
Department of Chemistry, University of Adelaide, SA 5005, Australia.
E-mail: Stephen.bell@adelaide.edu.au
† Electronic supplementary information (ESI) available: Additional data, chro-
matograms, MS and NMR data. See DOI: 10.1039/c7cy00088j
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have activity with aromatic substrates.2,16 CYP102A1, from
Bacillus megaterium, has been modified via protein engineer-
ing to facilitate the oxidation of aromatic substrates such as
naphthalene, phenanthrene and pyrene.2,22 Class I CYP en-
zymes, such as CYP101A1 (P450cam), from a Pseudomonas
sp., whose electron transfer systems consist of a flavin-
dependent ferredoxin reductase and a ferredoxin, are also ca-
pable of high monooxygenase activities and selective oxida-
tions.11,23,24 The wild-type forms of various class I CYP en-
zymes and mutants of CYP101A1, have been reported to be
biocatalysts for the oxidation of aromatic molecules includ-
ing polyaromatic hydrocarbons and biphenyl.25–29
Naphthalene, methylnaphthalenes and substituted biphe-
nyls are toxic molecules which are ubiquitous in the environ-
ment.30 Substituted naphthalene and biphenyl derivatives are
also found in a range of molecules with pharmacological ac-
tivity including drug molecules such as diflunisal, naftifine,
terbinafine and tolnaftate. The enzyme catalysed oxidation of
these aromatic substrates has potential applications in envi-
ronmental bioremediation and fine chemical synthesis. The
Phe87Val mutant form of CYP102A1 has been used to oxidise
naphthalene derivatives, including the methyl and dimethyl
substituted forms.31 CYP110E1, from Nostoc sp. strain
PCC7120, oxidised substituted naphthalenes and biphenyls.32
The reducing equivalents were provided to CYP110E1 by fus-
ing the enzyme to the RhFred reductase domain of CYP116B2
from Rhodococcus sp. NCIMB 9784.33 The turnovers of
substituted naphthalenes by both of these monooxygenase
enzymes were generally not selective with at least two prod-
ucts being generated in low yields for each substrate.31,32 The
fungal peroxygenases from Agrocybe aegerita and Coprinellus
radians efficiently oxidised 1- and 2-methylnaphthalene unse-
CYP108D1.11,20,37,40,41 The product formation activity with the
best substrates is in excess of 1000 nmol nmol P450−1 min−1.
A whole-cell system containing CYP101B1, ArR and Arx,
which is capable of product formation on the gram-per-liter
scale in shake flasks, has been constructed.37 Therefore
CYP101B1 is a promising monooxygenase system for biocata-
lytic applications involving C–H bond oxidation reactions.
Aromatic molecules including phenylcyclohexane and
p-cymene have been oxidised by CYP101B1 with the activity
of phenylcyclohexane oxidation being five times higher than
the oxidation of p-cymene.41 Phenylcyclohexane was selec-
tively oxidised to trans-4-phenylcyclohexanol while p-cymene
was hydroxylated at the benzylic carbons to yield a mixture of
isopropylbenzyl alcohol and p-α,α-trimethylbenzylalcohol. De-
spite inducing a lower shift to the high spin form (≤20%)
compared to norisoprenoids, phenylcyclohexane binds to
CYP101B1 with comparable affinity to β-damascone
suggesting that larger two ring aromatic systems may be good
substrates for this enzyme.41 Many drug molecules contain
multiple aromatic rings and the identification and synthesis
of drug metabolites using soluble and highly active P450 en-
zymes could simplify the drug development process. Here we
report that the substrate range of CYP101B1 includes aro-
matic substrates and demonstrate it is capable of acting as a
biocatalyst for the oxidation of substituted naphthalenes,
biphenyls and the drug molecule diclofenac. During these
investigations we determined that the oxidation products of
naphthalene could interfere with the transfer of the electrons
from the ferredoxin to the P450 enzyme which provided an
alternate mechanism for uncoupling via unproductive redox
cycling in these systems.
lectively to numerous products including those arising from Experimental
multiple oxidations.34
General
Novosphingobium bacteria are able to degrade a variety of
General reagents and organics were from Sigma-Aldrich, TCI,
Acros or VWR. Buffer components (Tris–HCl) NADH, and
isopropyl-β-D-thiogalactopyranoside (IPTG) were from
Anachem (Astral Scientific, Australia) or Biovectra, (Scimar,
Australia). General DNA manipulations and microbiological
experiments and the expression, purification and quantita-
tion of CYP101B1 and the electron transfer proteins ArR and
Arx from N. aromaticivorans were each performed as de-
scribed previously.37,40 All the proteins were purified to
≥90% by two ion-exchange steps.37,40
aromatic hydrocarbons and therefore the oxygenase enzymes
from these species have the potential to be biocatalysts for
the oxidation of these hydrophobic substrates.35,36 The bacte-
rium Novosphingobium aromaticivorans DSM12444 contains
many monooxygenase and dioxygenase enzyme coding
genes.35 The cytochrome P450 monooxygenase CYP108D1
from N. aromaticivorans has been shown to bind biphenyl,
naphthalene, phenanthrene and phenylcyclohexane.20
CYP101B1 from the same bacterium, which is related to
CYP101A1, CYP101C1, CYP101D1 and CYP101D2 (the last
three also being from N. aromaticivorans), oxidised indole
generating indigo.11,37–39 We have reported that the
CYP101B1 enzyme is an active biocatalyst for the oxidation of
norisoprenoids and can turnover other structurally diverse
substrates including phenylcyclohexane and various
esters.37,40–44 Importantly a class I electron transfer system,
consisting of a flavin-dependent ferredoxin reductase, ArR,
and a [2Fe–2S] ferredoxin, Arx, has been identified from this
bacterium. This electron transfer system supports the activity
of CYP101B1, as well as those of CYP101D1, CYP101D2,
Substrate binding and kinetic analysis
UV/vis spectroscopy was performed on Varian Cary 5000 or
Agilent Cary 60 spectrophotometers. For substrate binding
the P450 enzymes were diluted to ∼3.5–10 μM using 50 mM
Tris, pH 7.4. After addition of the substrate (as 1 μL aliquots
from a 50 mM stock in DMSO or ethanol) the high spin heme
content was estimated, to approximately 5%, by comparison
with a set of spectra generated from the sum of the appropri-
ate percentages of the spectra of the substrate-free form
(>95% low spin, Soret maximum at 418 nm) and camphor-
CYP101C1
and
CYP111A2
but
not
that
of
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bound form (>95% high spin, Soret maximum at 392 nm) of
wild-type CYP101A1.
