Improving the Monooxygenase Activity and the Regio- and Stereoselectivity of Terpenoid Hydroxylation Using Ester Directing Groups

Article abstract of DOI:10.1021/acscatal.6b01882

The monooxygenase enzyme CYP101B1, from Novosphingobium aromaticivorans DSM12444, binds norisoprenoids more tightly than monoterpenoids and oxidized these substrates with high regioselectivity. Ionols bound less tightly to CYP101B1 than ionones, but the levels of product formation remained high and the selectivity of oxidation was similar to that observed for the parent norisoprenoid. The structurally related sesquiterpene lactone (+)-sclareolide (9) was stereoselectively hydroxylated by CYP101B1 to (S)-(+)-3-hydroxysclareolide (9a). The turnover of monoterpenoid derivatives showed low levels of product formation and selectivity despite promising binding data. CYP101B1 catalyzed the selective oxidation of (1R)-(-)-nopol (14) and cis-jasmone (15), generating >90% (1R)-(-)-5-hydroxynopol (14a) and 4-hydroxy-cis-jasmone (15a), respectively. To develop strategies for the efficient and selective oxidation of monoterpenoid-based substrates using CYP101B1, we investigated the binding and catalytic properties of terpenoid acetates. The ester functional group of these substrates mimicked the carbonyl moiety of norisoprenoids and anchored the monoterpenoid acetates in the active site of CYP101B1 with high affinity for the monoterpenoid acetates. The oxidation of these substrates by CYP101B1 occurred with product formation rates in excess of 1000 min^-1 and total turnover numbers of greater than 5000 being observed in all but one instance. Critically, the oxidations were regioselective, with several being stereoselective. (-)-Myrtenyl acetate (20) was oxidized regioselectively (>95%) to yield cis-4-hydroxy-myrtenyl acetate (20a), which was further oxidized to 4-oxomyrtenyl acetate (20b) using a whole-cell system, providing a biocatalytic route to generate intermediates used in the production of cannabinoid derivatives. The ester carbonyl moiety could also be used as a directing group also to enhance the activity and control the selectivity of P450-catalyzed reactions; for example, the turnover of l-(-)-bornyl acetate (18) and isobornyl acetate (19) by CYP101B1 generated 9-hydroxybornyl acetate (18a) and 5-exo-hydroxyisobornyl acetate (19a), respectively, as the sole products.

Full text of DOI:10.1021/acscatal.6b01882

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Article
Improving the monooxygenase activity and the regio- and
stereoselectivity of terpenoid hydroxylation using ester directing groups
Emma A Hall, Md Raihan Sarkar, Joel Hoong Zhang Lee, Samuel D Munday, and Stephen G Bell
ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.6b01882 • Publication Date (Web): 09 Aug 2016
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Improving the monooxygenase activity and the regioꢀ
and stereoselectivity of terpenoid hydroxylation using
ester directing groups
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Emma A. Hall, Md. Raihan Sarkar, Joel H. Z. Lee, Samuel D. Munday and Stephen G. Bell*
Department of Chemistry, University of Adelaide, SA 5005, Australia
* To whom correspondence should be addressed.
Stephen G. Bell (stephen.bell@adelaide.edu.au)
Abstract
The monooxygenase enzyme CYP101B1, from Novosphingobium aromaticivorans DSM12444,
binds norisoprenoids more tightly than monoterpenoids and oxidized these substrates with high
regioselectivity. Ionols bound less tightly to CYP101B1 than ionones but the levels of product
formation remained high and the selectivity of oxidation was similar to that observed for the
parent norisoprenoid. The structurally related sesquiterpene lactone (+)ꢀsclareolide, 9, was
stereoselectively hydroxylated by CYP101B1 to (S)ꢀ(+)ꢀ3ꢀhydroxysclareolide, 9a. The turnover of
monoterpenoid derivatives showed low levels of product formation and selectivity despite
promising binding data. CYP101B1 catalyzed the selective oxidation of (1R)ꢀ(−)ꢀnopol, 14, and
cisꢀjasmone, 15, generating >90% (1R)ꢀ(−)ꢀ5ꢀhydroxynopol, 14a, and 4ꢀhydroxyꢀcisꢀjasmone, 15a,
respectively. To develop strategies for the efficient and selective oxidation of monoterpenoid based
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substrates using CYP101B1 we investigated the binding and catalytic properties of terpenoid
acetates. The ester functional group of these substrates mimicked the carbonyl moiety of
norisoprenoids and anchored the monoterpenoid acetates in the active site of CYP101B1 with high
affinity for the monoterpenoid acetates. The oxidation of these substrates by CYP101B1 occurred
with product formation rates in excess of 1000 minꢀ1 and total turnover numbers of greater than
5000 being observed in all but one instance. Critically, the oxidations were regioselective with
several being stereoselective. (−)ꢀMyrtenyl acetate, 20, was oxidized regioselectively (> 95%) to
yield cisꢀ4ꢀhydroxyꢀmyrtenyl acetate, 20a, which was further oxidized to 4ꢀoxoꢀmyrtenyl acetate,
20b, using a wholeꢀcell system providing a biocatalytic route to generate intermediates used in the
production of cannabinoid derivatives. The ester carbonyl moiety could also be used as a directing
group also to enhance the activity and control the selectivity of P450 catalyzed reactions; for
example, the turnover of Lꢀ(−)ꢀbornyl acetate, 18, and isobornyl acetate, 19, by CYP101B1
generated 9ꢀhydroxybornyl acetate, 18a, and 5ꢀexoꢀhydroxyisobornyl acetate, 19a, respectively, as
the sole products.
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Keywords; biocatalysis, norisoprenoids, terpenoids, esters, substrate engineering, directing groups,
C−H bond oxidation.
