The efficient and selective biocatalytic oxidation of norisoprenoid and aromatic substrates by CYP101B1 from Novosphingobium aromaticivorans DSM12444

Article abstract of DOI:10.1039/c4ra14010a

CYP101B1 from Novosphingobium aromaticivorans DSM12444 is a homologue of CYP101A1 (P450cam) from Pseudomonas putida and the CYP101D1, CYP101D2 and CYP101C1 enzymes from the same bacterium. CYP101B1 binds norisoprenoids more tightly than camphor and efficiently hydroxylates substrates in combination with ferredoxin reductase, ArR, and [2Fe-2S] ferredoxin, Arx, electron transfer partners. The norisoprenoids, α-ionone and β-damascone are both oxidised by CYP101B1 with high product formation activity, >500 min^-1. α-Ionone oxidation occurred regioselectively at the allylic C3 position while β-damascone was hydroxylated predominantly at C3, 86%, with the main competing minor product arising from oxidation at the allylic C4 position (11%). When incorporated into a whole-cell oxidation system, with ArR and Arx, CYP101B1 is also capable of oxidising the aromatic compound indole. Other aromatic molecules including phenylcyclohexane and p-cymene were tested and both were hydroxylated by CYP101B1. Phenylcyclohexane was selectively 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. Trans-4-Phenylcyclohexanol was formed with a product formation rate of 141 min^-1 and was five times more active than the oxidation of p-cymene. This journal is

Full text of DOI:10.1039/c4ra14010a

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The efficient and selective biocatalytic oxidation of norisoprenoid and aromatic
substrates by CYP101B1 from Novosphingobium aromaticivorans DSM12444
Emma A. Hall and Dr. Stephen G. Bell*,
School of Chemistry and Physics, University of Adelaide, SA 5005, Australia
* To whom correspondence should be addressed.
Stephen G. Bell (stephen.bell@adelaide.edu.au) Tel: +61 8 83134822; Fax: +61 8 83034380
TOC
O
O
CYP101B1
CYP101B1
HO
3-hydroxy- -ionone
-ionone
OH
phenylcyclohexane
trans-4-phenylcyclohexanol
CYP101B1 from Novosphingobium aromaticivorans oxidises ionone derivatives and
phenylcyclohexane with high activity and regioselectivity.
1

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Abstract
CYP101B1 from Novosphingobium aromaticivorans DSM12444 is a homologue of CYP101A1
(P450cam) from Pseudomonas putida and the CYP101D1, CYP101D2 and CYP101C1 enzymes
from the same bacterium. CYP101B1 binds norisoprenoids more tightly than camphor and
efficiently hydroxylates substrates in combination with ferredoxin reductase, ArR, and [2Feꢀ2S]
ferredoxin, Arx, electron transfer partners. The norisoprenoids, αꢀionone and βꢀdamascone are both
oxidised by CYP101B1 with high product formation activity, > 500 min−1. αꢀIonone oxidation
occurred regioselectively at the allylic C3 position while βꢀdamascone was hydroxylated
predominantly at C3, 86 %, with the main competing minor product arising from oxidation at the
allylic C4 position (11 %). When incorporated into a wholeꢀcell oxidation system, with ArR and
Arx, CYP101B1 is also capable of oxidising the aromatic compound indole. Other aromatic
molecules including phenylcyclohexane and pꢀcymene were tested and both were hydroxylated by
CYP101B1. Phenylcyclohexane was selectively oxidised to transꢀ4ꢀphenylcyclohexanol while
pꢀcymene was hydroxylated at the benzylic carbons to yield a mixture of isopropylbenzyl alcohol
and pꢀα,αꢀtrimethylbenzylalcohol. transꢀ4ꢀPhenylcyclohexanol was formed with a product
formation rate of 141 min−1 and was five times more active than the oxidation of pꢀcymene.
2

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Introduction
Cytochrome P450 (CYP) enzymes constitute a superfamily of hemeꢀcontaining monooxygenases
which, among other activities, are able to insert one oxygen atom from dioxygen into chemically
inert carbonꢀhydrogen bonds with high regioꢀ and stereoselectivity.1, 2 They are of great interest due
to their myriad of physiological roles and potential application in the biocatalytic synthesis of fine
chemicals under mild conditions.3ꢀ5 Dioxygen activation requires two electrons that are usually
derived from NAD(P)H and delivered to the CYP enzymes by electron transfer proteins.6 Numerous
CYP enzymes with potentially interesting and desirable activities have been discovered but many of
these are orphaned, with no electron transfer partners located nearby in the genome, and their
activities are often compromised in hybrid electron transfer systems.7, 8 Therefore, the identification
and characterisation of CYP enzymes with functional and highly active electron transfer chains is
imperative in order to optimise the catalytic activity of this class of enzymes across a broad range of
substrates and reaction types.
We have reported the heterologous production and purification of the majority of the sixteen
P450 enzymes from Novosphingobium aromaticivorans DSM12444.5, 9 Potential substrates for
many of these enzymes have been identified and these span a broad range of organic molecules
including terpenoids, linear alkanes and polyaromatic hydrocarbons.5, 10, 11 The CYP enzymes of
this bacterium are presumably involved in metabolic processes which enable it to survive in the
oligotrophic environments where it lives.12, 13 A class I electron transfer system, consisting of a
flavinꢀdependent ferredoxin reductase, ArR, and a [2Feꢀ2S] ferredoxin, Arx, has been identified
which is able to reconstitute the monooxygenase activity of at least five of these enzymes
(CYP101B1, CYP101C1, CYP101D1, CYP101D2 and CYP111A2).5, 9, 14 Wholeꢀcell systems
capable of product formation on the gramꢀperꢀlitre scale in shake flasks have also been
constructed.9 Like P450cam (CYP101A1), CYP101D1 and CYP101D2 both bind and oxidise
camphor (yielding ≥98 % 5ꢀexoꢀhydroxycamphor). While CYP101B1 catalyses the unselective
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oxidation of camphor generating four products, CYP101C1 does not. CYP101C1 and CYP101B1
are able to bind and oxidise βꢀionone. Crystal structures of CYP101D1, CYP101D2 and CYP101C1
and the ArR and Arx electron transfer partners reveal striking similarities between these CYP101
family enzymes and CYP101A1.15ꢀ18 The enzymes also provide templates in order to understand
how variations in the amino acid sequence of closely related P450 enzymes can lead to different
substrate binding preferences, altered product selectivity and a prediliction for specific electron
transfer partners.15ꢀ17
CYP101B1 when combined with ArR and Arx in a wholeꢀcell oxidation system was found
to turn the culture medium blue indicating that it is able to hydroxylate indole to 3ꢀhydroxyindole
which can then dimerise to form indigo.9, 19 Novosphingobium species are able to degrade a wide
variety of aromatic hydrocarbons and therefore CYP101B1 has the potential to be a biocatalyst for
the oxidation of these hydrophobic substrates.9, 12, 13 Here we report our investigations into the
substrate range of CYP101B1 and show it is a highly active and regioselective biocatalyst for the
oxidation of αꢀionone and βꢀdamascone. We also investigate the ability of CYP101B1 to oxidise
hydrophobic aromatic ring containing substrates such as phenylcyclohexane and pꢀcymene.