The concentration of hydrogen peroxide formed during
NADH oxidation in the absence of CYP101B1 was determined
by the horseradish peroxidase/phenol/4-aminoantipyrine as-
say.45 The total assay volume was 800 μL and contained 200
μL of 50 mM Tris, pH 7.4, 400 μL of a reaction mixture, 200
μL of 50 mM phenol and 200 μL of 5 mM 4-aminoantipyine
(both in 50 mM Tris pH 7.4). The absorbance at 510 nm was
set to zero, 1 μL of a 20 mg mL−1 solution of horseradish per-
oxidase in water was then added, and the absorbance
recorded (ε510 = 6580 M−1 cm−1).
To determine the dissociation constants of CYP101B1 with
the aromatic substrates the enzyme was diluted to 1.6–3.7
μM in a total volume of 2.5 mL in 50 mM Tris, pH 7.4 and
used to baseline the spectrophotometer. Aliquots of the sub-
strate (0.5–2 μL) were added using a Hamilton syringe from a
1, 10, 50 or 100 mM stock solution in DMSO or EtOH. The so-
lution was mixed and the peak-to-trough difference in absor-
bance recorded between 700 nm and 250 nm. Further aliquots
of substrate were added until the peak-to-trough difference of
the Soret band did not shift further. The apparent dissocia-
tion constant, Kd, was obtained by fitting the peak-to-trough
difference against substrate concentration to a hyperbolic
function (eqn (1)):
Product formation
Where authentic product standards were available they were
utilised for identification purposes via HPLC/GC-MS
coelution experiments. The product concentration in the in-
cubation mixtures was calculated by calibrating the UV detec-
tor response of the HPLC detector or GC-MS to the total ion
count (TIC) to the products. The coupling efficiency was the
percentage of NADH consumed that led to product forma-
tion. HPLC analyses were performed using an Agilent 1260
Infinity pump equipped with an Agilent Eclipse Plus C18 col-
umn (250 mm × 4.6 mm, 5 μm), an autoinjector and UV de-
tector (monitored at 280 nm for naphthalene turnovers and
254 nm for biphenyl and diclofenac assays). A gradient, 20–
95%, of acetonitrile (with trifluoroacetic acid, 0.1%) in water
(TFA, 0.1%) was used. The HPLC retention times were as
follows; 1-methylnaphthalene, 22.4 min; 1-naphthylmethanol,
where ΔA is the peak-to-trough absorbance difference, ΔAmax
is the maximum absorbance difference and [S] is the sub-
strate concentration.
NADH turnover assays were performed with mixtures (1.2
mL) containing 50 mM Tris, pH 7.4, 0.5 μM CYP101B1, 5 μM
Arx, 0.5 μM ArR and 100 μg mL−1 bovine liver catalase. The
buffer component was oxygenated by bubbling oxygen
through the solution before addition of the other compo-
nents and equilibration at 30 °C for 2 min. NADH was added
to ∼320 μM, final A340 = 2.00, and the absorbance at 340 nm
was monitored. Substrates were added from a 100 mM stock
solutions in ethanol or DMSO to a final concentration of
0.25–0.5 mM. The rate of NADH turnover was calculated
using ε340 = 6.22 mM−1 cm−1 (Table 1). Where required turn-
overs using 4 mM NADH were undertaken in order to gener-
ate sufficient product for GC coelution experiments (the rate
of NADH oxidation was not monitored).
15.1
min;
2-methylnaphthalene,
22.4
min;
2-naphthylmethanol, 15.3 min; 2,7-dimethylnaphthalene, 24.4
min; 2-(7-methylnaphthyl)methanol, 17.0 min; 7-methyl-2-
naphthoic acid, 17.6 min; 1-ethylnaphthalene, 23.7 min;
1-naphthyl-1-ethanol, 16.6 min; 2-ethylnaphthalene, 24.1 min;
2-naphthyl-1-ethanol, 16.5 min; 2-methylbiphenyl, 23.7 min;
2-biphenylmethanol, 17.4 min; 3-methylbiphenyl, 23.7 min;
3-biphenylmethanol, 17.2 min; 4-methylbiphenyl, 23.7 min;
4-biphenylmethanol, 17.1 min; 4-(4-methylphenyl)phenol, 19.2
min; 4-(4-hydroxyphenyl)benzyl alcohol, 13.0 min; diclofenac,
20.0 min and 4′-hydroxydiclofenac, 16.7 min. The internal
standard retention times were 9-fluorenone, 16.6 min and
4-methoxycinnamic acid, 12.6 min.