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Introduction
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Cytochrome P450 (CYP) enzymes constitute a superfamily of hemeꢀcontaining monooxygenases
which have a myriad of physiological roles and have many potential applications in the catalytic
synthesis of fine chemicals under mild conditions.1ꢀ5 There is great interest in applying them as
biocatalysts for the insertion of an oxygen atom from dioxygen into chemically inert
carbonꢀhydrogen bonds with high regioꢀ and stereoselectivity.6, 7 Cytochrome P450 activity
requires two electrons that are usually derived from NAD(P)H and these are delivered individually
to the CYP enzymes by electron transfer proteins.8 Electron transfer is often the rate determining
step in cytochrome P450 catalysis and the identification of complete systems which can catalyze
the selective oxidation of hydrocarbons and other organic molecules is a prominent field of
research.9ꢀ11
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Many bacterial CYP enzymes have been investigated as potential biocatalysts for the
selective oxidation of C−H bonds. Selfꢀsufficient enzymes in which the P450 is fused to the
electron transfer partners have been studied.7, 12 The CYP102 family is highly active for fatty acid
substrates but modifications of the enzyme are required to selectively oxidize other desirable target
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substrates.7,
Class I CYP enzymes, whose electron transfer systems consist of a
flavinꢀdependent ferredoxin reductase and a ferredoxin or flavodoxin, are also capable of high
monooxygenase activities and selective hydroxylations.18ꢀ22 Biocatalytic oxidations using these
systems have been reported on a range of substrates using either the wildꢀtype or mutant forms of
the CYP enzyme.1, 5, 9, 10 The activities of these class I systems or artificial fusion enzymes are
often compromised when nonꢀphysiological electron transfer partners are used which can hamper
the development of efficient biocatalytic procedures.19, 23ꢀ26
We have reported that the CYP101B1 enzyme from Novosphingobium aromaticivorans
DSM12444 is a highly active catalyst for the oxidation of norisoprenoids and can oxidize other
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structurally diverse classes of substrates.27ꢀ29 A class I electron transfer system, consisting of a
flavinꢀdependent ferredoxin reductase, ArR, and a [2Feꢀ2S] ferredoxin, Arx, has been identified
which supports the high activity of CYP101B1.19, 27, 28 To date CYP101B1 is one of only two
members of this subfamily of P450 enzymes to be identified the other member being found in a
different strain of Novosphingobium.29 A wholeꢀcell system capable of product formation on the
gramꢀperꢀliter scale in shake flasks has also been constructed.27 As a result, CYP101B1 is a
promising monooxygenase system for biocatalytic applications involving C−H bond oxidation
reactions.
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CYP101B1 is related to CYP101A1 (P450cam), CYP101D1 and CYP101D2 all of which
bind and oxidize the stereoselective oxidation of camphor (yielding ≥98%
5ꢀexoꢀhydroxycamphor).19, 27, 30, 31 It is also related to CYP101C1 which is able to bind and
catalyze the hydroxylation of norisoprenoids.32 CYP101B1 also catalyzes the oxidation of
(1R)ꢀ(+)ꢀcamphor but the reaction is not selective, generating five products;
5ꢀexoꢀhydroxycamphor, 5ꢀendoꢀhydroxycamphor, 6ꢀendoꢀhydroxycamphor, 9ꢀhydroxycamphor
and an isomer of 3ꢀhydroxycamphor.28, 33 The CYP101B1 catalyzed oxidation of the related
terpenoid 1,8ꢀcineole was more regioselective with >90% of the products arising from oxidation at
C3.33 The oxidation of αꢀionone was regioselective, generating isomers of 3ꢀhydroxyꢀαꢀionone.
The oxidation of βꢀionone and βꢀdamascone was selective for oxidation at C3 (90%).29 Therefore
CYP101B1 has the potential to catalyze routes to high value compounds without the requirement
for extensive protein engineering. Here we report an investigation into the substrate range of
CYP101B1 and show it is a capable of acting as a highly active and regioselective biocatalyst for
the oxidation of norisoprenoids and structurally related terpenoids. We also demonstrate that
monoterpenoid acetates can use the ester moiety as a directing group to facilitate and control
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substrate binding leading to selective and rapid oxidation of unactivated C−H bonds in terpenoid
frameworks. As such we show that the simple addition of an ester group could be used as chemical
auxiliary or directing group in substrate engineering experiments, as described by Griengl and
others, to improve the activity and selectivity of C−H bond oxidation reactions.25, 34ꢀ36
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Experimental Section
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General
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General reagents and organics, including all the substrates, were from SigmaꢀAldrich, TCI, Acros
or VWR. 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.
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General DNA manipulations and microbiological experiments and the expression,
purification and quantitation of CYP101B1 and the electron transfer proteins ArR and Arx from N.
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aromaticivorans were performed as described previously.27,
concentration was calculated using ε419 = 113 mM−1 cm−1.27, 28
Substrate binding analysis
The CYP101B1 protein
For substrate binding CYP101B1 was diluted between 1 and 6 µM using 50 mM Tris, pH 7.4.
Substrate was added (in 200 ꢀM) aliquots until no shift in the Soret band was observed. 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% 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 to
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between 0.5 and 4.5 µM 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 or DMSO. The solution was mixed and the peakꢀtoꢀtrough difference in
absorbance of the Soret band was recorded between 700 nm and 250 nm. Further aliquots of
substrate were added until the maximum peakꢀtoꢀtrough difference (A386 to A419) did not shift
further. The apparent dissociation constants, Kd, were obtained by fitting the peakꢀtoꢀtrough
difference of the Soret band against substrate concentration to a hyperbolic function (Eqn. 1):
ꢁAmax × [S]
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ꢁA =
Eqn. 1
Kd +[S]
Where ꢁA is the peakꢀtoꢀtrough absorbance difference, ꢁAmax is the maximum absorbance
difference and [S] is the substrate concentration.
Certain norisoprenoid and monoterpenoid acetate substrates exhibited tight binding, with
Kd < 5[E], and in these instances the data were fitted to the tight binding quadratic equation (Eqn.
2):37
([E]+[S]+ Kd ) − {([E]+[S]+ Kd )2 − 4[E][S]}
ꢁA
=
Eqn. 2
ꢁAmax
2[E]
where ꢁA is the peakꢀtoꢀtrough absorbance difference, ꢁAmax is the maximum absorbance
difference, [S] is the substrate concentration and [E] is the enzyme concentration.
Enzyme turnover and product formation analysis
NADH turnover assays were performed with mixtures (1.2 mL) containing 50 mM Tris, pH 7.4,
0.5 ꢂM CYP101B1, 5 ꢂM Arx, 0.5 ꢂM ArR and 100 ꢂg mL–1 bovine liver catalase. The mixtures
were oxygenated and then equilibrated at 30 °C for 2 min. Substrates were added from a 100 mM
stock solutions in ethanol or DMSO to a final concentration of 0.5 ꢀ 1 mM. NADH was added to
~320 ꢂM, final A340 ∼ 2.00, and the absorbance at 340 nm was monitored. The rate of NADH
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turnover was calculated using ε340 = 6.22 mM–1 cm–1. The total turnover number was determined
using assays set up like those above with the same ratio of the enzymes but with 0.1 ꢂM CYP
enzyme, 2 mM substrate and 4 mM NADH.