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Results
Phylogenetic analysis of CYP101B1
CYP101B1 shares 46 % sequence identity with CYP101D2 and 44 % with CYP101A1, and
CYP101D1 (Fig. 1 and Table S1). A BLAST search revealed only one CYP enzyme in the NCBI
database, from Novosphingobium tardaugens NBRC 1675, with a greater than 50 % identity to
CYP101B1 (59 %). Other CYP enzymes identified in the 45ꢀ50 % range all have greater sequence
homology with CYP101A1 or CYP101D2. As a result there are currently only two identified
members of the CYP101B subfamily and both are found in species of Novosphingobium bacteria.
CYP101B1 has little sequence similarity with its closest structurally characterised relative outside
of the CYP101 family, CYP176A1 (P450cin) or other ionone binding P450 monooxygenases such
as CYP109D1 and CYP264B1, or those from Streptomyces species which are known to oxidise
norisoprenoids (Table S1, vide infra).20ꢀ23
Despite the relatively low sequence similarity with CYP101B2 from N. tardaugens a
comparison of the residues that are likely to make up the active site and substrate access channel
revealed the almost complete conservation of amino acids (Fig. 1 and Table S2). The only
difference was the conservative replacement of Ile236 in CYP101B1 with Val244 in CYP101B2.
This is the equivalent position to Val247 in CYP101A1 a residue located in the active site which is
important for substrate binding.24 This comparison also revealed important differences in the active
sites of the two CYP101B subfamily enzymes when compared to the other members of the CYP101
family. For example sequence alignment reveals that, Val91 aligns with Phe and Trp residues
(Phe87; CYP101A1 numbering given) and His100 with Tyr residues (Tyr96). However there are
similarities with Thr173 aligning with Thr residues (Thr185) and Val284 and Val385 with Val
residues (Val295 and Val396; Table S2).25, 26 Additionally residues such as Thr85 and Ile384
(Thr101 and Ile395) align with other Thr and Ile residues in all the CYP101 family members except
CYP101C1.16
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In CYP101B1 there are also changes to the residues that comprise the potassium binding
site and salt bridge network of CYP101A1. Glu84 and Tyr96 of the CYP1011A1 potassium binding
site are replaced with Arg72 and His80, respectively in CYP101B1. In the salt bridge network most
of the residues are conserved but there are modifications with Asp97 being replaced by Gly81 and
Lys178 by Arg166. These differences when taken together with those in active site are likely to
account for the lower affinity for camphor and the altered substrate profile and CꢀH bond oxidation
selectivity of CYP101B1.5 It is of note that the number of different active site residues in
CYP101B1 compared to CYP101A1 is greater than in both CYP101D enzymes but fewer when
compared to CYP101C1 (Table S2 and Fig. 1 and 2).16
The acid alcohol pair of the Iꢀhelix, which is involved in proton delivery and oxygen
activation, and the glutamate and arginine pair (EXXR), which is proposed to be involved in heme
incorporation, are conserved in CYP101B1.27 The normally conserved phenylalanine amino acid
seven residues towards the Nꢀterminus from the heme iron ligating cysteine, Cys346, is also present
(Fig. 1).28 One potentially interesting point of difference in the CYP101B1 sequence is the presence
of a proline residue, Pro347, one residue to the the Cꢀterminal side of the proximal cysteine. Proline
occurs naturally at this position in chloroperoxidase and P450s such as CYP7A1, and CYP121A1.27,
29, 30
The introduction of proline residues at this position is known to have distinctive effects in
other CYP enzymes, with the Leu358Pro mutant of CYP101A1 exhibiting the same spectral
32
perturbations induced by putidaredoxin binding.31,
The Ile401Pro mutant of CYP102A1
(P450Bmꢀ3) displays enhanced oxidation rates with nonꢀnatural substrates and exhibits unusual
electronic properties and structural changes.33, 34
The substrate range of CYP101B1
Previously we have shown that βꢀionone induces a large type I spin state shift (≥ 95 %) on substrate
binding (Kd, 0.23 ± 0.1 µM) to CYP101B1.5, 9 The activity of CYP101B1 has been reconstituted
using Arx, which is genomically associated with CYP101D2, in conjunction with ArR. Under
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standard assay conditions (0.5 µM CYP: 5 µM Arx) CYP101B1 oxidised βꢀionone with high
NADH consumption (1600 ± 100 nmolꢀnmol P450−1ꢀmin−1, henceforth abbreviated to min−1) and
product formation rates (1010 ± 60 min−1). This indicates that βꢀionone may be, or is at least a close
structural analogue of, the physiological substrate of CYP101B1. In this work similar spin state
shifts and dissociation constants are observed with the closely related substrate αꢀionone, ≥ 95 %
and 0.26 ± 0.04 µM (Fig. 3 and 4). The norisoprenoid βꢀdamascone, which switches the position of
the ketone and alkene moieties on the butenone side chain, displayed a lower spin state shift and
weaker binding, 80 % and 8.3 ± 0.9 µM (Fig. 3 and 4). Both substrates yielded fast NADH turnover
frequencies with CYP101B1, αꢀionone (1380 ± 140 min–1) and βꢀdamascone (931 ± 13 min–1),
though both are slower than βꢀionone (Table 1).
The oxidation of βꢀdamascone generated one major product (∼86 %) and two minor
products (Scheme 1 and Fig. 5). GC analysis revealed two major products from αꢀionone oxidation
in 63:37 % ratio (Fig. 5). The product formation rate of αꢀionone and βꢀdamascone were similar,
663 ± 56 min−1 and 562 ± 12 min−1, with the coupling of NADH to product formation for
βꢀdamascone (60 %) compensating for its lower NADH turnover frequency when compared to
αꢀionone (Table 1).
Using a wholeꢀcell oxidation system which produced CYP101B1, Arx and ArR in E. coli we
were able to generate 100 – 200 mg of a mixture of the products formed from each norisoprenoid.
The wholeꢀcell biotransformation of αꢀionone generates three products all in roughly equal amounts
(30:37:33 % ratio, Fig. 5). The first two of these correspond to the two products observed in vitro
and have masses indicating they are hydroxylated monooxygenase products (Mass 208.2 AMU).
The third product, which was not observed in the in vitro turnovers, has a mass indicating it arises
from further oxidation of an alcohol product to a ketone (Mass 206.1 AMU). The wholeꢀcell
oxidation of βꢀdamascone and βꢀionone yielded predominantly one main product (Fig. 5) which
corresponded to the products of the in vitro turnover systems. βꢀDamascone oxidation produced two
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minor products in low yields. Purification and isolation of the individual products was carried out
using silica gel chromatography with only the fractions containing predominantly a single
compound being taken forward for characterisation. We isolated between 2 ꢀ 10 mg pure product
from each turnover of βꢀionone, αꢀionone and βꢀdamascone after column chromatography, with the
remaining product being found in mixed fractions. These pure samples were used to identify the
products using NMR and mass spectrometry (Experimental section and ESI). The products of
αꢀionone oxidation were identified as cisꢀ and transꢀ3ꢀhydroxyꢀαꢀionone and 3ꢀoxoꢀαꢀionone
(Scheme 1). The lack of significant quantities of the further oxidation product 3ꢀoxoꢀαꢀionone in
the in vitro enzyme assays suggests it is only produced when the amount of αꢀionone substrate is
limited relative to the two hydroxylated products or that the increased solubility of the alcohols
allow them to enter the cells more readily than the less soluble substrate. Furthermore the
proportion of the cis and trans isomers were different in the wholeꢀcell oxidation turnover
suggesting that the trans isomer is more readily converted to 3ꢀoxoꢀαꢀionone. The regioselective
oxidation of αꢀionone at C3 and the absence of any evidence of the epoxidation of the double bond
suggests that the substrate must be held in the active site with both the C3 pseudoequatorial and
pseudoaxial C−H bonds held close to the heme iron.