Gas chromatography-mass spectrometry (GC-MS) analyses
were carried out on a Shimadzu GC-17A instrument coupled
to a QP5050A MS detector using a Zebron DB-5 MS fused sil-
ica column (Phenomenex; 30 m × 0.25 mm, 0.25 μm) and
helium as the carrier gas. The retention times were as follows
1-naphthol 11.0 min; 1,4-naphthoquinone, 9.8 min; 2,6-
dihydroxynaphthalene, 14.7 min; 2-naphthol, 11.2 min; biphe-
nyl, 9.6 min; 2-phenylphenol, 11.1 min; 4-phenylphenol, 13.2
min; 3-methylbiphenyl, 10.8 min; 3-phenylbenzaldehyde, 13.0
min; 3-biphenylmethanol, 13.8 min; 4-methylbiphenyl, 10.9
min; 4-biphenylmethanol, 13.9 min and 4-(4-methylphenyl)-
phenol, 14.3 min. Chiral and additional GC analysis was
performed on a Shimadzu Tracera GC coupled to Barrier dis-
charge Ionization Detector (BID) detector using a RT®-
BDEXse chiral silica column (Restek; 30 m × 0.32 mm × 0.25
Table 1 Substrate binding and turnover data for CYP101B1 with naph-
thalene, biphenyl, naphthols and phenylphenols. The NADH oxidation fre-
quencies were measured using a ArR : Arx : CYP101B1 concentration ratio
of 1 : 10 : 1 (0.5 μM CYP enzyme, 50 mM Tris, pH 7.4). Rates are reported
a
as mean S.D. (n ≥ 3) and given in nmol nmol P450−1 min−1
CYP101B1/substrate
% HS heme
NADH oxidation ratea (min−1
)
Naphthalene
1-Naphthol
2-Naphthol
15%
65%
40%
15%
35%
40%
40%
∼90 25b
2090 90
560 60
200 10
Biphenyl
2-Phenylphenol
3-Phenylphenol
4-Phenylphenol
90
80
1
8
110 10
a
The NADH oxidation rate is the frequency of NADH oxidation,
including product formation and uncoupling reactions. Note for ease
of comparison the NADH oxidation rates provided in the main text in
the absence of the CYP101B1 enzyme are given in the same units as
b
if the P450 enzyme was present. An estimate of the initial rate of
NADH oxidation as the rate increased over time (Fig. 2).
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1
um) or a SH-Rxi-5 ms fused silica column (Shimadzu, 30 m ×
0.25 mm, 0.25 μm) and helium as the carrier gas. Retention
times are given in the ESI† (Fig. S2).
Data for 4′-(4-hydroxyphenyl)benzyl alcohol. H NMR (500
MHz, d6-acetone) δ 7.54 (m, 2H), 7.50 (m, 2H), 7.40 (m, 2H),
6.92 (m, 2H), 4.65 (s, 2H).
Data for 4′-hydroxydiclofenac46,47 1H NMR (600 MHz, d6-
.
DMSO) δ 7.10 (m, 1H, H4), 7.00–6.96 (m, 1H, H6), 6.93 (s,
2H, 2 × H3′), 6.71 (m, 1H, H5), 6.10 (m, 1H, H7), 3.60 (s, 2H,
H2). 13C NMR (151 MHz, d6-DMSO) δ 176.84 (C1), 155.67
(C4′), 144.68 (C8), 133.08 (C1′), 131.04 (C4), 128.63 (C2′),
127.66 (C6), 123.59 (C3), 119.31 (C5), 116.26 (C3′), 113.97
(C7), 35.98 (C2).
Product isolation and characterisation
To isolate and identify products where standards were not
available
a
whole-cell system utilising the plasmids
pETDuetArx/ArR and pRSFDuetArx/CYP101B1 was used to oxi-
dise substrates. These were grown in LB broth, the cells were
harvested by centrifugation and resuspened in E. coli mini-
mal media (EMM; K2HPO4 7 g, KH2PO4 3 g, Na3 (citrate) 0.5
g, (NH4)2SO4 1 g, MgSO4 0.1 g, 20% glucose (20 mL), and glyc-
erol (1% v/v) per liter) as described previously.27,37 Two ali-
quots of 0.5 mM substrate were added, one at the beginning
of the turnover and the second after 6 h.27,37 The supernatant
(200 mL) was extracted in ethyl acetate (3 × 100 mL), washed
with brine (100 mL) and dried with magnesium sulfate. The
organic extracts were pooled and the solvent was removed by
vacuum distillation and then under a stream of nitrogen. The
products were purified using silica gel chromatography using
a hexane/ethyl acetate stepwise gradient ranging from 80 : 20
to 50 : 50 hexane to ethyl acetate using 2.5% increases every
100 mL. The composition of the fractions was assessed by
TLC and GC-MS and those containing single products
(≥95%) were combined for characterisation. The solvent was
removed under reduced pressure.
Results and discussion
The oxidation of naphthalene and biphenyl by CYP101B1
To investigate the ability of CYP101B1 to oxidise aromatic
compounds we analysed naphthalene and biphenyl, each of
which contain two aromatic benzene rings (Fig. 1). Substrate
binding and enzyme turnover activity were measured using
in vitro assays. Neither biphenyl nor naphthalene induced a
large type I spin-state shift on binding to CYP101B1, both
≤20% high spin (HS) (Table 1, Fig. S1†). The low solubility
and relatively weak binding precluded the accurate determi-
nation of the dissociation constant for both of these
substrates.
Despite this, the addition of naphthalene and biphenyl en-
hanced the rate of NADH oxidation of the ArR/Arx/CYP101B1
system above the leak rate (the rate of NADH oxidation in the
absence of substrate, Table 1). The NADH oxidation activity
of the CYP101B1 system with naphthalene was unusual in
that it accelerated from an initial rate of 90 nmol nmol P450−1
min−1 (henceforth abbreviated to min−1) over the course of
the reaction (Fig. 2a). HPLC and GC-MS analysis of the turn-
overs revealed minimal levels of the expected naphthol prod-
ucts or any others (Fig. S2†). Therefore it appears that the
coupling efficiency, which is the productive use of the NADH
reducing equivalents, of naphthalene oxidation by CYP101B1
must be very low. In vitro turnovers conducted with a large
excess of NADH (4 mM, twelve times the amount in the
The purified products (ranging from ∼3–15 mg) were
dissolved in CDCl3 or d6-acetone and the organics
characterised by NMR spectroscopy. NMR spectra were ac-
quired on an Agilent DD2 spectrometer operating at 500 MHz
1
for H and 126 MHz for 13C or a Varian Inova-600 spectrome-
1
ter operating at 600 MHz for H and 151 MHz for 13C. A com-
1
bination of H, 13C, COSY and HSQC experiments were used
to determine the structures of the products. Assignments of
minor products were made via GC coelution or comparison
of MS spectra of standards published by others (Fig. S5†).