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The turnovers assays were extracted and product analysis by GC was performed as
described previously.27 The product concentration in incubation mixtures was calculated by
calibrating the total ion count response of the GCꢀMS 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. 4ꢀhydroxyꢀβꢀionol and 3ꢀhydroxyꢀβꢀionol were assumed to
have equal detector responses. The coupling efficiency was the percentage of NADH consumed
that led to product formation. The product formation rate (PFR) was calculated assuming the
NADH was used to form product as the ratio determined by the coupling efficiency. The turnover
number (TON) and total turnover numbers (TTN) which are the number of moles of product per
mole of P450) were calculated by quantitating the product in the turnovers as above. The analysis
methods and retention times are given in the supporting information.
Product isolation and characterization
To isolate and identify products where standards were not available a wholeꢀcell oxidation system
utilizing the plasmids pETDuetArx/ArR and pRSFDuetArx/CYP101B1 was used to oxidize
substrates as described previously.27 The plasmids were transformed into competent BL21(DE3)
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cells and grown on LB plates containing ampicillin, 100 ꢂg mL− , and kanamycin, 30 ꢂg mL−
(LBamp/kan). A single colony was inoculated into 500 mL broth (2xYTamp/kan) and grown at 37 ºC
overnight with shaking at 110 rpm. Protein expression was induced by the addition of 100 ꢂM
IPTG (from a 0.5 M stock in H2O) and the temperature and the shaker speed were reduced to 20
ºC and 90 rpm. The growths were allowed to continue for another 24 hours before the cell pellet
was harvested by centrifugation and washed in E. coli minimal media (EMM; K2HPO4 7 g,
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KH2PO4 3 g, Na3citrate 0.5 g, (NH4)2SO4 1 g, MgSO4 0.1 g, 20 % glucose (20 mL) and glycerol
(1 % v/v) per litre).38 The cell pellet was resuspended in an equal the volume EMM (~6 g cell wet
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weight.L− ) and split into 200 mL aliquots in 2L flasks. The substrates were added in 2 and 1 mM
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aliquots up to 8 mM over 24 hours and the reactions were run for 30 hours. Samples (1mL) were
taken for GCꢀMS analysis and were prepared as described above. To isolate product the
supernatant (200 mL) was extracted in ethylacetate (3 x 100 mL), washed with brine (100 mL) and
dried with magnesium sulfate 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
characterization. The solvent was removed under reduced pressure.
The purified products (ranging from 2 to 20 mg) after purification were dissolved in CDCl3
or acetoneꢀd6 and the organics characterized by NMR spectroscopy and GCꢀMS. NMR spectra
were acquired on a Varian Inovaꢀ600 spectrometer operating at 600 MHz for 1H and 151 MHz for
13C or an Agilent DD2 spectrometer operating at 500 MHz for 1H and 126 MHz for 13C. A
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combination of H, 13C, COSY, HSQC and HMBC experiments was used to determine the
structures of the products. NOESY was used to confirm the stereochemistry were required. Other
assignments of minor products produced in low yields were made via coꢀelution experiments or
comparison of MS spectra of standards published by others
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Results
The oxidation of norisoprenoids and analogous substrates by CYP101B1
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The norisoprenoids, αꢀ and βꢀionone, have been shown to induce a large type I spin state shift (≥
95%) on binding to CYP101B1, the binding is tight and these substrates are efficiently
hydroxylated by the enzyme solely or predominantly at C3.19, 27, 28 The related norisoprenoid
βꢀdamascone, which switches the position of the carbonyl and alkene moieties on the butenone
side chain, induced a lower spin state shift after addition to the enzyme. The binding was also
weaker but the efficiency and regioselectivity of CYP101B1 catalyzed oxidation were maintained
(Table 1).29 In order to investigate the importance of different functional groups within the
structural framework of norisoprenoids which result in effective binding and turnover efficiency
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by CYP101B1 we analyzed a series of substrates related to αꢀ and βꢀionone (1 and 2, Scheme 1).
Substrate binding and enzyme turnover activity were measured using in vitro enzyme assays and
oxidized metabolites were isolated using a wholeꢀcell oxidation system which produced
CYP101B1, Arx and ArR in E. coli.
The substrates αꢀ and βꢀionol (3 and 4) differ from the equivalent ionones by simply
replacing the carbonyl with an alcohol functional group (Scheme 1). Both ionols induced a large
type I spin state shift when added to CYP101B1 (70% and 90%, Fig. S1) but they bound thirtyꢀ to
fortyꢀfold less tightly than the ionone equivalents (Table 1, Fig. S2). The turnover and coupling
efficiency of CYP101B1 with both were high (product formation rate (PFR); 670 to 764
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nmol.nmolꢀCYP)–1.min–1; henceforth abbreviated to min− ). This is approximately twoꢀthirds that
of βꢀionone with that of αꢀionol being greater than αꢀionone (Table 1). αꢀIonol oxidation by
CYP101B1 generated a mixture of two products arising from monooxygenase activity (Fig. S3(a)
and Fig. S4(a)). Using a wholeꢀcell oxidation system generated the ketone, 3ꢀoxoꢀαꢀionol (3a), as
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the major product which was isolated and identified by NMR (Fig. S3(a) and Fig. S5(a)). The two
alcohols (3b and 3c) were isolated as mixtures after silica chromatography but the MS and NMR
analysis were consistent with oxidation at C3 and the data of D’Abrosca et al (Fig. S4(a)).39 There
was an approximate 2:1 excess of the cis isomer in the in vitro turnover (Scheme 1) which was
consistent with that found for the parent ionone.29 Two major products were obtained from the
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CYP101B1 catalyzed oxidation of βꢀionol, 4. Wholeꢀcell oxidation generated three products, two
of which were isolated and identified as 3ꢀhydroxyꢀβꢀionol (4a) and 4ꢀoxoꢀβꢀionol (4b) by
matching the NMR spectra and MS data with those previously reported.39 The other product was
assigned as 4ꢀhydroxyꢀβꢀionol (4c) based on its NMR and MS data (Fig. S4(b) and S5(b)). The
regioselectivity of oxidation of βꢀionol is lower for the C3 position (60%) when compared with the
oxidation of βꢀionone, which was 90% selective for oxidation at the equivalent position (Scheme 1
and Fig. S3 (a)).29
Methylionone (≥85% αꢀisomer,
butenone side chain (Scheme 1), induced a slightly higher spin state shift in CYP101B1 compared
to αꢀionone, , but bound threeꢀfold less tightly (Table 1, Fig. S1 and Fig. S2). The rate of NADH
5), which introduces an extra methyl group to the
1
oxidation was slower but comparable to that induced by αꢀionone. However, both the coupling
efficiency and the product formation rate were higher. Three products were identified, one of
which was isolated in a pure form and characterized by NMR as 3ꢀoxoꢀαꢀmethylionone (5a
,
Scheme 1, Fig. S3 (a) and Fig S5 (c)). A second product was purified which had NMR and MS
spectra consistent with an isomer of 3ꢀhydroxyꢀαꢀmethylionone (5b, Fig. S4(c) and supporting
information).40 The uncharacterized third product was not isolated in a pure form but the
spectroscopic and MS data (Fig. S4 (c)) were consistent with those of another isomer of
3ꢀhydroxyꢀαꢀmethylionone (5c). The main product from the wholeꢀcell oxidation was
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3ꢀoxoꢀαꢀmethylionone, 5a while the cisꢀ and transꢀhydroxy products were formed in roughly equal
quantities in the in vitro turnovers (Fig. S3 (a), Fig. S7).