βꢀDamascone oxidation by CYP101B1 yielded 86 % of the majority product,
3ꢀhydroxyꢀβꢀdamascone, and 11 % of the more abundant minor product, 4ꢀhydroxyꢀβꢀdamascone.
(Scheme 1). The other minor product that was produced (≤ 3% of an in vitro reaction) was not
generated in a sufficiently pure enough form in high enough yields for NMR characterisation.
GCꢀMS analysis indicated that it is likely to be a further oxidation product of one of the alcohols
(Mass 206.2 AMU). These results are similar to those obtained previously with βꢀionone. The major
product from βꢀionone was confirmed by NMR as 3ꢀhydroxyꢀβꢀionone and the minor product
coꢀeluted with 4ꢀhydroxyꢀβꢀionone produced by CYP101C1 and CYP102A1.9, 16, 35 The ratio of
3ꢀhydroxyꢀ to 4ꢀhydroxyꢀβꢀdamascone is similar to that observed for the equivalent products of
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βꢀionone suggesting these two substrates are bound in a similar orientation in the active site with
the C3 carbon being closest to the heme iron followed by C4.
When using the CYP101B1 wholeꢀcell oxidation system we previously observed the
formation of a blue coloured pigment in cells after induction, which can be the result of indigo
formation from indole oxidation.9 Here we show that the addition of indole to this system resulted
in increased levels of blue pigment (Fig. S1). This indicates that indole and related aromatic
molecules may be viable substrates for oxidation by CYP101B1. Therefore we tested a small
selection of aromatic substrates for their ability to bind to and be oxidised by CYP101B1 (indole,
pꢀcymene and phenylcyclohexane). pꢀCymene resulted in virtually no shift to the high spin form
compared to indole which induced a small spin state shift (15 %). Phenylcyclohexane induced a
slightly larger shift of 20 % but bound to CYP101B1 with comparable affinity to βꢀdamascone
(Table 1, Fig. 3 and 4).
Both substrates induced moderate activity with CYP101B1 as measured by the NADH
turnover frequency, but both are lower when compared to the norisoprenoids. Phenylcyclohexane
was more active, 293 ± 9.0 min–1, than pꢀcymene, 197 ± 6.0 min–1. Indole did not induce any
enzyme activity above that of the leak rate. HPLC and GC analysis of phenylcyclohexane turnover
indicated that a single product was formed (Fig. 6). This was isolated using the wholeꢀcell oxidation
system, purified (∼10 mg) and characterised using NMR as transꢀ4ꢀphenylcyclohexanol. Coupling
of the NADH consumption to product formation was calculated to be 48 % resulting in a product
formation rate of 141 ± 17 min–1. Two products were observed in small quantities in the turnover of
pꢀcymene. These were identified via GC and HPLC coꢀelution experiments as
pꢀα,αꢀtrimethylbenzyl alcohol (25 %) and isopropylbenzyl alcohol (75 %). The coupling of NADH
to product formation and the product formation rates are low (13 % and 25.3 ± 6.0 min–1).
Overall the data suggests certain aromatic substrates such as phenylcyclohexane are good
substrates for CYP101B1 resulting in moderate activity and regioselective hydroxylations. However
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the norisoprenoid type substrates, ionone and damascone, show high affinities and activities for
CYP101B1 and are efficiently oxidised with high regioselectivity and rates of product formation.
αꢀIonone is solely oxidised at the allylic C3 C−H compared to βꢀionone and βꢀdamascone where
C3 oxidation dominates but with a contribution from allylic C4 hydroxylation.
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Discussion
To date the CYP101B1 enzyme from N. aromaticivorans is one of only two members of this
subfamily of P450 enzymes known with the other member only having 59 % sequence identity. The
low number of CYP101B subfamily members and the presence of both in Novosphingobium species
hint at a potentially unique functional role. It is of note that CYP101B1 is located on a plasmid,
whereas the electron transfer partners, ArR and Arx are located on the chromosomal genomic DNA
with CYP101D1, CYP101D2 and CYP101C1.5, 9 The active site residues of CYP101B1 vary when
compared to those of the camphor oxidising CYP101A1, CYP101D1 and CYP101D2 and these
differences will modify the substrate selectivity away from camphor towards alternative substrates
such as norisoprenoids. The most significant change is the absence of an equivalent residue to
tyrosine 96 of CYP101A1 which in CYP101B1 is replaced with a histidine residue.
Potassium ions have been shown to increase the strength of camphor binding to CYP101A1.
The behaviour of CYP101 family enzyme from N. aromaticivorans is different due to changes in
the residues that comprise the camphor binding site between the CYP101 family members. The
K+ꢀbinding site in CYP101A1 is octahedral, with the backbone carbonyl oxygens of Glu84, Gly93,
Glu94 and Tyr96, and two water molecules, as ligands.36 These residues are conserved in
CYP101D2, but not in CYP101D1, CY101C1 and CYP101B1. As well as the change at Tyr96,
Glu84, is replaced with Arg residues in CYP101B1 and CYP101D1 and Gln in CYP101C1 which
significantly reduces the effect of potassium on substrate binding.14We have previously reported
that, in common with these other CYP101 enzymes, CYP101B1 shows decreased activity at higher
ionic strength predominantly due to an increasing KM.14 The proximal face of CYP101B1 is likely
to consist of more positively charged than negatively charged residues, similar to CYP101C1,
CYP101D1 and CYP101D2, allowing for strong interactions with the negatively charged surface
potential of Arx in the vicinity of the [2Feꢀ2S] cluster.14
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The sideꢀchains of Lys178, Asp182, Thr185, Arg186, Asp251, and Asn255 are involved in
the extensive salt bridge interactions in camphorꢀbound CYP101A1.37, 38 In addition the Asp97 (B'
helix) and Lys197 (G helix) interaction has been proposed to play a role in camphor entry and
product exit. Structural analysis of CYP101D1, CYP101D2 and CYP101C1 revealed that this salt
bridge network was less extensive in these CYP101 family members. In CYP101B1 the changes of
Lys178 to an arginine and the more dramatic alteration of Asp97 to a glycine would also be
expected to alter this salt bridge network. The modifications in these regions along with those in the
active site will alter the substrate range and CꢀH bond oxidation selectively of CYP101B1, relative
to the other CYP101 subfamily members. Overall these changes in CYP101B1, when compared
with CYP101A1, are fewer than those of CYP101C1 which agrees with the observation that
CYP101B1 retains some binding affinity for camphor while binding of this substrate is virtually
abolished in CYP101C1.5, 9
CYP101B1 is able to bind norisoprenoids and camphor as well as selected substituted
aromatics. It is tempting to speculate that the proline residue one position to the Cꢀterminus of the
proximal iron binding cysteinate residue contributes to the seemingly broader substrate range of this
enzyme, though this remains to be confirmed. This would agree with data from the Ile401Pro
variant of CYP102A1 but the CYP121A1 enzyme from Mycobacterium tuberculosis which has a
proline in the same position has been reported to have a very specific substrate and reaction type,
the C−C coupling of a cyclodipeptide.39
The physiological substrate of CYP101B1 is yet to be confirmed. However, the
norisoprenoid class of compounds such as βꢀ and αꢀionone and βꢀdamascone are efficiently
oxidised, the binding affinity for these substrates is relatively strong and they induce large type I
spin state shifts. Norisoprenoids can be derived from the breakdown of carotenoids and N.
aromaticivorans contains at least one carotenoid dioxygenase, though this was identified as a
13,14ꢀdioxygenase.40 The relative location of the ketone and alkene functionalities in the butenone
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side chain has more of an effect on the binding affinity than the location of the alkene double bond
in the cyclohexyl ring with αꢀ and βꢀionone being better substrates than βꢀdamascone. The location
of the double bond in the cyclohexyl ring does have an effect on the selectivity of the hydroxylation.