NMR data
Data for 2-(7-methylnaphthyl)methanol. 1H NMR (500
MHz, d6-acetone) δ 7.79 (s, 1H), 7.77 (m, 1H), 7.75 (m, 1H),
7.63 (s, 1H), 7.46 (m, 1H), 7.33 (m, 1H), 4.77 (s, 2H), 2.48 (s,
3H).
Data for 7-methyl-2-naphthoic acid. 1H NMR (500 MHz,
d6-acetone) δ 8.64 (s, 1H), 8.07 (m, 1H), 8.02 (m, 1H), 7.93
(m, 1H), 7.80 (s, 1H), 7.50 (m, 1H), 2.57 (s, 3H). 13C NMR
(126 MHz, d6-acetone) δ 168.38 (C11), 139.78 (C9), 137.26
(C10), 132.31 (C7), 132.06 (C1), 130.52 (C5), 130.36 (C6),
128.85 (C4), 128.51 (C2), 128.09 (C8), 126.83 (C3), 22.35
(C12).
1
Data for 3-biphenylmethanol. H NMR (500 MHz, CDCl3) δ
7.64–7.57 (m, 3H), 7.53 (m, 1H), 7.64–7.57 (m, 3H), 7.39–7.31
(m, 2H), 4.77 (s, 2H).
Data for 4′-(4-methylphenyl)phenol. 1H NMR (500 MHz,
CDCl3) δ 7.45 (m, 2H), 7.43 (m, 2H), 7.22 (m, 2H), 6.88 (m,
2H), 2.38 (s, 3H).
Fig. 1 Substrates tested with CYP101B1.
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Fig. 2 NADH oxidation assays of CYP101B1 with (a) naphthalene and
(b) 1-naphthol (red), 1-naphthol no P450 control (blue) and 1-naphthol,
no Arx and P450 control (black).
normal turnover assays) allowed the identification of low
levels of 1-napthol as a product from the CYP101B1 catalysed
turnover of naphthalene (Scheme 1, Fig. S2a†). Under these
conditions biphenyl was oxidised to 4-phenylphenol but the
level of product formation was also very low (Fig. S2b†).
Scheme 1 The products identified from the CYP101B1 turnovers of
the naphthalene substrates.
To investigate if the increasing NADH oxidation rate ob-
served in the CYP101B1 turnovers of naphthalene arose from
further oxidation of naphthol products, and to determine if
we could identify any product arising from such activity, we
tested 1- and 2-naphthol as substrates. 1-Naphthol induced a
larger spin state shift (65%, HS, Kd 165 μM) on binding to
CYP101B1 than naphthalene, and that induced by 2-naphthol
(40%, HS, Kd 195 μM) lay in between (Table 1, Fig. S1 and
S3†). The NADH oxidation activity of both naphthols was sig-
nificantly greater than naphthalene. That of 1-naphthol (2090
min−1) being almost 4-fold more active than 2-naphthol (560
min−1, Table S1 and Fig. S4†). Analysis of the standard
in vitro assays was inconclusive for product formation with
naphthol substrates due to the low level of metabolites gener-
ated. Using excess NADH (4 mM) we were able to detect low
levels of product from the oxidation of 1- and 2-naphthol
(Fig. S2c and d†). 2-Naphthol oxidation resulted in a single
metabolite which was assigned as 2,6-dihydroxynaphthalene
by GC coelution experiments with an authentic product stan-
dard and analysis of the mass spectrum (Fig. S5†).48 There
was no evidence of either 2-naphthol or the further oxidation
product, 2,6-dihydroxynaphthalene, in the CYP101B1 turn-
overs of naphthalene (Scheme 1, Fig. S2†). The product from
1-naphthol oxidation was assigned as 1,4-naphthoquinone
based on GC coelution and the mass spectrum (Fig.
S2c†).48,49
A small peak, which coeluted with the 1,4-
naphthoquionone, was observed in the turnovers of naphtha-
lene and was also present in the 1-naphthol standard as an
impurity (Fig. S2a†).
Importantly the quantities of naphthalene and naphthol
remaining in these turnovers when excess NADH was used
were also high. Further oxidation products, such as those
arising from oxidative aryl coupling also do not appear signif-
icant.31,50,51 This and the low levels of product formation in
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the above assays indicated that further oxidation of 1-napthol
or 2-napthol is unlikely to be the major reason for oxidation
of NADH resulting in the acceleration in the activity observed
in the naphthalene turnovers (Scheme 1, Fig. S2a†). Control
experiments were performed in which the CYP101B1 enzyme
was omitted from the turnover. The rate of NADH oxidation
in the presence of just the ArR and Arx electron transfer pro-
teins with 1-naphthol (1 mM) was 1560 min−1 (Fig. 2). The
rate of NADH oxidation in the presence of 2-naphthol in
these control reactions, was lower but was similar to that ob-
served in the fully reconstituted in vitro turnover (Table 1).
When both Arx and CYP101B1 were absent the rate of NADH
oxidation dropped dramatically and was similar to the leak
rate in the absence of substrate (Fig. 2). We also tested 1,4-
naphthoquinone with the Arx and ArR electron transfer part-
ners. The addition of 1,4-naphthoquinone resulted in the
rapid oxidation of NADH even at concentrations as low as
8 μM (∼2600 min−1) with lower concentrations resulting in
significant activity (4 μM, 1300 min−1, 50 nM; 320 min−1).
Analysis of these naphthol and naphthoquinone turnovers in
the absence and presence of CYP101B1 showed that the ma-
jority, >95%, of the reducing equivalents ended up as hydro-
gen peroxide.