7
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9
10
11
12
13
14
15
16
17
18
19
20
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24
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40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 1 Substrate binding, kinetic and coupling efficiency data for CYP101B1 with
norisoprenoids and related substrates.
CYP101B1/
substrate
%HS
heme
Kd
(µM)
NADH
(min–1)
PFR
C
%
TON TTN
TTN
vivo
(min–1)
vitro
βꢀionone29 2
0.23 ± 0.1 1600 ± 100 1010 ± 60
63
418/
682
5240
620027
≥95%
0.26 ± 0.04 1380 ± 140
660 ± 60
48
272/
566
8660
14750[a]
αꢀionone29 1
≥95%
βꢀdamascone29
80%
90%
70%
8.3 ± 0.9
7.4 ± 0.2
11.4 ± 0.5
930 ± 13
1030 ± 55
1030 ± 50
1270 ± 10
180 ± 30
73 ± 5
562 ± 12
670 ± 84
764 ± 76
980 ± 70
103 ± 9.0
n.d.
60
424/
700
440/
677
424/
565
500/
667
ꢀ
ꢀ
ꢀ
7700
6320
βꢀionol
4
3
65
ꢀ
5680
75
ꢀ
6740
1800
ꢀ
ꢀ
αꢀionol
90% 0.8 ± 0.01
77
7790[a]
αꢀmethylionone
Pseudoionone
Isophorone
5
6
90%
40%
55%
5.4 ± 0.1
n.d.
58
6050
8
n.d.
n.p.
ꢀ
ꢀ
transꢀ4ꢀphenylꢀ3ꢀ
butenꢀ2ꢀone
n.d.
71 ± 28
n.p.
ꢀ
7
Steady state turnover activities were measured using a ArR:Arx:CYP101B1 concentration ratio of
1:10:1 (0.5 ꢂM CYP enzyme, 50 mM Tris, pH 7.4). Coupling is the percentage efficiency of
NADH utilization for the formation of products, NADH is the NADH oxidation rate and PFR the
product formation rate. C is the coupling efficiency, TON the average turnover number for the
NADH consumption assay (with the maximum TON possible given in italics). TTN vitro is the
total turnover number observed using in vitro assays set up like those above with the same ratio of
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enzymes but with 0.1 ꢂM CYP enzyme, 2 mM substrate and 4 mM NADH. TTN vivo is the total
turnover number obtained from wholeꢀcell turnover assays (concentration of the P450 enzyme in
this system was 0.65 ꢂM).27 The Rates are reported as mean ± S.D. (n ≥ 3) and given in
nmol.(nmolꢀCYP)–1.min–1. n.p no observable product, n.d. not determined. [a] The in vivo TTN
includes further oxidation to the ketone. Some of the data for the norisoprenoid substrates was
reported previously and are included for comparison.
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Scheme 1 The products formed from CYP101B1 turnovers with norisoprenoid analogues. The
turnovers of αꢀ and βꢀionone were as reported previously.29 Unless otherwise stated the
regioselectivities of the turnovers conducted in this manuscript were 100%.
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Pseudoionone (6), which is a linear analogue of the norisoprenoids, also induced a
5
6
7
8
highꢀspin state shift when added to CYP101B1 (Scheme 1). The binding was weaker than those of
the cyclic ionones but stronger than ionols and βꢀdamascone (Table 1 and Fig. S2). The activity of
CYP101B1 with pseudoionone, as measured by the NADH oxidation and product formation rates,
was significantly lower when compared to all the cyclic norisoprenoids and ionols though the
coupling efficiency was comparable (Table 1). Two monooxygenase products were generated
from the in vitro turnovers in roughly equal amounts (Fig. S3(a)). These products were generated
using the CYP101B1 wholeꢀcell system but were not separated by silica gel chromatography
preventing the detailed spectroscopic characterization of the monooxygenase products. The
pseudoionone substrate is a mixture of two isomers (cis and trans) and the MS fragmentation
patterns of the two metabolites were similar (Fig. S4 (d)). Both cisꢀ and transꢀpseudoionone are
selectively oxidized by perbenzoic acids (e.g. mꢀCPBA) at the alkene furthest from the carbonyl
group, to an epoxide (6a, 3,5ꢀoctadienꢀ2ꢀone, 8ꢀ(3,3ꢀdimethyloxiranyl)ꢀ6ꢀmethyl, (Z,E) and
(E,E)).41ꢀ43 The epoxidation of pseudoionone using this method generated two products with
identical GC retention times and matching MS spectra and fragmentation patterns to those formed
by the CYP101B1 catalyzed oxidation of the same substrate allowing characterization (Scheme 1).
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We also tested transꢀ4ꢀphenylꢀ3ꢀbutenꢀ2ꢀone (7) with CYP101B1. This substrate has the
same butenone side chain as αꢀ and βꢀionone but replaces the trimethylcyclohexene ring with an
aromatic one (Scheme 2). The proportion of highꢀspin heme induced by substrate binding to
CYP101B1 was lower (55%) than the ionone and ionol substrates (Table 1 and Fig. S1). The lower
solubility and weaker binding of this substrate prevented accurate measurement of the dissociation
constant but it has a lower affinity than any of the norisoprenoid substrates. The activity of
CYP101B1 with this substrate was slower than even pseudoionone,
6 (Table 1) and no product
arising from monooxygenase activity could be identified by GCꢀMS or HPLC analysis of the in
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vitro turnovers. Wholeꢀcell oxidation turnovers resulted in trace amounts of three potential
products (all m/z = 164.1 AMU, data not shown) but their identity could not be determined.
Given the high affinity of CYP101B1 for molecules with the ionone framework we
decided to assess how active it would be for larger and smaller substrates with similar structures.