The allylic C3 C−H bonds of αꢀionone being exclusively oxidised but for βꢀionone and
βꢀdamascone allylic C4 hydroxylation is observed alongside C3 oxidation. The C−H bonds of the
C3 in the norisoprenoid substrates, which are oxidised in the major products, are presumably held
closer to the heme iron than any other C−H bond. The lower regioselectivity of βꢀionone and
βꢀdamascone is presumably due to the more reactive hydrogens on the allylic C4 being in close
enough proximity to the heme iron enabling them to compete with oxidation at C3. In αꢀionone the
C4 hydrogen is vinylic and the reactive alkene double bond must be too far away from the heme
iron for epoxidation to occur.
Microbial oxidative biotransformations of αꢀ and βꢀionones have been reported using
different bacteria including Streptomyces.21 P450 enzymes from S. griseolus and S. griseus were
shown to oxidise αꢀ and βꢀionone at the allylic C3 and C4 positions, respectively.41 Variants of the
selfꢀsufficient cytochrome P450, CYP102A1, are able to selectively oxidise βꢀionone at the allylic
C4 position.35 Others have reported the stereoselective oxidation of the different enantiomers of
αꢀionone at the C3 position.42 The best reported product formation rates and coupling of these
variants of the highly efficient and selfꢀsufficient CYP102A1 enzyme with norisoprenoids are 280
min−1 and 35 % for βꢀionone, 152 min−1 and 39 % for (R)ꢀαꢀionone and 98 and 44 % for
(S)ꢀαꢀionone, which are lower than those observed with the CYP101B1/ArR/Arx system.42
Other researchers have reported the binding and oxidation of ionones by the CYP264B1,
CYP109B1 and CYP109D1 bacterial enzymes. The CYP109 enzymes were regioselective for
oxidation at the allylic position while CYP264B1 oxidised both αꢀ and βꢀionones solely at C3.20, 23,
43
Nonꢀphysiological electron transfer partners were used for all three of these cytochrome P450
enzymes and the catalytic rates were slow compared to those reported for CYP102A1 and
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CYP101B1 (< 5 min−1).
CYP101C1 from the same Novosphingobium bacterium is interesting in that it can rapidly
oxidise βꢀionone (PFR ∼2000 min−1) to a mixture of 4ꢀhydroxyꢀ (75 %) and 3ꢀhydroxyꢀβꢀionone
(25 %) but the turnover with αꢀionone is much slower (PFR ∼220 min−1). CYP101C1 oxidation of
αꢀionone is also regioselective for allylic C3 oxidation. It can also oxidise βꢀdamascone to a
mixture of 3ꢀ and 4ꢀhydroxy products as well as further oxidation products. CYP101B1 is more
active than all the other P450s tested so far for αꢀionone oxidation. Whether this reaction is
stereoselective with enantiomerically pure αꢀionones remains to be determined. With βꢀionone and
βꢀdamascone CYP101B1 is less active than CYP101C1 but alters the selectivity to favour oxidation
predominantly at the C3 rather than the allylic C4 position. Of the other enzymes utilised to oxidise
ionones, CYP102A1 mutants and the CYP109 enzymes family are less active but are more selective
for allylic hydroxylation at C4 while CYP264B1 is very selective for oxidation at C3 but has very
low activity.
The selective oxidation of the unrelated and more hydrophobic aromatic substrate
phenylcyclohexane to transꢀ4ꢀphenylcyclohexanol by CYP101B1 on the aliphatic ring at moderate
activity is interesting. Tyrosine variants of CYP101A1 have been used to oxidise
phenylcyclohexane at predominantly the 4ꢀposition though the activities were lower. Other
mutations in the active site were also shown to alter the selectivity though none were completely
regioselective.44ꢀ46 CYP108D1, also from N. aromaticivorans, has also been reported to tightly bind
phenylcyclohexane and oxidise it to transꢀ4ꢀphenylcyclohexanol, though the activity using
nonꢀphysiological electron transfer partners is very low.47 The oxidation of pꢀcymene was
significantly slower than phenylcyclohexane suggesting the smaller size of this substrates is
detrimental to binding and productive and selective turnover. pꢀCymene can bind with either methyl
or isopropyl substituent closest to the heme iron. The combination of selective oxidations, high
monooxygenase activities and the large total number of turnovers obtained using the wholeꢀcell
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oxidation system make CYP101B1 ideally suited to the biocatalytic generation of hydroxylated
products.
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Experimental Section
General
General reagents and organics were from SigmaꢀAldrich or VWR, Australia. Buffer components,
NADH, and isopropylꢀβꢀDꢀthiogalactopyranoside (IPTG) were from Anachem (Astral Scientific,
Australia) or Biovectra, (Scimar, Australia). UV/Vis spectroscopy was performed on Varian Cary
5000 or Agilent Cary 60 spectrophotometers. Gas chromatography (GC) analyses were carried out
on a Shimadzu GCꢀ17A instrument coupled to a QP5050A MS detector using a DBꢀ5 MS fused
silica column (30 m x 0.25 mm, 0.25 µm) and helium as the carrier gas. Analytical liquid
chromatography was performed using an Agilent 1260 Infinity pump equipped with an Agilent
Eclipse Plus C18 column (250 mm x 4.6 mm, 5 µm), an autoinjector and UV detector. A gradient,
20 ꢀ 95 %, of acetonitrile (with trifluoroacetic acid, 0.1 %) in water (TFA, 0.1 %) was used. NMR
spectra were recorded using a Varian Inovaꢀ600 spectrometer
Phylogenetic Analysis
BLAST searches were performed using the databases at the National Center for Biotechnology
Information. Sequence alignments were performed using ClustalW.48 The evolutionary history was
inferred by using the Maximum Likelihood method based on the Jones Taylor Thornton (JTT)
matrixꢀbased model.49 The tree with the highest log likelihood is shown. Initial tree(s) for the
heuristic search were obtained automatically by applying NeighborꢀJoin and BioNJ algorithms to a
matrix of pairwise distances estimated using a JTT model, and then selecting the topology with
superior log likelihood value. The tree is drawn to scale, with branch lengths measured in the
number of substitutions per site. All positions containing gaps and missing data were eliminated.