Similar experiments were performed with the three possi-
ble phenylphenol products of biphenyl oxidation. The addi-
tion of polyphenols to CYP101B1 shifted the spin state to a
greater degree than biphenyl (35–40%, Table 1) but the
NADH oxidation activities were all lower than that of biphe-
nyl and much lower than those of the naphthols (≤110
min−1, Table 1). The NADH oxidation rate in the absence of
CYP101B1 was also similar to those of the phenylphenols in
the normal turnover assays but the levels of hydrogen perox-
ide generated were low for 2- and 4-phenylphenol (<10 μM).
The levels were slightly higher for 3-phenylphenol (∼20 μM).
The data suggests that the unusual behavior of naphtha-
lene oxidation by the ArR/Arx/CYP101B1 system is predomi-
nantly due to an increase in the NADH oxidation activity of
the system in the presence of small quantities of 1,4-
naphthoquinone. This arises from hydroxylation of naphtha-
lene to 1-napthol followed by further oxidation to the qui-
none. The increased rate observed with 1-naphthol is most
likely due to low levels of a quinone impurity. Rather than
chondrial
electron
transfer
systems
containing
ferredoxins.5,12,56–58 This redox cycling competes with
electron transfer to CYP101B1 and therefore reduces the
levels of metabolite formation. This resulted in low levels of
productive P450 catalysis despite high levels of NADH oxida-
tion activity and should be considered an additional form of
uncoupling in P450 systems. Further investigations are re-
quired to determine the exact mechanism of the transfer of
the electrons from the ferredoxin to the naphthoquinone and
the subsequent generation of hydrogen peroxide. It would
also be important to ascertain if this behaviour is generic
across the class I electron transfer systems and to understand
the structural features of the molecules which can interfere
with the electron transfer process in this way.57,58 The in-
crease in the rate of futile redox cycling during the turnovers
was greatest for the oxidation products arising from naphtha-
lene oxidation to 1-naphthol and 1,4-naphthoquinone with
minimal activity of this type being observed with
phenylphenols, biphenylmethanols or naphthylmethanols
(vide infra). We note that the 1,4-naphthoquinone and 2,6-
dihydroxynaphthalene metabolites which arose from the turn-
overs of the naphthols are the same as those observed in the
metabolism of these substrates by mammalian P450 en-
zymes.59,60 This phenomenon is likely to have important im-
plications for understanding the toxicity of polyaromatic hy-
drocarbons and their oxygenated metabolites such as
quinones.61,62
Oxidation of substituted two ring aromatic molecules by
CYP101B1
The low productive monooxygenase activity of CYP101B1 with
naphthalene and biphenyl, led us to investigate similar sub-
strates which contain more reactive benzylic aliphatic C–H
bonds (Fig. 1). 1-Methylnaphthalene bound to CYP101B1,
with a spin state shift of 55% HS (Fig. S1†) which is greater
than naphthalene and almost as high as 1-naphthol. We were
able
to
measure
the
dissociation
constant
of
1-methylnaphthalene binding to CYP101B1, Kd 63 μM (Fig. 3).
1,4-naphthoquinone acting as
a substrate or promoting
established P450 uncoupling pathways it accepts electrons
from Arx and in the aerobic environment of the turnover was
able to reduce oxygen. Oxygen is likely to be reduced to
superoxide which dismutates to hydrogen peroxide and sin-
glet oxygen. This would be similar to reactivity reported for
NADPH cytochrome P450 reductase (FAD/FMN cofactors)
with quinones and nitroaromatics and that observed with the
leaking of reducing equivalent in the adrenodoxin mitochon-
drial system.52–54 The reduction of oxygen by the mitochon-
drial electron transfer system in the absence of substrate and
the P450 enzyme has been described before.54,55 The acceler-
ation of this uncoupling pathway by exogenous substrates
has not been described in great detail with bacterial or mito-
Fig.
3
Dissociation
constant
analysis
of
CYP101B1
with
1-methylnaphthalene (2.1 μM enzyme/Kd 63
A390 − A420).
5 μM, peak to trough,
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This was weaker than that of norisoprenoids, monoterpenoid
acetates and phenylcyclohexane but tighter than most mono-
terpenoids.41 The NADH oxidation activity (240 min−1) was
higher than the initial rate observed with naphthalene and it
was linear throughout the turnover (Table 2, Fig. S4†). HPLC
analysis revealed the formation of a single product, which
coeluted with an authentic standard of 1-naphthylmethanol
(Scheme 1 and Fig. 4). The amount of 1-naphthylmethanol
was quantitated and both the product formation rate, 38
min−1, and coupling efficiency, 16%, for the turnovers were
determined.
methylnaphthalenes, the coupling of reducing equivalents to
product formation decreased (both <10%, Table 2). The
lower coupling efficiencies and product formation rates ob-
served for both of these substrates compared to the methyl-
naphthalenes is consistent with the reduced spin state shifts
observed on substrate binding.
Chiral GC analysis showed that the turnover of
2-ethylnaphthalene generated a single enantiomer which
coeluted with (R)-(+)-1-(2-naphthyl)ethanol (Fig. S2(e)†). The
enantioselectivity of 1-ethylnaphthalene oxidation was lower
with both enantiomers of 1-naphthyl-1-ethanol were gener-
ated though there was a significant excess of one stereo-
isomer (∼84% ee, Fig. S2(f)†). Overall the larger ethyl naph-
thalenes bind tightly but do not appear to be positioned as
well in the active site of CYP101B1 for efficient C–H bond oxi-
dation when compared to the methyl naphthalenes. However
they must be positioned in such a way that there is a strong
preference for abstraction of one of the enantiotopic hydro-
gens of the methylene group.