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Isophorone (8, 3,5,5ꢀTrimethylꢀ2ꢀcyclohexeneꢀ1ꢀone) has a similar ring system to αꢀionone, 1
((3E)ꢀ4ꢀ(2,6,6ꢀTrimethylcyclohexꢀ2ꢀenꢀ1ꢀyl)butꢀ3ꢀenꢀ2ꢀone, Scheme 2) but is missing the
butenone side chain of the isoprenoids. Isophorone binding to CYP101B1 only induced a 40%
shift to the highꢀspin form and the activity as measured by NADH oxidation was low (Table 1).
The level of product formation was meagre and the oxidation was unselective generating three
products in similar yields (Fig. S4(e)). Analysis of the MS fragmentation patterns of the three
products
suggested
they
were
isophorone
oxide
(
8a),
4ꢀhydroxyꢀ3,5,5ꢀtrimethylꢀ2ꢀcyclohexenꢀ1ꢀone
(8b
,
4ꢀhydroxyisophorone)
and
3ꢀhydroxymethylꢀ5,5ꢀdimethylꢀ2ꢀcyclohexenꢀ1ꢀone (8c, 7ꢀhydroxyisophorone, Fig. S4(e), Scheme
2).44, 45 The results from a selection of other smaller monoterpenoid organic molecules are
discussed in the next section.
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Scheme 2 The products formed from CYP101B1 turnovers with transꢀ4ꢀphenylꢀ3ꢀbutenꢀ2ꢀone, 7,
5
6
isophorone, 8, and (+)ꢀsclareolide, 9. The portion of the (+)ꢀsclareolide structure which is related
7
8
9
to norisoprenoid framework is highlighted in red. * 3 products in very low yield could be detected
after wholeꢀcell oxidation.
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(+)ꢀSclareolide (
9
) is a sesquiterpene lactone with structural similarities to the
, bound to CYP101B1 with
norisoprenoids but is significantly larger (Scheme 2). (+)ꢀSclareolide,
9
high affinity (Kd, 20 ± 4 ꢀM) and induced a reasonable shift to the highꢀspin state (50%).
Therefore the active site of CYP101B1 can accommodate substrates larger than norisoprenoids
(Table 1, Fig. S1 and S2). The oxidation activity was slow but a single product was isolated using
the wholeꢀcell oxidation system (Scheme 2 and Fig. S4(f)). The product was identified as
(S)ꢀ(+)ꢀ3ꢀhydroxysclareolide (9a) by matching the NMR signals with those reported in the
literature (Fig. S5d).46, 47
The oxidation of terpenes and related substrates by CYP101B1
We have previously shown that both enantiomers of camphor (10) are hydroxylated by CYP101B1
but the reactions are unselective generating five products (10a-e). 1,8ꢀCineole (11) induced a
greater shift to the highꢀspin form on binding to CYP101B1 when compared to the enantiomers of
camphor and the selectivity of oxidation is more favorable with >90% selectivity for the C3
position (11a-c). However the activity of product formation for all of these was lower than the
norisoprenoid substrates as was the regioselectivity.28,
To further investigate which
29, 33
monoterpenoid frameworks can be regioselectively oxidized by CYP101B1 we measured the
substrate binding and turnover activity data of a series of monoterpenes and related substrates.
(+)ꢀFenchone
(12),
which
is
vs.
an
analogue
of
camphor
(1,3,3ꢀTrimethylbicyclo[2.2.1]heptanꢀ2ꢀone
1,7,7ꢀTrimethylbicyclo[2.2.1]heptanꢀ2ꢀone,
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Scheme 3), bound less tightly to CYP101B1 but had a similar product formation rate and coupling
efficiency (Table 2, Fig. S1 and S2). The in vitro CYP101B1 turnover of (+)ꢀfenchone, 12, was
more selective than those of camphor with 75% of one major product being formed with two
minor products making up the remaining 25% (Scheme 3, Fig. 1(a), Fig. S3(b) and Fig. S4(g)).
The major product was isolated after a wholeꢀcell oxidation turnover and silica gel
chromatography and identified as 5ꢀexoꢀhydroxyfenchone (12a, Fig. S5(e)).48, 49 The two minor
products had MS fragmentation patterns which enabled tentative assignments of 6ꢀendoꢀ and
6ꢀexoꢀhydroxyfenchone (12b and 12c, Fig. S4(g)).50
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11
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41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 2 Substrate binding, kinetic and coupling efficiency data for CYP101B1 with
monoterpenoids and related substrates.
CYP101B1/
substrate
%HS
heme
Kd
(µM)
NADH
PFR
C
%
TON TTN TTN
vitro vitro
(1R)ꢀ(−)ꢀcamphor33 10r
(1S)ꢀ(−)ꢀcamphor33 10s
(+)ꢀfenchone 12
55%
334 ± 8
1040 ± 8 150 ± 20 15% 110/
ꢀ
4470
730
55%
262 ± 8 1260 ± 90 160 ± 40 13% 100/
ꢀ
ꢀ
770
50% 544 ± 23 1110 ± 80 200 ± 20 18% 122/ 920
1450
678
1,8ꢀcineole33 11
75% 576 ± 15
940 ± 2
355 ± 12 38% 260/ 420 7490[a]
684
1,4ꢀcineole 13
50% 609 ± 25 419 ± 35
42 ± 12 10% 50/
ꢀ
ꢀ
500
45%
64 ± 2
553 ± 87 165 ± 32 30% 206/ 620
2820
3390
ꢀ
(1R)ꢀ(−)ꢀnopol 14
cisꢀjasmone 15
687
35% 473 ± 17
65% 506 ± 15
133 ± 2
140 ± 5
28 ± 11 21% 118/
ꢀ
ꢀ
562
75 ± 18 54% 362/
670
5ꢀnorbornenꢀ2ꢀyl
acetate 16
See Table 1 for experimental details. The data for the camphor and 1,8ꢀcineole substrates was
reported previously and are included for comparison. [a] The in vivo TTN includes further
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oxidation to the ketone and hydroxyketone products.