There were a total of 386 positions in the final dataset. Evolutionary analyses were conducted in
MEGA6.50
Protein production and purification
General DNA manipulations and microbiological experiments and the expression, purification and
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quantitation of the electron transfer proteins ArR and Arx from N. aromaticivorans were performed
as described elsewhere.5, 9 The CYP101B1 gene has been cloned into the pET26 vector and the
encoded CYP101B1 protein was produced in E. coli BL21(DE3).5, 9, 51 A single colony was
inoculated into 20 mL LuriaꢀBertani broth (LB) containing kanamycin (25 mg L–1; LBkan) and
grown at 37 °C for 6 hours. This culture was then inoculated into LBkan (1 ml per 500 ml) medium
and grown at 37 °C for 6 h to an OD600 of ∼1. Protein production was induced by the addition of
IPTG (0.1 mM, from a 0.5 M stock in water). The incubator temperature was lowered to 25 °C, and
the culture was grown for a further 16 h. The red cell pellet, harvested from the culture by
centrifugation (9000 g), was resuspended in 200 ml of buffer T (50 mM Tris, pH 7.4). The cells
were lysed by sonication (30 x 20 s pulses with 40 s gap in between each pulse) and the cell debris
was removed by centrifugation 27000 g for 30 min at 4 °C. The supernatant was loaded onto a
DEAE Fastflow Sepharose column (200 mm × 50 mm; GE Healthcare) and the protein eluted using
a linear salt gradient of KCl (80 – 350 mM) in buffer T developed over eight column bedꢀvolumes
at a flow rate of 8 mL min–1. The red proteinꢀcontaining fractions were collected and concentrated
by ultrafiltration. The protein was desalted using a gel filtration step on a G25 Sephadex column
(600 mm × 10 mm) using Buffer T. The final purification step was anionꢀexchange chromatography
using a Sourceꢀ15Q column (120 mm × 26 mm; GE Healthcare) and eluting with a linear gradient
of 50 – 250 mM KCl in buffer T developed over 20 column bedꢀvolumes at a flow rate of 6 mL
min–1. Purified fractions (A418/A280 ≥ 1.2) were combined and concentrated by ultrafiltration. An
equal volume of glycerol was added, the solution filtered through a 0.22ꢀꢁm sterile syringe filter
and stored at –20 ºC. CYP101B1 was quantitated using ε
417 = 113 mM–1 cm–1.9
Substrate binding and kinetic analysis
For substrate binding the P450 enzymes were diluted to ∼2.0 µM using 50 mM Tris, pH 7.4. The
high spin heme content was estimated, to approximately ±5 %, by comparison with a set of spectra
generated from the sum of the appropriate percentages of the spectra of the substrateꢀfree form (>95
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% low spin, Soret maximum at 418 nm) and camphorꢀbound form (>95 % high spin, Soret
maximum at 392 nm) of wildꢀtype CYP101A1.
To determine the dissociation constant the CYP101B1 enzyme was prepared as above in a
total volume 2.5 mL and used to baseline the spectrophotometer. Aliquots of substrate (0.5–2 µL)
were added using a Hamilton syringe from a 1, 10 or 100 mM stock solution in ethanol. The
solution was mixed and the peakꢀtoꢀtrough difference in absorbance recorded between 700 nm and
250 nm. Further aliquots of substrate were added until the peakꢀtoꢀtrough difference did not shift
further. The apparent dissociation constants, Kd, were obtained by fitting the peakꢀtoꢀtrough
difference against substrate concentration to a hyperbolic function (Eqn. 1):
ꢀAmax × [S]
ꢀA =
Eqn. 1
Kd +[S]
Where ꢂA is the peakꢀtoꢀtrough absorbance difference, ꢂAmax is the maximum absorbance
difference and [S] is the substrate concentration.
Both ionone substrates exhibited tight binding, with Kd < 5[E], and in these instances the
data were fitted to the tight binding quadratic equation (Eqn. 2):
([E]+[S]+ Kd ) − {([E]+[S]+ Kd )2 −4[E][S]}
ꢀA
=
Eqn. 2
ꢀAmax
2[E]
where ꢂA is the peakꢀtoꢀtrough absorbance difference, ꢂAmax is the maximum absorbance
difference, [S] is the substrate concentration and [E] is the enzyme concentration.
NADH turnover assays were performed with mixtures (1.2 mL) containing 50 mM Tris, pH
7.4, 0.5 ꢁM CYP101B1, 5 ꢁM Arx, 1 ꢁM ArR and 100 ꢁg mL–1 bovine liver catalase. The mixtures
were oxygenated and then equilibrated at 30 °C for 2 min. Substrates were added as 100 mM stock
solutions in ethanol to a final concentration of 1 mM. NADH was added to ~320 ꢁM, final A340
=
2.00, and the absorbance at 340 nm was monitored. The rate of NADH turnover was calculated
using ε340 = 6.22 mM–1 cm–1 (Table 1).
Product formation and isolation
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Where authentic product standards were available they were utilised for identification purposes via
HPLC and GC coꢀelution experiments. Extraction of the products from activity assays and product
analysis by GC were performed as before.9 The pꢀcymene turnover products isopropylbenzyl
alcohol and thymol eluted together on the GCꢀMS and HPLC. The alcohol products were
derivatised using BSTFA/TMCS and analysed via GCꢀMS to identify the product as isopropyl
benzyl alcohol rather than thymol. The product concentration in incubation mixtures was calculated
by calibrating the total ion count response of the GC detector to products when available (or the
substrate if the product was not available). Where more than one product was produced the detector
response was assumed to be equal, e.g. 4ꢀhydroxyꢀβꢀdamascone and 3ꢀhydroxyꢀβꢀdamascone were
assumed to have equal detector responses. The coupling efficiency was the percentage of NADH
consumed that led to product formation.
GCꢀMS analyses were carried out with the injector held at 250 °C and the interface at
280 °C. The oven temperature was varied as follows: for norisoprenoids and phenylcyclohexane,
1
120 °C for 3 minutes followed by a gradient of 10 °C min− for 10 min and held at 220 °C for a
1
further 7 minutes; pꢀcymene 80 °C for 3 minutes followed by a gradient of 10 °C min− for 14 min
and held at 220 °C for a further 3 minutes. The GC retention times were as follows: βꢀionone, 7.5
min; 4ꢀhydroxyꢀβꢀionone, 9.9 min; 3ꢀhydroxyꢀβꢀionone, 10.3 min; αꢀionone, 6.7 min;
transꢀ3ꢀhydroxyꢀαꢀionone, 9.4 min; cisꢀ3ꢀhydroxyꢀαꢀionone, 9.5 min; 3ꢀoxoꢀαꢀionone, 9.8 min;
βꢀdamascone, 6.5 min; 4ꢀhydroxyꢀβꢀdamascone, 9.1 min; 3ꢀhydroxyꢀβꢀdamascone, 9.3 min;
phenylcyclohexane, 5.1 min; transꢀ4ꢀhydroxyphenylcyclohexane, 7.7 min; pꢀcymene 4.4 min;
isopropylbenzyl alcohol, 8.9 min (14.0 after derivitisation); pꢀα,αꢀtrimethylbenzylalcohol, 7.2 min;
thymol, 8.9 min (11.9 after derivatisation); carvacrol, 9.1 min.
To isolate and identify products where standards were not available a wholeꢀcell oxidation
system utilising the plasmids pETDuetArx/ArR and pRSFDuetArx/CYP101B1 was used to oxidise
βꢀionone, αꢀionone, βꢀdamascone and phenylcyclohexane, as described previously.9 The
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supernatant (200 mL) was extracted in ethylacetate (3 x 100 mL), washed with brine (100 mL) and
dried with magnesium sulphate and 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 GCꢀMS and those containing single products (≥95 %) were combined for
characterisation. The solvent was removed under reduced pressure. The purified product was
dissolved in CDCl3 and the organics characterised by NMR spectroscopy and GCꢀMS. NMR
1
spectra were acquired on a Varian Inovaꢀ600 and spectrometer operating at 600 MHz for H and
150 MHz for 13C.
A combination of 1H, 13C, COSY, HSQC and HMBC experiments was used to determine the
structures of the products. The cis/trans configuration of 3ꢀhydroxyꢀαꢀionone diastereomers was
attributed from the assignments of Yamazaki52 and the selective nOe difference measurements in
ROESY experiments. The diagnostic nOe was between the H3 and H6 protons in the cis
diastereomer with the C11 methyl 3H signal.