2,7-Dimethylnaphthalene induced a lesser spin state shift
on binding to CYP101B1, 30% HS, than either 1- or
2-methylnaphthalene. However the binding affinity of this
substrate was higher than any other tested (Kd 14 μM,
Table 2). The activity of CYP101B1 with 2,7-
dimethylnaphthalene was high as measured by NADH oxida-
tion (448 min−1, Table 1). The product formation rate was the
greatest of all the substituted naphthalenes (79 min−1) and
the coupling efficiency (18%) was comparable to that of the
methylnaphthalenes. The in vitro turnover produced a single
oxidation product (Fig. 4d). The most likely product, which
could arise from benzylic C–H bond hydroxylation of 2,7-
dimethylnaphthalene, was not available for coelution so a
whole-cell oxidation turnover was conducted to generate me-
tabolites for characterisation. The in vivo turnover converted
all the added substrate (1 mM, Fig. 4d) into a single oxida-
tion product after 20 hours. The metabolite from the whole-
cell oxidation was isolated and identified by NMR as
7-methyl-2-naphthoic acid. The characteristic signal was the
chemical shift of 168.38 ppm in the 13C NMR, corresponding
to a carboxylic acid, and this combined with the lack of
2-Methylnaphthalene induced a smaller spin state shift
with a lower binding affinity, 40% HS and Kd 135 μM, com-
pared to 1-methylnaphthalene (Fig. S1 and S3†). The NADH
oxidation activity of CYP101B1 in the presence of
2-methylnaphthalene was 212 min−1.
A single product,
2-naphthylmethanol, identified by HPLC coelution, was
formed at a product formation rate of 57 min−1 (Scheme 1
and Fig. 4). The higher activity of 2-methylnaphthalene oxida-
tion compared to 1-methylnaphthalene arose as a result of
greater coupling efficiency of the reducing equivalents to
product formation, 26% (Table 2).
The effect of the size of the alkyl substituent on the bind-
ing and biocatalytic parameters of CYP101B1 was investigated
by testing 1- and 2-ethylnaphthalene. Addition of both sub-
strates induced spin state shifts of only 20% HS, which was
lower than the equivalent methylnaphthalenes but compara-
ble to naphthalene. The dissociation constants indicated that
binding was tighter than the methylnaphthalenes (Table 2,
Fig. S3†). The NADH oxidation activity of CYP101B1 with
1-ethylnaphthalene (482 min−1) was greater than that of
2-ethylnaphthalene (284 min−1) and both methylnaphthalenes
(Table 2). A single product was generated from each of the
turnovers of 1- and 2-ethylnaphthalene. These coeluted with
1-naphthyl-1-ethanol and 2-naphthyl-1-ethanol, respectively
(Scheme 1, Fig. 4 and Fig. S6†). The product formation rate
with 1-ethylnaphthalene (18 min−1) was lower than
2-ethylnaphthalene (20 min−1). Despite the higher NADH oxi-
dation rates and the greater reactivity of the benzylic methy-
lene group of these substrates, versus the methyl group of the
Table 2 Substrate binding, turnover and coupling efficiency data for the turnovers of CYP101B1 with alkyl substituted naphthalene and biphenyl sub-
strates. The turnover activities were measured using a ArR : Arx : CYP101B1 concentration ratio of 1 : 10 : 1 (0.5 μM CYP enzyme, 50 mM Tris, pH 7.4). N is
the NADH oxidation rate (as defined in Table 1), PFR the product formation rate and C is the coupling efficiency, which is the percentage of NADH
utilised for the formation of products. Rates are reported as mean S.D. (n ≥ 3) and given in nmol nmolCYP−1 min−1
a
CYP101B1/substrate
% HS
Kd (μM)
N
PFR
C %
1-Methylnaphthalene
2-Methylnaphthalene
2,7-Dimethylnaphthalene
1-Ethylnaphthalene
2-Ethylnaphthalene
2-Methylbiphenyl
3-Methylbiphenyl
4-Methylbiphenyl
4-Biphenylmethanol
55%
40%
30%
20%
20%
20%
20%
20%
10%
63
135
14
40
45
29
17
30
—
240 17
212 24
448 44
38 10
57 18
79 10
16
26
18
4
482
284
257
7
5
9
18
20
35
3
1
8
7
14
23
22
12
127 10
176 10
30 12
39
11
7
4
106
8
a
See Fig. S3 for details.
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Fig. 4 HPLC analysis of the in vitro CYP101B1 turnovers of (a) 1-methylnaphthalene overlaid with 1-naphthylmethanol, (b) 2-methylnaphthalene
overlaid with 2-naphthylmethanol, (c) 1-ethylnaphthalene overlaid with 1-naphthyl-1-ethanol and (d) 2,7-dimethylnaphthalene overlaid with the
whole-cell oxidation turnover of the same substrate. Turnovers are shown in black the internal standard (IS), impurities (*) and substrate are la-
belled. The HPLC runs of the authentic product standards (A) are shown in red for (a–c). In (d) the whole-cell product turnover which generated
7-methyl-2-naphthoic acid (B) is in red. A different internal standard, 4-methoxycinnamic acid, was used in the turnover of 1-ethylnaphthalene as
9-fluorenone coeluted with the 1-naphthyl-1-ethanol product.
1
CH2OH peak and presence of all six aromatic protons in H
NMR allowed identification (Fig. S7†). However this
naphthoic acid metabolite did not coelute with the product
from the in vitro enzyme turnovers. 7-Methyl-2-naphthoic acid
was reduced using an excess of lithium aluminium hydride
and the single product generated was found to coelute with
that obtained in the enzyme turnover assays. NMR analysis of
this product confirmed it was the expected metabolite,
2-(7-methylnaphthyl)methanol (Fig. S8†). In the in vitro turn-
over the limiting amount of NADH compared to substrate,
precludes the further oxidation of 2-(7-methylnaphthyl)-
methanol but in the whole-cell oxidation system all of the
substrate was converted to the alcohol product, which is then
further oxidised to the carboxylic acid (Scheme 1). Presum-
ably this occurs via the aldehyde, though this intermediate
product was not observed in either the in vivo or in vitro turn-
overs. The total conversion of 1 mM substrate into the car-
boxylic acid in the whole-cell oxidations corresponds to ap-
proximately 4500 turnovers based on an enzyme
concentration of 0.65 μM.37
Methylbiphenyls with the substituent at the 2-, 3- and
4-positions were also investigated (Fig. 1). All three com-
pounds induced a small shift to the high spin state upon
binding to CYP101B1 (∼20%, HS, Fig. S1†). This was greater
than biphenyl but lower than the equivalent phenylphenols
(Table 1). The binding affinities were higher than the mono-
substituted naphthalenes with that of 3-methylbiphenyl
approaching that of 2,7-dimethylnaphthalene (Table 2, Fig.
