P450BM3 on steroids: The Swiss Army Knife P450 enzyme just gets better

Full text of DOI:10.1002/cbic.201100606

DOI: 10.1002/cbic.201100606
P450 on Steroids: The Swiss Army Knife P450 Enzyme
BM3
Just Gets Better
[a]
Luet-Lok Wong*
[
13]
The selective oxidation of an unactivated, aliphatic CÀH bond
tase domain fused to the haem monooxygenase domain.
[
1]
to an alcohol is a challenging problem in synthesis. Catalytic
systems for this reaction will have wide application in the
chemical and pharmaceutical industries. With rising energy
costs and increasing environmental awareness and concerns,
the ability to oxidise a targeted CÀH bond in a synthetic inter-
mediate would allow multistep routes to be shortened and
new routes to be developed. Processes would gain atom effi-
ciency, with attendant reductions in solvent and energy usage,
and compounds from sustainable sources might become
viable feedstocks.
The crystal structure (Figure 1) of the N-palmitoylglycine (NPG)-
bound form of the haem domain shows a spacious active
[14]
site.
Discrete molecule systems for CÀH oxyfunctionalisation are
mostly based on transition metal compounds. Of the metal-
free systems, dioxiranes are the most common and effective
[
2]
for aliphatic CÀH bond oxidation, and the oxidation of meth-
[
3]
II
ane to methanol was reported recently. Apart from Co -medi-
ated oxidation by oxygen, metal-based catalysts utilise a highly
reactive compound such as hydrogen peroxide, an alkyl hydro-
peroxide or an imine oxide, to generate a high-valency species,
typically a M=O complex, that inserts the oxygen atom into
[
4–6]
the CÀH bond through a radical mechanism.
Regioselectivi-
ty is observed in some instances due to the steric demands of
the supporting ligand, but the majority of molecular catalysts
show low selectivity.
Monooxygenase enzymes provide an alternative approach
Figure 1. Active-site residues in the X-ray crystal structure of the N-palmi-
[
7]
to CÀH bond oxidation. In biological systems, the cyto-
chrome P450 (CYP) haem monooxygenases catalyse the hy-
droxylation of a wide range of organic molecules by dioxy-
toylglycine (NPG)-bound form of the haem domain of wild-type P450BM3
[
14]
(PDB ID: 1jpz ). The secondary structure elements in which the residues
are located are highlighted, as are the three groups of residues targeted for
CAST (R47/T49/Y51 in green, V78/A82 in cyan, and M85/L188 in orange), as
well as A330, which was subjected to site-saturation mutagenesis, and F87,
which was mutated to an alanine to serve as the starting mutant.
[
8]
gen. The P450 enzymes involved in the biosynthesis of en-
dogenous compounds such as steroids and antibiotics are sub-
strate specific and display high product selectivity, whereas
those that metabolise and degrade xenobiotics have a broad
substrate range and often show low selectivity. Human P450
enzymes play a central role in drug metabolism.
Numerous site-directed, random and chimera mutagenesis
and screening strategies have been applied to the directed
Native and engineered P450 enzymes have been explored
evolution of P450
for the oxidation of a vast array of mole-
BM3
[
9]
[11,12]
for applications in synthesis, for example, in synthetic-biology
approaches to the production of the antimalarial agent artemi-
cules.
High-throughput methods include indigo formation
through whole-cell oxidation of indole from tryptophan me-
tabolism by P450BM3 mutants with altered substrate specificity.
[
10]
sinin.
P450BM3 (CYP102A1) from Bacillus megaterium is the
/
best-characterised P450 enzyme and the most studied for pro-
This procedure led to the discovery of the A74G/F87VL 188Q
[
11,12]
[15]
tein engineering and directed evolution.
A medium-chain
mutant that showed enhanced activity for the oxidation of
[16]
fatty acid hydroxylase, P450BM3 is highly active due to its cata-
lytically self-sufficient nature, with the electron-transfer reduc-
non-natural substrates including polyaromatic hydrocarbons,
and has been an excellent template for further engineering.
[12]
Mutations that enhanced substrate oxidation activity without
affecting product selectivity were found by coupling indigo
[
a] L.-L. Wong
Department of Chemistry, University of Oxford
Inorganic Chemistry Laboratory, South Parks Road
Oxford OX1 3QR (UK)
[17]
formation to a substrate oxidation screen. The formation of
+
NADP from NADPH oxidation induced by non-natural sub-
[18]
E-mail: luet.wong@chem.ox.ac.uk
strates can be detected in strong alkali; resorufin from O-
ChemBioChem 2011, 12, 2537 – 2539
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2537