NMR and GC MS data
1
Data for 3ꢀhydroxyꢀ
β
ꢀionone: m+/z = 208.2, H NMR (600 MHz, CDCl3) δ 7.21 (d, J = 16.4 Hz,
1H, H8), 6.11 (d, J = 16.4 Hz, 1H, H7), 3.99 (s, 1H, H3), 2.43 (d, J = 17.5 Hz, 1H, H4), 2.29 (s, 3H,
H10), 2.09 (dd, J = 17.3, 9.7 Hz, 1H, H4), 1.83 – 1.78 (m, 1H, H2), 1.77 (s, 3H, H13), 1.49 (t, J = 12.0
Hz, 1H, H2), 1.11 (s, 3H, H11), 1.10 (s, 3H, H12); 13C NMR (151 MHz, CDCl3) δ 198.68 (C9),
142.47 (C8), 135.52 (C6), 132.50 (C5), 132.19 (C7), 64.27 (C3), 48.31 (C2), 42.70 (C4), 36.82 (C1),
30.03 (C11), 28.51 (C12), 27.22 (C10), 21.54 (C13).
1
Data for trans
ꢀ
3ꢀhydroxyꢀαꢀionone: m+/z = 208.1; H NMR (500 MHz, CDCl3) δ 6.54 (dd, J =
15.8, 10.1 Hz, 1H, H7), 6.10 (d, J = 15.8 Hz, 1H, H8), 5.65 – 5.61 (m, 1H, H4), 4.27 (s, 1H, H3), 2.50
(d, J = 10.1 Hz, 1H, H6), 2.26 (s, 3H, H10), 1.84 (dd, J = 13.5, 5.9 Hz, 1H, H2), 1.62 (d, J = 0.6 Hz,
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3H, H13), 1.41 (dd, J = 13.6, 6.2 Hz, 1H, H2), 1.03 (s, 3H, H11), 0.89 (s, 3H, H12); 13C NMR (126
MHz, CDCl3) δ 198.03 (C9), 147.09 (C7), 135.43 (C5), 133.61 (C8), 125.83 (C4), 65.47 (C3), 54.30
(C6), 43.83 (C2), 33.86 (C1), 29.31 (C11), 27.20 (C10), 24.70 (C12), 22.66 (C13).
1
Data for cis
ꢀ
3ꢀhydroxyꢀαꢀionone: m+/z = 208.1; H NMR (600 MHz, CDCl3) δ 6.64 (dd, J = 15.8,
9.6 Hz, 1H, H7), 6.08 (d, J = 15.8 Hz, 1H, H8), 5.59 (s, 1H, H4), 4.25 (t, J = 7.6 Hz, 1H, H3), 2.29 –
2.25 (m, 4H, H6 & H10), 1.69 (dd, J = 12.9, 6.5 Hz, 1H, H2), 1.63 (s, 3H, H13), 1.40 (dd, J = 12.8, 9.9
Hz, 1H, H2), 0.98 (s, 3H, H11), 0.89 (s, 3H, H12); 13C NMR (151 MHz, CDCl3) δ 198.46 (C9),
147.83 (C7), 135.30 (C5), 132.68 (C8), 126.47 (C4), 66.37 (C3), 54.32 (C6), 40.65 (C2), 34.97 (C1),
29.10 (C11), 27.61 (C10), 26.41 (C12), 22.41 (C13).
1
Data for 3ꢀoxoꢀαꢀionone: m+/z = 206.1; H NMR (600 MHz, CDCl3) δ 6.69 (dd, J = 15.8, 9.5 Hz,
1H, H7), 6.20 (d, J = 15.8 Hz, 1H, H8), 5.98 (s, 1H, C4H), 2.73 (d, J = 9.6 Hz, 1H, H6), 2.37 (d, J =
16.9 Hz, 1H, H2), 2.29 (s, 3H, H10), 2.15 (d, J = 17.0 Hz, 1H, H2), 1.91 (s, 3H, H13), 1.09 (s, 3H,
H11), 1.02 (s, 3H, H12); 13C NMR (151 MHz, CDCl3) δ 198.20 (C3), 197.49 (C9), 159.12 (C5),
143.57 (C7), 133.74 (C8), 126.88 (C4), 55.40 (C6), 47.33 (C2), 36.67 (C1), 27.89 (C10), 27.55 (C11),
27.31 (C12), 23.48 (C13).
1
Data for 3ꢀhydroxyꢀ
β
ꢀdamascone: m+/z = 208.2; H NMR (600 MHz, CDCl3) δ 6.74 (dq, J = 13.8,
6.9 Hz, 1H, H9), 6.16 (dd, J = 15.7, 1.3 Hz, 1H, H8), 4.11 – 4.04 (m, 1H, H3), 2.36 (dd, J = 16.8, 5.8
Hz, 1H, H4), 2.04 (dd, J = 17.1, 9.3 Hz, 1H, H4), 1.93 (dd, J = 6.9, 1.4 Hz, 3H, H10), 1.79 – 1.73 (m,
1H, H2), 1.54 (s, 3H, H13), 1.51 (t, J = 12.0 Hz, 1H, H2), 1.15 (s, 3H, H11), 0.99 (s, 3H, H12); 13C
NMR (151 MHz, CDCl3) δ 201.85 (C7), 146.41 (C9), 139.87 (C6), 134.40 (C8), 128.23 (C5), 64.65
(C3), 47.78 (C2), 40.87 (C4), 36.31 (C1), 29.70 (C12), 29.08 (C11), 21.04 (C13), 18.41 (C10).
Data for 4ꢀhydroxyꢀβ
ꢀdamascone: m+/z = 208.2; 1H NMR (600 MHz, CDCl3) δ 6.77 (dq, J = 15.6,
6.9 Hz, 1H, H9), 6.15 (dd, J = 15.7, 1.5 Hz, 1H, H8), 4.00 (t, J = 5.0 Hz, 1H, C4H), 2.03 – 1.96 (m,
1H, H3), 1.93 (dd, J = 6.9, 1.5 Hz, 3H, H10), 1.80 – 1.74 (m, 1H, H3), 1.69 – 1.65 (m, 1H, H2), 1.65
(s, 3H, H13), 1.45 (ddd, J = 13.4, 7.7, 3.1 Hz, 1H, H2), 1.04 (s, 6H, H11 & H12); 13C NMR (151
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MHz, CDCl3) δ 200.89 (C7), 146.46 (C9), 143.59 (C6), 133.92 (C8), 130.98 (C5), 69.01 (C4) 34.64
(C2), 33.98 (C1), 28.82 (C11), 28.64 (C3), 27.64 (C12), 18.46 (C10), 17.96 (C13).