5
6
7
8
1,4ꢀCineole
(13
,
1ꢀisopropylꢀ4ꢀmethylꢀ7ꢀoxabicyclo[2.2.1]heptane) has structural
9
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similarities to 1,8ꢀcineole, 11 (1,3,3ꢀtrimethylꢀ2ꢀoxabicyclo[2.2.2]octane) and pꢀcymene
(1ꢀisopropylꢀ4ꢀmethylbenzene, Scheme 3). It induced a lower spin state shift upon binding to
CYP101B1 than the 1,8ꢀcineole, 11, but greater than the nonꢀoxygen containing substrate
pꢀcymene (Fig. S1).29, 33 The product formation rate of 1,4ꢀcineole, 13, was similar to that of
pꢀcymene but tenꢀfold lower than 1,8ꢀcineole, 11 (Table 2). At least seven monooxygenase
products were generated from the oxidation of 1,4ꢀcineole with low selectivity (Fig. S3(c)). All
seven had masses indicating they were monooxygenase products with one further product which
has a mass consistent with that of a ketone further oxidation product (Fig. S4(h)). The low levels
of each of the products generated prevented detailed characterization though the major product
was isolated and identified as 6ꢀendoꢀhydroxyꢀ1,4ꢀcineole (13a, or its enantiomer,
2ꢀendoꢀhydroxyꢀ1,4ꢀcineole, or a mixture of both, Scheme 3, Fig. S5(f)).51 The MS fragmentation
pattern was also in agreement with that reported in the literature (Fig. S4(h)).51
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Scheme 3 The products formed from CYP101B1 catalyzed oxidation of (+)ꢀfenchone, 1,4ꢀcineole.
The products arising from camphor (R and S enantiomers, 10r and 10s) and 1,8ꢀcineole, 11, are
shown for comparison and their selectivity of oxidation were as reported previously.33
We tested a range of other monoterpenoid molecules and these resulted in minimal spin
state shifts (≤20%) on addition to CYP101B1. These included; (−)ꢀαꢀpinene (20%), (+)ꢀαꢀpinene
(5%), βꢀpinene (5%), carvone (20%), αꢀterpineol (10%), carvacrol (<5%) and thymol (10%). The
enzyme catalyzed turnovers with these substrates were very unselective generating multiple
products in very low yield or no detectable product. As a result these substrates were not
investigated further (data not shown).
We also chose to study other molecules which share structural features with norisoprenoids
and terpenes. (1R)ꢀ(−)ꢀNopol (14) and cisꢀjasmone (15) are chemicals used in the flavor and
fragrance industry and both contain an alkene containing aliphatic ring system and an alkyl side
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chain (Scheme 4). (1R)ꢀ(−)ꢀNopol, 14, is related to αꢀpinene and the terpenoid myrtenol but has a
primary alcohol functionality at the end of an ethyl sideꢀchain. cisꢀJasmone, 15, has a pentenyl
side chain and contains a carbonyl group on the ring system (Scheme 4). Both induced a type I
spin state change upon binding to CYP101B1, with that of (1R)ꢀ(−)ꢀnopol, 14, 45%, being greater
10
11
12
13
14
15
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than that of cisꢀjasmone, 15, 35% (Fig. S1). (1R)ꢀ(−)ꢀNopol, 14, bound to CYP101B1 with a
dissociation constant almost an order of magnitude lower, 64 ꢀM, than any of the monoterpene
substrates tested thus far (Table 2, Fig. S1 and Fig. S2). The activity and coupling efficiency of the
enzyme turnovers with both substrates was moderate generating observable levels of a single
major product in vitro (Fig. S3(d) and (e)). The product formation rate of cisꢀjasmone, 15, 28 minꢀ1,
was lower than that of (1R)ꢀ(−)ꢀnopol, 14 (Table 2). With both substrates additional minor
products were formed in the wholeꢀcell oxidation reactions (Fig. S3(d) and (e)).
After purification of the wholeꢀcell oxidation turnover of (1R)ꢀ(−)ꢀnopol, 14 two out of
three products were identified as (1R)ꢀ(−)ꢀ5ꢀhydroxynopol (14a, major product) and
(1R)ꢀ(−)ꢀ4ꢀoxonopol (14b, Fig. S4(i)). (1R)ꢀ(−)ꢀ4ꢀOxonopol, 14b was only formed in the
wholeꢀcell turnovers and this suggested that (1R)ꢀ(−)ꢀ4ꢀhydroxynopol, (14c) must be the other
product observed (Scheme 2, GC RT 11.0 min, Fig. S3(d)). The major product
(1R)ꢀ(−)ꢀ5ꢀHydroxynopol, 14a and (1R)ꢀ(−)ꢀ4ꢀOxonopol, 14b were identified by NMR (Fig. S5(g)
and (h)) with the data for both closely matching that reported previously.52 For cisꢀjasmone, 15, the
major product of the enzyme turnover was characterized via NMR as 4ꢀhydroxyꢀcisꢀjasmone (15a
)
with 11ꢀhydroxyꢀcisꢀjasmone (15b) being the minor product formed in vivo (Fig. S4(j), Fig. S5(i)
and (j)).53
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Scheme 4 The products formed from CYP101B1 catalyzed oxidation of (1R)ꢀ(−)ꢀnopol (14),
cisꢀjasmone (15) and 5ꢀnorborneneꢀ2ꢀyl acetate (16).
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5ꢀNorborneneꢀ2ꢀyl acetate (16) was also investigated as it has an acetate side chain
attached to a bicyclic ring structure (Scheme 4). The acetate side chain could, in principle, mimic
the butenone side chains of norisoprenoids and result in improved binding and turnover by
CYP101B1. The spin state shift on binding to CYP101B1 was high (65%, as a comparison that of
norcamphor was 30%, Fig. S1). The dissociation constant of CYP101B1 for 5ꢀnorborneneꢀ2yl
acetate, 16, was comparable to that of (+)ꢀfenchone, 12, but the activity as measured by NADH
oxidation was low (Table 2 and Fig. S2). However, the coupling of the reducing equivalents to
product formation in the enzyme turnovers was 54%. This is greater than any of the
monoterpenoid like substrates and was comparable to those of the norisoprenoid substrates (Tables
1 and 2). CYP101B1 catalyzed oxidation of this substrate, which is a mixture of the endoꢀ and
exoꢀisomers, generated two products (Fig. S3(f)). These were not separated after silica
chromatography but the major product could be assigned as an isomer of 2,3ꢀepoxyꢀ5ꢀnorbornanyl
acetate by MS and NMR (16a, Fig. S4(k) and S5(k)).