S3†).
The NADH oxidation activity of CYP101B1 after addition
of the methylbiphenyls was similar to that of biphenyl but
faster than all the phenylphenols (Table 1) and varied as
follows; 2-methylbiphenyl
>
1-methylnaphthalene
>
2-methylnaphthalene > 4-methylbiphenyl > 3-methylbiphenyl.
That of 3-methylbiphenyl (127 min−1) was approximately half
that of 2-methylbiphenyl (257 min−1, Table 2). Overall the
product formation activity of 2-methylbiphenyl and
3-methylbiphenyl were similar (30 to 35 min−1; Table 2). The
in vitro turnovers of 2- and 3-methylbiphenyl generated 2- and
3-biphenylmethanol, respectively, as the sole products
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(Scheme 2 and Fig. 5). 2-Biphenylmethanol was identified by
HPLC coelution experiments with an authentic product stan-
dard (Fig. 5a). 3-Biphenylmethanol was generated using the
whole-cell oxidation system, purified by silica chromatogra-
phy and identified by NMR, the 2H signal at 4.77 ppm and
the signals of nine hydrogens in the aromatic region allowed
characterisation (Fig. S9†). There was no evidence of further
oxidation of either product when using the whole-cell oxida-
tion system, despite only a small amount of 3-methylbiphenyl
substrate remaining. Low levels of 3-phenylbenzaldehyde were
detected by GC-MS analysis of the in vitro turnovers of
3-methybiphenyl when excess NADH (4 mM) was used (Fig. S5†).
The turnover of 4-methylbiphenyl was more complex and
resulted in the formation of two metabolites. The product
formation rate of the 4-methylbiphenyl (39 min−1) and the ef-
ficiency (22%) resembled those of 2- and 3-methylbiphenyl
despite the different P450 mechanisms involved in aromatic
versus
aliphatic
oxidation.63
The
minor
product
Scheme
2 The products formed from the CYP101B1 catalysed
4-biphenylmethanol (30%) was identified via HPLC coelution
with an authentic product standard (Fig. 5c). As with 2,7-
dimethylnaphthalene we considered that the major product
oxidation of biphenyl substrates.
Fig. 5 HPLC analysis of the in vitro CYP101B1 turnovers of (a) 2-methylbiphenyl overlaid with 2-biphenylmethanol, (b) 3-methylbiphenyl, (c)
4-methylbiphenyl overlaid with 4-biphenylmethanol and (d) 4-methylbiphenyl overlaid with the turnover of 4-biphenylmethanol. Turnovers are
shown in black the internal standard (IS), impurities (*) and substrate are labelled. The HPLC chromatograms of the authentic product standards (A)
are shown in red for (a) and (b). In (c) and (d) the peak labelled A is the 4′-(4-methylphenyl)phenol major product. In (c) the peak labelled B is
4-biphenylmethanol and in (d) it is 4′-(4-hydroxyphenyl)benzyl alcohol.
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(70%) may arise from further oxidation of the
4-biphenylmethanol product generating the aldehyde or the
carboxylic acid. Whole-cell oxidation turnovers were
conducted with 4-biphenylmethanol and 4-methylbiphenyl.
The product from the oxidation of 4-biphenylmethanol did
not coelute with any peaks in the 4-methylbiphenyl turnover
(Fig. 5d). Therefore the unidentified product from the whole-
cell turnover of 4-methylbiphenyl was isolated by silica col-
umn chromatography and characterised. The 3H methyl sin-
glet (2.38 ppm) and the distinctive signals of the two para-
substituted rings (8H) in the aromatic region allowed identifi-
cation as 4′-(4-methylphenyl)phenol (Scheme 2, Fig. S10†).
The product of CYP101B1 catalysed oxidation of
4-biphenylmethanol was also isolated and characterised by
NMR as 4′-(4-hydroxyphenyl)benzyl alcohol (Fig. S11†). The
NADH oxidation rate, product formation rate and the cou-
pling efficiency of the turnover of 4-biphenylmethanol were
also measured and found to be lower than those of the
methylbiphenyls (Table 2).
4-Methylbiphenyl is unusual in that the major product
arises from oxidation at an aromatic C–H bond with the mi-
nor product being hydroxylation at the more reactive benzylic
methyl group. In addition, unlike 2,7-dimethylnaphthalene
further oxidation to the carboxylic acid is less favoured when
compared to aromatic oxidation on the other benzene ring.
This substrate must be bound in at least two orientations
with the most favourable leading to oxidation of the aromatic
C–H bond. In the second orientation the methyl group would
be located close enough to the heme iron-oxo intermediate to
favour C–H bond abstraction at the sp3 hydrogens.
The alcohol oxygen of 4-biphenylmethanol, may favour
binding of the substrate in an orientation with the para aro-
matic ring closest to the heme iron. This would be in broad
agreement with the regioselectivity observed with the oxygen
containing norisoprenoids and monoterpenoid acetates. It is
of note that 4-methylbiphenyl and 4-biphenylmethanol are the
only two substrates tested so far with CYP101B1 where oxida-
tion at an aromatic C–H bond is preferred over aliphatic oxi-
dation. The CYP101B1 catalysed oxidation of phenylcyclo-
hexane, p-cymene and the other methyl substituted
naphthalenes and biphenyls favoured aliphatic hydroxylation.