Data for transꢀ4ꢀphenylcyclohexanol: m+/z = 176.1
1H NMR (600 MHz, CDCl3) δ 7.31 – 7.16 (m, 5H, H 8ꢀ12), 3.70 (tt, J = 10.7, 4.3 Hz, H1ax),
2.50 (tt, J = 12.1, 3.4 Hz, 1H, H4ax), 2.13 – 2.07 (m, 2H, H3eq & H5eq), 1.96 – 1.90 (m, 2H, H2eq
& H6eq), 1.50 – 1.58 (m, 2H, H3ax & H5ax), 1.48 – 1.40 (m, 2H, H2ax & H6ax).
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Conclusion
CYP101B1 is a valuable addition to the field of monooxygenase biocatalysis. It is stable, produced
in high yield and its electron transfer partners are known. It has product formation rates that
approach, and in certain cases exceed, those of the selfꢀsufficient CYP102A1. The coupling of
reducing equivalents to product formation is also high. Using a wholeꢀcell oxidation system we
were able to generate significant quantities of hydroxylated metabolites. CYP101B1 was capable of
oxidising a broad range of substrates including norisoprenoids, monoterpenes and hydrophobic
aromatic compounds, which is distinct to the other members of the CYP101 family and therefore
offers a specialised substrate binding profile. Taken together these factors, and the additional
potential to engineer CYP101B1 for more efficient and selective turnovers, will enable the
development of biocatalytic routes to as yet unobtainable fine chemicals.
Acknowledgements
The work was supported by the School of Chemistry & Physics at the University of Adelaide (to
S.G.B.). E.A.H. thanks the University of Adelaide for an M. Phil Scholarship.
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References
1.
P. R. Ortiz de Montellano, ed., Cytochrome P450: Structure, Mechanism, and Biochemistry
3rd edn., Kluwer Academic/Plenum Press, New York, 2005.
2.
A. Sigel, H. Sigel and R. Sigel, eds., The Ubiquitous Roles of Cytochrome P450 Proteins,
1st edn., John Wiley & Sons, 2007.
3.
4.
5.
6.
R. Bernhardt and V. B. Urlacher, Appl. Microbiol. Biotechnol., 2014, 98, 6185ꢀ6203.
T. Furuya and K. Kino, Appl. Microbiol. Biotechnol., 2010, 86, 991ꢀ1002.
S. G. Bell and L. L. Wong, Biochem. Biophys. Res. Commun., 2007, 360, 666ꢀ672.
F. Hannemann, A. Bichet, K. M. Ewen and R. Bernhardt, Biochim. Biophys. Acta, 2007,
1770, 330ꢀ344.
7.
J. A. Peterson and S. E. GrahamꢀLorence, in Cytochrome P450: Structure, Mechanism, and
Biochemistry, ed. P. R. Ortiz de Montellano, Plenum Press, New York, 2nd edn., 1995, pp.
151ꢀ182.
8.
S. G. Bell, F. Xu, E. O. Johnson, I. M. Forward, M. Bartlam, Z. Rao and L. L. Wong, J. Biol.
Inorg. Chem., 2010, 15, 315ꢀ328.
9.
S. G. Bell, A. Dale, N. H. Rees and L. L. Wong, Appl. Microbiol. Biotechnol., 2010, 86,
163ꢀ175.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
C. Hong, S. G. Bell, W. Yang, H. Wang, Y. Hao, X. Li, W. Zhou, M. Bartlam and L. L. Wong,
Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun., 2009, 65, 364ꢀ367.
R. Zhou, C. Huang, A. Zhang, S. G. Bell, W. Zhou and L. L. Wong, Acta Crystallogr. Sect. F
Struct. Biol. Cryst. Commun., 2011, 67, 964ꢀ967.
J. K. Fredrickson, D. L. Balkwill, G. R. Drake, M. F. Romine, D. B. Ringelberg and D. C.
White, Appl. Environ. Microbiol., 1995, 61, 1917ꢀ1922.
J. K. Fredrickson, F. J. Brockman, D. J. Workman, S. W. Li and T. O. Stevens, Appl. Environ.
Microbiol., 1991, 57, 796ꢀ803.
W. Yang, S. G. Bell, H. Wang, W. Zhou, N. Hoskins, A. Dale, M. Bartlam, L. L. Wong and Z.
Rao, J. Biol. Chem., 2010, 285, 27372ꢀ27384.
S. G. Bell, W. Yang, A. Dale, W. Zhou and L. L. Wong, Appl. Microbiol. Biotechnol., 2013,
97, 3979ꢀ3990.
M. Ma, S. G. Bell, W. Yang, Y. Hao, N. H. Rees, M. Bartlam, W. Zhou, L. L. Wong and Z.
Rao, Chembiochem, 2011, 12, 88ꢀ99.
W. Yang, S. G. Bell, H. Wang, W. Zhou, M. Bartlam, L. L. Wong and Z. Rao, Biochem. J.,
2011, 433, 85ꢀ93.
S. Vohra, M. Musgaard, S. G. Bell, L. L. Wong, W. Zhou and P. C. Biggin, Protein Sci.,
2013, 22, 1218ꢀ1229.
E. M. Gillam, L. M. Notley, H. Cai, J. J. De Voss and F. P. Guengerich, Biochemistry, 2000,
39, 13817ꢀ13824.
Y. Khatri, M. Girhard, A. Romankiewicz, M. Ringle, F. Hannemann, V. B. Urlacher, M. C.
Hutter and R. Bernhardt, Appl. Microbiol. Biotechnol., 2010, 88, 485ꢀ495.
S. LutzꢀWahl, P. Fischer, C. SchmidtꢀDannert, W. Wohlleben, B. Hauer and R. D. Schmid,
Appl. Environ. Microbiol., 1998, 64, 3878ꢀ3881.
Y. T. Meharenna, K. E. Slessor, S. M. Cavaignac, T. L. Poulos and J. J. De Voss, J. Biol.
Chem., 2008, 283, 10804ꢀ10812.
T. T. Ly, Y. Khatri, J. Zapp, M. C. Hutter and R. Bernhardt, Appl. Microbiol. Biotechnol.,
2012, 95, 123ꢀ133.
X. Chen, A. Christopher, J. P. Jones, S. G. Bell, Q. Guo, F. Xu, Z. Rao and L. L. Wong, J.
Biol. Chem., 2002, 277, 37519ꢀ37526.
F. Xu, S. G. Bell, J. Lednik, A. Insley, Z. Rao and L.ꢀL. Wong, Angew. Chem. Int. Ed., 2005,
44, 4029ꢀ4032.
S. G. Bell, C. F. HarfordꢀCross and L.ꢀL. Wong, Protein Eng., 2001, 14, 797ꢀ802.
24

Page 25 of 34
RSC Advances
View Article Online
DOI: 10.1039/C4RA14010A
27.
28.
T. L. Poulos, Chem. Rev., 2014, 114, 3919ꢀ3962.
J. P. Clark, C. S. Miles, C. G. Mowat, M. D. Walkinshaw, G. A. Reid, S. N. Daff and S. K.
Chapman, J. Inorg. Biochem., 2006, 100, 1075ꢀ1090.
29.
30.
W. Tempel, I. Grabovec, F. MacKenzie, Y. V. Dichenko, S. A. Usanov, A. A. Gilep, H. W.
Park and N. Strushkevich, J. Lipid Res., 2014.
K. J. McLean, P. Carroll, D. G. Lewis, A. J. Dunford, H. E. Seward, R. Neeli, M. R.
Cheesman, L. Marsollier, P. Douglas, W. E. Smith, I. Rosenkrands, S. T. Cole, D. Leys, T.
Parish and A. W. Munro, J. Biol. Chem., 2008, 283, 33406ꢀ33416.