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The oxidation of monoterpene acetates by CYP101B1
5
6
7
8
Monoterpenoid oxidation with CYP101B1 resulted in lower activity and levels of product
formation compared with norisoprenoids. Given the tight binding of (1R)ꢀ(−)ꢀnopol, 14, and the
increased levels of coupling with 5ꢀnorborneneꢀ2ꢀyl acetate, 16, we decided to investigate if
monoterpenoid acetates, which contain the carbonyl containing acetate group, could interact with
the amino acid residues of the active site of CYP101B1 in a similar fashion to the butenone side
chain of the norisoprenoids. The oxygen atom of the carbonyl group on the acetate ester would
therefore act as an anchor and hold the substrate more tightly in the active site of CYP101B1
allowing only specific C−H bonds to be abstracted. If this is case, monoterpenoid acetates would
show improved substrate binding to CYP101B1 over the parent terpenes. The acetate ester could
function as a directing group and result in enhanced biocatalyst characteristics including turnover
activity and product selectivity. Fenchyl acetate (17), Lꢀ(−)ꢀbornyl acetate (18), isobornyl acetate
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(19) and (−)ꢀmyrtenyl acetate (20, Scheme 5) were chosen and all induced shifts to the high spin
state when added to CYP101B1 (80% to ≥ 95%). These are higher than those obtained with the
related monoterpenoids (fenchone, 12, camphor, 10, isoborneol, 21, borneol, 22, (−)ꢀmyrtenyl, 23
and (1R)ꢀ(−)ꢀnopol, 14; Tables 2 and 3, Fig. S1). The binding affinities of fenchyl, bornyl,
isobornyl and myrtenyl acetates (17-20) for CYP101B1 were also significantly tighter than those
of the corresponding monoterpenoids (Table 3). The tightest binding monoterpenoid acetate to
CYP101B1 was Lꢀ(−)ꢀbornyl acetate, 18 (Kd = 0.71 ꢀM) which binds more strongly than all of the
norisoprenoids bar αꢀ and βꢀionone,
1 and 2 (Tables 1 and 3, Fig. S2).
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Scheme 5 The products formed from CYP101B1 catalyzed oxidation of monoterpenoid acetates.
Isoborneol, 21, and borneol, 22, oxidation yielded at least three products in low yield (Fig. S3(h)).
The activity of the CYP101B1 catalyzed oxidation of the monoterpenoid acetates was also
considerably higher than the parent monoterpenoids. (Table 2 and Table 3) The product formation
1
rate of CYP101B1 with Lꢀ(−)ꢀbornyl acetate, 18, was the lowest of the esters at 600 min− which
significantly higher than those of camphor, 10, borneol, 22, and isoborneol, 21. The other three
1
acetates (17
,
19 and 20) induced product formation rates greater than 1000 min− , exceeding those
of the norisoprenoids (Table 2). The highest product formation activity was observed for
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(−)ꢀmyrtenyl acetate, 20; 1520 min− which was significantly greater than those of (−)ꢀmyrtenol,
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6
23, and (−)ꢀnopol, 14 (Table 2 and Table 3).
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Table 3 Substrate binding, kinetic and coupling efficiency data for CYP101B1 with
monoterpenoid acetates. The turnovers were regioselective with the exception of myrtenol acetate
which yielded 5% of the epoxide.
CYP101B1/
substrate
%HS
heme
Kd
(µM)
NADH
PFR
C
%
TON TTN TTN
vitro vivo
fenchyl acetate 17
isobornyl acetate 19
Lꢀ(−)ꢀbornyl acetate 18
(−)ꢀmyrtenyl acetate 20
borneol 22
80%
7.3 ± 0.8
14 ± 0.2
1220 ± 5
1110 ± 30 90% 566/ 1790 10810
628
≥95%
2000 ± 70 1250 ± 80 61% 360/ 6360
5200
2720
590
≥95% 0.71 ± 0.04 3080 ± 70
600 ± 50 19% 130/ 1870
684
≥95%
50%
55%
30%
5.1 ± 0.8
140 ± 24
140 ± 8
87 ± 4
1700 ± 14 1520 ± 30 90% 660/ 3220 8970[a]
733
410 ± 2
337 ± 15
336 ± 2
11 ± 3
32 ± 1
44 ± 2
3%
9%
28/
646
59/
115
220
75
<250
<250
2020
isoborneol 21
620
13% 88/
(−)ꢀmyrtenol 23
638
See Table 1 for experimental details. [a] The in vivo TTN includes further oxidation to the ketone.
The coupling efficiency of product formation to the NADH reducing equivalents in the
CYP101B1 turnovers was also considerably greater (50ꢀ100%) than the monoterpenoids with the
exception of Lꢀ(−)ꢀbornyl acetate, 18, which had lower coupling efficiency (19%). GCꢀMS
analysis of the in vitro and wholeꢀcell turnovers showed that CYP101B1 oxidized all of the
monoterpenoid acetate substrates regioselectively in contrast to the turnovers of camphor, 10 and
(+)ꢀfenchone, 12, which generated multiple products (Fig. 1 and Fig. S3(g)). The turnovers of
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(−)ꢀmyrtenol, 23, was selective for a single product while those of borneol, 22 and isoborneol, 21
,
4
5
6
generated very little product and were not selective (Table 3 and Fig. S3(h)).
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8
9
CYP101B1 turnovers with (−)ꢀmyrtenyl acetate, 20, generated one major product (∼95%,
Fig. S3(g)). Wholeꢀcell oxidation generated two major products which were isolated and identified
as, 4ꢀcisꢀhydroxymyrtenyl acetate (20a) and the further oxidation product 4ꢀoxomyrtenyl acetate
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(20b, Fig. S4(l), S5(l) and S5(m)). Small levels of a third product (<5%) which has a mass
spectrum indicating it arose from monooxygenase activity were also observed (Fig. S4(l)). This
metabolite coꢀeluted (RT 7.5 min, Fig. S3(g)) with the epoxide product obtained from the reaction
of (−)ꢀmyrtenyl acetate and mꢀCPBA (20c). A single major product was obtained in low yield from
the turnovers of (−)ꢀmyrtenol, 23, which was characterized as 4ꢀcisꢀhydroxymyrtenol, 23a (Fig.
S4(m) and S5(n)).
Fenchyl acetate, 17, and isobornyl acetate, 19, were oxidized stereoselectively by
CYP101B1 (Scheme 3, Fig. 1(b), Fig. 1(c) and Fig. S3(f)) and the products were identified using
NMR as 5ꢀexoꢀhydroxyfenchyl acetate (17a) and 5ꢀexoꢀisobornyl acetate (19a), respectively
(Scheme 3, Fig. S4(l), Fig. S5(o) and S5(p)). Enzyme catalyzed turnover of Lꢀ(−)ꢀbornyl acetate,
18, also generated a single product but in significantly lower yield (Fig. 1(d)). The product was
isolated and identified by NMR as 9ꢀhydroxybornyl acetate (18a, Fig. S4(l) and Fig. S5(q))
suggesting that the orientation of the acetate group controls which C−H bond is held closest to the
heme iron.