L.-L. Wong
[
38]
dealkylation of alkoxyresorufin allows fluorescence screen-
97% perillyl alcohol.
Steroid oxidation presents an even
[
19]
ing; and p-nitrophenolate from the O-dealkylation of p-nitro-
phenoxycarboxylic acids and p-nitrophenoxyalkanes is detect-
greater challenge to selectivity engineering. An eight-mutation
enzyme variant that metabolised a range of drugs was found
to oxidise testosterone to a 53:29:18 mixture of 15b-, 16b- and
[
20,21]
ed spectrophotometrically.
[
39]
P450BM3 engineering efforts have been directed at various
synthetic targets. Alkane oxidation systems have been sought.
Variants with high activity and regio- and stereo-selectivity for
2b-hydroxytestosterone.
This mutant contained the F87V
mutation; site-saturation mutagenesis revealed that the F87I
variant gave 84% 16b-OH, whereas F87N gave 100% 2b-OH,
[
22]
[40]
octane oxidation have been reported, as have mutants for
and F87A was the most selective for 15b-OH (57%).
[
23–26]
[27]
propane
an E. coli whole-cell system for propane oxidation has been
and ethane oxidation, and the optimisation of
Recently the broader approach of the iterative combinatorial
active-site saturation test (CAST) was applied by Reetz and co-
workers to engineer P450BM3 variants with high regio- and ste-
[
28]
described. Methane oxidation was reported recently, but by
the wild-type enzyme with a perfluorocarboxylic acid acting as
an inert filler substrate that constrained methane molecules to
[41]
reoselectivity for testosterone and progesterone oxidation.
Twenty active-site residues (Figure 1) were divided into nine
groups. Residues in three groups were subjected to saturation
mutagenesis or a set of mutations that cover polar/nonpolar,
charged/uncharged and aromatic/nonaromatic substitutions.
Whole-cell steroid oxidation reactions for 8700 mutants were
analysed by HPLC. Starting with the F87A single-site mutant,
which gave a 52:45 mixture of 2b- and 15b-hydroxytestoster-
one (Scheme 1), Reetz et al. found that mutations in one
group of residues (R47/T49/Y51) favoured the 2b-OH (up to
94%), but those in another (V78/A82) favoured the 15b-OH,
while changes in a third group (M185/L188) had minimal
effect. A second round of CAST, combining mutations in the
first two groups, raised the 15b-OH selectivity to 96%. With
progesterone, the starting F87A mutant gave a 18:82 mixture
of 2b- and 16b-hydroxyprogesterone (Scheme 1). Of the three
mutant libraries, one gave a mutant with 100% selectivity for
the 2b-OH, but no variant gave higher 16b-OH selectivity than
[
26]
bind closer to the haem iron. Chemo-enzymatic synthesis
was demonstrated by the treatment of hydroxylated substrates
with diethylaminosulfur trifluoride to prepare fluorinated phar-
[
29]
maceuticals.
Wild-type P450
has low activity towards a
BM3
[
30]
number of drugs. Panels of variants from a variety of screen-
ing methods give product arrays broadly similar to those given
by drug-metabolising human P450s, though in altered propor-
[
31–34]
tions.
The role of active-site residues in substrate binding has been
exploited. Residues close to the haem have been targeted for
product selectivity engineering by site-directed mutagene-
[
35,36]
sis.
The screening of focused libraries of compounds with
mutations at two or three residues known to affect product
selectivity has identified mutants with high regioselectivity for
terpene oxidation; this could be used for the synthesis of fine
chemicals. Valencene has been oxidised to a mixture of noot-
[
37]
[24]
katol and nootkatone with >95% C selectivity, while anoth-
the F87A mutant. Site saturation at A330 gave the A330W/
2
er library gave a mutant that oxidised (R)-limonene to give
F87A mutant, which was the most selective for 16b-hydroxy-
Scheme 1. Hydroxylation of testosterone and progesterone mediated by P450BM3 mutants. The products from the oxidation of these two compounds in
steroid biosynthesis are also shown.
2
538
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ChemBioChem 2011, 12, 2537 – 2539

CASTing P450BM3
progesterone (91%) and, interestingly, also for 2b-hydroxytes-
tosterone (97%). Importantly, these increases in regioselectivity
were obtained without loss of diastereoselectivity (no a-alco-
hols were detected) and were accompanied by increased turn-
over activity and coupling efficiency compared to the starting
template mutant.
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Keywords: directed evolution · heme enzymes · oxidation ·
P450 monooxygenases · steroids
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[
Received: September 26, 2011
Published online on October 13, 2011
1
ChemBioChem 2011, 12, 2537 – 2539
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chembiochem.org
2539