4-Methylbiphenyl and 4-biphenylmethanol must have a shape
which allows them to bind in a different orientation in the
substrate binding pocket of CYP101B1.
The majority of the substrates are presumably held in ori-
entations such that that the initial formation of the arene ox-
ide intermediate in the NIH shift mechanism64,65 or Shaik's
porphyrin mediated proton-shuttle mechanism,63 required
for aromatic oxidation, must be impeded (Scheme S1†). If ar-
omatic oxidation does occur via an arene oxide intermediate
this pathway could also generate 3-(4-methylphenyl)phenol.
This product was not observed, perhaps as it would be formed
via a less favoured cationic intermediate (Scheme S1†).
methyl or ethyl substituent. This is in accordance with the re-
activity of the C–H bonds in these substrates. The preference
for aliphatic versus aromatic oxidation, may reflect the differ-
ent mechanisms and energies of aromatic versus aliphatic
C–H bond oxidation. The size and planar nature of the naph-
thalene and biphenyl molecules may limit the motion of the
substrate in the active site. The alkyl group of the substrates
is likely to be held close to the heme in at least one of the
preferred binding orientations in order to allow the genera-
tion of the observed products.
Finally we used CYP101B1 to oxidise the nonsteroidal
anti-inflammatory drug molecule diclofenac. Diclofenac con-
sists of two aromatic rings linked through an amine group
(diphenylamine like), and contains a carboxylic acid moiety
as well as two chlorines (Fig. 1). The carboxylate group oxy-
gen could potentially mimic the ketone functionality of
norisoprenoids, which bind efficiently to CYP101B1. The
binding of diclofenac induced a minimal shift in the spin
state of CYP101B1, ∼5% HS, upon binding. The NADH oxida-
tion rate could not be accurately determined as the substrate
absorbs strongly at 340 nm. However HPLC analysis of a
whole-cell oxidation turnover of diclofenac indicated that a
single product was formed (Fig. S12†). This was generated in
larger quantities using the whole-cell oxidation system and
purified by silica gel chromatography. The product was iden-
tified as 4′-hydroxydiclofenac by matching its NMR data to
that reported in the literature (Scheme 3).46,47
As
a control we tested diphenylmethane with the
CYP101B1 enzyme. This was a poor substrate for CYP101B1
but showed low activity with hydroxylation occurring at the
benzylic C–H bonds (Fig. S5 and S13†). The selective oxida-
tion of the drug molecule diclofenac by “wild-type” CYP101B1
is a promising step towards using this system for drug me-
tabolite production from molecules with similar structural
features to naphthalene, biphenyl and diclofenac. CYP101B1
could be used to mimic the drug oxidising properties of
mammalian P450 enzymes and thus allow the facile genera-
tion of hydroxylated drug metabolites in larger quantities.
Previously we have shown than CYP101B1 has a strong
preference for substrates containing a carbonyl oxygen group
such as norisoprenoids and monoterpenoid esters which pre-
sumably arises through some hydrophilic interactions with
the residues that line the active site of the enzyme. The bind-
ing affinity of the more apolar aromatic substrates tested
here could therefore be improved using protein engineering
to increase the hydrophobicity of the active site. The size of
the substrate binding pocket could also be modified to better
With the exception of 4-methylbiphenyl the alkyl
substituted aromatic substrates hydroxylated by CYP101B1
with high regioselectivity for the benzylic C–H bonds of the
Scheme 3 The product formed from the CYP101B1 turnover with
diclofenac. The numbering system used for diclofenac is the same as
that from Marco-Urrea et al.46
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currently no structural information available for the
CYP101B1 enzyme. These approaches would lead to signifi-
cant improvement in the coupling efficiency and product for-
mation rate leading to more efficient biocatalytic oxidation of
naphthalene and biphenyl based substrates.
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Conclusions
We have shown that CYP101B1 is capable of oxidising a
range of molecules containing two aromatic rings. The oxida-
tion of naphthalene and biphenyl were inefficient and one of
the products generated during the turnover of naphthalene
could compete with CYP101B1 for electrons from the ArR/Arx
electron transfer system via redox cycling. This form of
uncoupling of reducing equivalents would make regular cyto-
chrome P450 enzymes unsuitable biocatalysts for naphtha-
lene oxidation and using alternate systems such as
perooxygenases may be more effective. CYP101B1 was able to
efficiently and selectively oxidise a range of alkyl substituted
naphthalene and biphenyl substrates. The product formation
activities are comparable or in excess to other available sys-
tems and the selective oxidation and total turnovers achieved
using a whole-cell oxidation system with CYP101B1 with
these hydrophobic substrates is suited to the biocatalytic
generation of hydroxylated products. The reactions are, in
general, regioselective for oxidation at the more reactive
benzylic carbons. 4-Methylbiphenyl was the exception being
preferably oxidised to 4′-(4-methylphenyl)phenol with
4-biphenylmethanol being formed as a minor product. The
oxidation of both ethylnaphthalenes by CYP101B1 was also
highly enantioselective. The aromatic drug molecule
diclofenac, which also contains two aromatic rings, was
regioselectively hydroxylated to 4′-hydroxydiclofenac. When
incorporated into a whole-cell oxidation system, with the
physiological electron transfer partners, ArR and Arx,
CYP101B1 was capable of generating the oxidised metabolites
of the substituted naphthalenes and biphenyls in sufficient
amounts for characterisation. In certain instances further oxi-
dation products were formed using the whole-cell oxidation
system for example 2,7-dimethylnaphthalene oxidation gener-
ated the further oxidation product 7-methyl-2-naphthoic acid
as the only metabolite.
Acknowledgements
21 M. J. Pecyna, R. Ullrich, B. Bittner, A. Clemens, K. Scheibner,
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