(a)
(b)
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(c)
(d)
Figure 1 GCꢀMS analysis of the CYP101B1 turnovers of (a) wholeꢀcell oxidation of (+)ꢀfenchone
with product A, 6ꢀendoꢀhydroxyfenchone and 6ꢀexoꢀhydroxyfenchone and
B
C
5ꢀexoꢀhydroxyfenchone. In all traces impurities are labelled (*) (b) In vitro turnovers of fenchyl
acetate with the product marked A, 5ꢀexoꢀhydroxyfenchyl acetate, (c) In vitro turnovers of
isobornyl acetate with the product marked A, 5ꢀexoꢀhydroxyisobornyl acetate and (d) In vitro
turnovers of Lꢀ(−)ꢀbornyl acetate with the product marked A, 9ꢀhydroxybornyl acetate.
The turnover data (PFR and coupling efficiency) and the linear NADH oxidation time
courses indicate that CYP101B1 is a good biocatalyst for use synthetic applications (Table 1ꢀ3 and
Fig. S6). The in vitro turnover assays have turnover numbers limited by the amount of NADH
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added (TON up to a maximum of ∼730, Table 1ꢀ3). In order to measure the efficiency of the
enzyme over a longer period of time we measured the total turnover number (TTN) of the enzyme
with selected substrates. In vitro the TTN followed a similar trend to the PFR and the TON with
the cyclic norisprenoids and terpenoid acetates having high values, 1790 to 8660 compared to the
monoterpenoids < 920 (Tables 1ꢀ3). The TTN of isobornyl acetate, 19, 6360, approached than of
the best norisoprenoids (5240ꢀ8660), and was greater than that of Lꢀ(−)ꢀbornyl acetate, 18, as
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expected from the PFR (Tables 1 and 3). The TTN of (−)ꢀmyrtenyl acetate, 20, was lower due to
competing ester hydrolysis over the longer duration of these in vitro turnovers while that of
fenchyl acetate was lower than expected and requires further optimization (Table 3).
The products generated using the shake flask wholeꢀcell oxidations were the same as those
from the in vitro turnovers, though more ketone and further oxidation products were obtained
(Tables 1ꢀ3, Fig. S7 and Fig. S8). The product yields and total turnovers from these oxidation were
also high. In contrast to the in vitro turnovers over 7 mM product (∼ 87% conversion in a 200 ml
1
reaction) was generated during the oxidation fenchyl acetate, 17 (TTN 10810, ∼1.4 g L− ). The
TTNs of the other monoterpenoid acetates were also high (>5000), though that of Lꢀ(−)ꢀbornyl
acetate, 18, was lower presumably due to its lower PFR (Table 3). These were comparable to those
obtained for the norisoprenoids (5680 – 14750, Table 1). The conversions were also high ranging
from 50ꢀ87% of the 8 mM product added to the 200 mL reaction during the turnover (Fig. S8). The
TTN and conversions of isoborneol, 21, and borneol, 22, were low but those of (−)ꢀmyrtenol, 22
,
and (1R)ꢀ(−)ꢀnopol, 14, were higher though still significantly lower than that of (−)ꢀmyrtenyl
acetate (Table 2 and 3). The TTN of (1R)ꢀ(−)ꢀcamphor, 10r, and 1,8ꢀcineole, 11, were 4470 and
7790, in line with what was reported previously, though the selectivity was lower than the
monoterpenoid acetates.33 The TTN and conversions of the other ketone compounds, (+)ꢀfenchone,
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12, and cisꢀjasmone, 15, were significantly lower than the norisoprenoids (Table 2).
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8
Overall the data suggests that the monoterpenoid acetates are a good fit for the active site
of CYP101B1 and as a consequence are efficiently and regioselectively, and in some instances,
stereoselectively hydroxylated. The altered regioselectivity of isobornyl and bornyl acetates, 18
and 19, shows that the carbonyl group of the acetate plays an important role in anchoring the
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substrate in the active site and can control the selectivity of oxidation. (−)ꢀMyrtenyl acetate, 20
,
turnover resulted in the oxidation of a C−H bond in a different position relative to the other
bicyclic terpenoid acetates. This could be due the different location of the acetate group, the
presence of a double bond or the modified bicyclic ring structure. The interaction of the carbonyl
group with the enzyme appears to regulate the orientation of the substrate in the active site. As a
result modification of the location of the carbonyl group in a molecule would allow chemists to
govern which C−H bonds are held closest to the heme ironꢀoxo intermediate and are therefore
abstracted.
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Discussion
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CYP101B1 is able to oxidize a broad range of substrates though optimal catalytic efficiency is
achieved for norisoprenoids and monoterpenoid acetates both of which have a carbonyl group on a
side chain attached to a substituted cyclohexyl or bicyclic ring systems. These substrates are
hydroxylated with high regioselectivity and in certain instances stereoselectivity. Terpene
oxidation by natural and modified P450 enzymes is a highly active area of research due to their
use as flavor and fragrance compounds and their biological activity.9, 47, 54 Microbial oxidative
biotransformations of norisoprenoids and related substrates have been reported using different
bacteria including Streptomyces55 and the fungus Aspergillus niger40 and P450 enzymes from
different strains of Streptomyces, Bacillus and Myxobacterium.56ꢀ60 The ability of CYP101B1 to
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regioselectively epoxidize the linear pseudoionone,
linear terpenoids and related derivatives.61
6, would allow it to be used as a biocatalyst for
The importance of the trimethylcyclohexene ring of the norisoprenoids can be clearly seen
by comparing their activities with CYP101B1 to those of the linear pseudoionone, and the
aromatic transꢀ4ꢀphenylꢀ3ꢀbutenꢀ2ꢀone, with the norisoprenoids. Previously we have shown that
6
7
the mixed phenyl and cyclohexyl ring substrate, phenylcyclohexane, was solely oxidized on the
aliphatic ring by CYP101B1.29 Cytochrome P450s are capable of efficiently oxidizing aromatic
molecules62ꢀ65 and phenylcyclohexane and transꢀ4ꢀphenylꢀ3ꢀbutenꢀ2ꢀone,
7 must be held with the
benzene ring held in such a fashion that the initial formation of the arene oxide or heme bound
intermediate required for the oxidation of aromatic C−H bonds was strongly disfavored.66
The carbonyl moiety in the butenone side chain of the norisoprenoids must also be
important for substrate binding as ionols,
3 and 4, bind less strongly than the equivalent ionones, 1
and . In addition both enantiomers of camphor, 10 and (+)ꢀfenchone, 12, are better substrates than
2
the isomers of pinene, borneol isomers, 21 and 22, and αꢀterpineol. The improved selectivity of
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