31.
32.
33.
34.
35.
36.
37.
S. Nagano, T. Tosha, K. Ishimori, I. Morishima and T. L. Poulos, J. Biol. Chem., 2004, 279
42844ꢀ42849.
,
T. Tosha, S. Yoshioka, K. Ishimori and I. Morishima, J. Biol. Chem., 2004, 279
,
42836ꢀ42843.
C. J. Whitehouse, S. G. Bell, W. Yang, J. A. Yorke, C. F. Blanford, A. J. Strong, E. J. Morse,
M. Bartlam, Z. Rao and L. L. Wong, ChemBioChem, 2009, 10, 1654ꢀ1656.
C. J. Whitehouse, W. Yang, J. A. Yorke, B. C. Rowlatt, A. J. Strong, C. F. Blanford, S. G.
Bell, M. Bartlam, L. L. Wong and Z. Rao, ChemBioChem, 2010, 11, 2549ꢀ2556.
V. B. Urlacher, A. Makhsumkhanov and R. D. Schmid, Appl. Microbiol. Biotechnol., 2006,
70, 53ꢀ59.
A. C. Westlake, C. F. HarfordꢀCross, J. Donovan and L. L. Wong, Eur. J. Biochem., 1999,
265, 929ꢀ935.
E. Deprez, N. C. Gerber, C. Di Primo, P. Douzou, S. G. Sligar and G. Hui Bon Hoa,
Biochemistry, 1994, 33, 14464ꢀ14468.
38.
39.
C. Di Primo, E. Deprez, S. G. Sligar and G. Hui Bon Hoa, Biochemistry, 1997, 36, 112ꢀ118.
P. Belin, M. H. Le Du, A. Fielding, O. Lequin, M. Jacquet, J. B. Charbonnier, A. Lecoq, R.
Thai, M. Courcon, C. Masson, C. Dugave, R. Genet, J. L. Pernodet and M. Gondry, Proc.
Natl. Acad. Sci. U. S. A., 2009, 106, 7426ꢀ7431.
40.
41.
42.
Y. S. Kim, E. S. Seo and D. K. Oh, Biotechnol. Lett., 2012, 34, 1851ꢀ1856.
A. Celik, S. L. Flitsch and N. J. Turner, Org. Biomol. Chem., 2005, 3, 2930ꢀ2934.
H. Venkataraman, S. B. A. d. Beer, D. P. Geerke, N. P. E. Vermeulen and J. N. M.
Commandeur, Adv. Synth. Catal., 2012, 354, 2172ꢀ2184.
43.
44.
45.
M. Girhard, T. Klaus, Y. Khatri, R. Bernhardt and V. B. Urlacher, Appl. Microbiol.
Biotechnol., 2010, 87, 595ꢀ607.
P. A. England, D. A. Rouch, A. C. G. Westlake, S. G. Bell, D. P. Nickerson, M. Webberley, S.
L. Flitsch and L. L. Wong, Chem. Commun., 1996, 357ꢀ358.
N. E. Jones, P. A. England, D. A. Rouch and L. L. Wong, Chem. Commun., 1996,
2413ꢀ2414.
46.
47.
S. G. Bell, D. A. Rouch and L. L. Wong, J. Mol. Catal. BꢀEnzym., 1997,
S. G. Bell, W. Yang, J. A. Yorke, W. Zhou, H. Wang, J. Harmer, R. Copley, A. Zhang, R.
3, 293ꢀ302.
Zhou, M. Bartlam, Z. Rao and L. L. Wong, Acta Crystallogr. D Biol. Crystallogr., 2012, 68
,
277ꢀ291.
48.
R. Chenna, H. Sugawara, T. Koike, R. Lopez, T. J. Gibson, D. G. Higgins and J. D.
Thompson, Nucleic Acids Res., 2003, 31, 3497ꢀ3500.
49.
50.
D. T. Jones, W. R. Taylor and J. M. Thornton, Comput. Appl. Biosci., 1992, 8, 275ꢀ282.
K. Tamura, G. Stecher, D. Peterson, A. Filipski and S. Kumar, Mol. Biol. Evol., 2013, 30
,
2725ꢀ2729.
51.
52.
E. M. Gillam, A. M. Aguinaldo, L. M. Notley, D. Kim, R. G. Mundkowski, A. A. Volkov, F.
H. Arnold, P. Soucek, J. J. DeVoss and F. P. Guengerich, Biochem. Biophys. Res. Commun.,
1999, 265, 469ꢀ472.
Y. Yamazaki, Y. Hayashi, M. Arita, T. Hieda and Y. Mikami, Appl. Environ. Microbiol., 1988,
54, 2354ꢀ2360.
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RSC Advances
Page 26 of 34
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DOI: 10.1039/C4RA14010A
Table and Figures
Table 1. Substrate binding, steady state kinetic data and coupling for CYP101B1 with different
substrates. Steady state turnover activities were measured using a ArR:Arx:CYP101B1
concentration ratio of 1:10:2 (0.5 ꢁM CYP enzyme, 50 mM Tris, pH 7.4). Coupling is the
percentage efficiency of NADH utilisation for the formation of products. Rates are reported as
mean ± S.D. (n ≥ 3) and given in nmolꢀnmol CYP–1ꢀmin–1. %HS is a measure of the shift in the spin
state from low spin to high spin on substrate binding and Kd is the dissociation constant.
CYP101B1/
substrate
%HS
heme
Kd
NADH
consumption formation
frequency
(min–1)
Product
Coupling
%
(ꢁM)
rate
(min–1)
βꢀionone
1600 ± 100
1010 ± 60
663 ± 56
63
48
≥95 %
≥95 %
0.23 ± 0.1
0.26 ± 0.04 1380 ± 140
αꢀionone
βꢀdamascone
phenylcyclohexane
pꢀcymene
80 %
20 %
5 %
8.3 ± 0.9
7.8 ± 0.9
23.0 ± 3.0
931 ± 13
293 ± 9.0
197 ± 27
562 ± 12
141 ± 17
25.3 ± 6.0
60
48
13
26

Page 27 of 34
RSC Advances
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DOI: 10.1039/C4RA14010A
Scheme 1. Substrate tested with CYP101B1 and the products isolated from the turnovers.
27

RSC Advances
Page 28 of 34
View Article Online
DOI: 10.1039/C4RA14010A
Figure 1 Protein sequence alignments of CYP101B1, CYP101D1, CYP101D2 and CYP101C1
from N. aromaticivorans, CYP101A1 (P450cam) from a Pseudomonas putida species and
CYP101B2 from Novosphingobium tardaugens NBRC 16725. Conserved residues are highlighted
in red boxes with similar residues in red font.
28

Page 29 of 34
RSC Advances
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DOI: 10.1039/C4RA14010A
Figure 2. A phylogenetic tree (phenogram) of the CYP101 family of P450 enzymes from N.
aromaticivorans DSM12444, CYP101B1, CYP101C1, CYP101D1 and CYP101D2, CYP101A1
(P450cam) from Pseudomonas putida, and CYP101B2 from Novosphingobium tardaugens NBRC
16725. Included for comparison are P450cin (CYP176A1) the closest structurally characterised
homologue to CYP101B1 outside of the CYP101 family. Also included are CYP109D1 and
CYP264B1 from Sorangium cellulosum and CYP105D1 from Streptomyces griseus all of which can
bind and catalyse the oxidation of norisoprenoids.
29