A self-sufficient peroxide-driven hydroxylation biocatalyst

Article abstract of DOI:10.1002/anie.200351434

Directed evolution of the heme domain of cytochrome P450 BM-3 has resulted in a versatile, highly active peroxide-driven hydroxylation catalyst (see picture) that requires neither NADPH nor reductase and functions in a cell-free reaction system. This simplified, biomimetic catalyst is amenable to further optimization, for example, to improve stability or alter its substrate range.

Full text of DOI:10.1002/anie.200351434

Angewandte
Chemie
Enzyme Engineering
inactivated after only a few turnovers, the F87A mutant
supports low levels of peroxygenase activity (ca. 70 total
turnovers). In a previous study we demonstrated that directed
evolution could increase the rate of peroxide-driven naph-
A Self-Sufficient Peroxide-Driven Hydroxylation
Biocatalyst**
[
9]
thalene hydroxylation by cytochrome P450cam. Since then,
our efforts have focused on improving the peroxygenase
activity of the more active P450 BM-3 as a step towards
engineering a “biomimetic” hydroxylation catalyst.
Patrick C. Cirino and Frances H. Arnold*
[
14]
Controlled, selective oxidations of unactivated CÀH bonds
The P450 BM-3 heme domain (BMP) supports peroxy-
[
1]
[12]
are among the most desired transformations in catalysis.
genase activity
and is more thermostable than the full-
Limitations to chemical oxidation catalysts include low
turnover numbers, low or no regioselectivity, poor reaction
specificity, their use of environmentally harmful components
length P450 BM-3 protein (unpublished data). Removal of
the reductase domain also results in an approximate fourfold
increased molar expression (ca. 70 mgL in shake-flask
À1
(
for example, heavy metals or halogens), or their require-
cultures). We used BMP mutant F87A (HF87A) as the
starting point (parent)to increase catalyst performance by
sequential rounds of random mutagenesis and screening for
H O -driven hydroxylation of 12-p-nitrophenoxycarboxylic
acid (12-pNCA). Experimental details and a description of
the evolutionary path are available in the Supporting
Information.
ments for harsh, expensive reaction conditions. Many oxy-
genase enzymes catalyze the insertion of oxygen into
[
2,3]
unactivated CÀH bonds
and are superior to synthetic
2
2
[
4]
[15]
catalysts with regard to selectivity and turnover rate.
Enzymes have found numerous industrial applications,
[
5]
and the use of enzymes in industrial biocatalysis is expected
[
6]
to grow significantly. One limitation to the application of
oxygenases is their requirement for an expensive cofactor
such as NAD(P)H, which precludes their use in vitro without
Fifth-generation mutant “21B3” is nearly 20-fold more
active than HF87A in 10 mm H O using 12-pNCA as the
2
2
[
16]
substrate. Figure 1 shows the activities of HF87A and 21B3
[
7]
addition of a coupled, cofactor-regenerating system. The
need for additional proteins to transfer electrons from the
cofactor to the oxygenase presents a further complication.
Here we describe a self-sufficient P450 BM-3 variant which
utilizes hydrogen peroxide (H O )to catalyze hydroxylation
2
2
and epoxidation at high rates.
The cytochromes P450 are heme-containing oxygenases
which collectively catalyze a variety of oxidations on a diverse
[
3,8]
array of substrates. The natural P450 catalytic cycle utilizes
two electrons from NAD(P)H to activate dioxygen, although
P450s are also capable of utilizing peroxides as a source of
oxygen through a peroxide “shunt” pathway. In principle, this
“
peroxygenase” activity offers an opportunity to employ cell-
free P450 catalysis without requiring NAD(P)H regeneration,
additional proteins, or dioxygen, and eliminates rate-limiting
electron-transfer steps. In practice, this non-natural pathway
is too inefficient for any practical application.
À1
À1
Figure 1. Initial rates ((mol pNP formed)(mol P450) min ) of per-
oxide-driven 12-pNCA hydroxylation by P450 BMP variants HF87A and
21B3 in different H O concentrations. Reactions were performed at
[
9]
2
2
The soluble P450 BM-3 enzyme catalyzes the hydroxyla-
tion of sub-terminal CÀH bonds of medium chain (C –C )
room temperature and contained 300 mm 12-pNCA, 6% DMSO, and
purified enzyme (0.5 mm HF87A or 0.2 mm 21B3) in 100 mm Tris-HCl,
pH 8.2. Tris=tris(hydroxymethyl)aminoethane.
1
2
18
[
10]
fatty acids and amides
with initial rates exceeding
À1 [11]
3
000 min .
The activities of P450 BM-3 and its corre-
sponding F87A mutant in reactions driven by H O were
at different peroxide concentrations. Activities are reported
2
2
[
12,13]
recently characterized.
Whereas the wild-type enzyme is
as initial rates of p-nitrophenolate (pNP)formation at room
[
17]
temperature. The peroxygenase reaction follows apparent
Michaelis–Menten kinetics. Whereas the apparent K value
[
*] Prof. Dr. F. H. Arnold, P. C. Cirino
Division of Chemistry and Chemical Engineering
California Institute of Technology
Pasadena, CA 91125 (USA)
Fax: (+1)626-568-8743
E-mail: frances@cheme.caltech.edu
m
for H O in HF87A is about 30 mm, in 21B3 the apparent
2
2
K value is reduced to about 8 mm. Total turnover numbers
m
(TON)of approximately 1000 are achieved.
Improved peroxygenase activity also extends to fatty acid
substrates, with product distributions that are the same as
those from HF87A, as demonstrated in Figure 2 for reactions
of HF87A and 21B3 with lauric (dodecanoic)acid and
myristic (tetradecanoic)acid. No products other than those
shown were identified from these reactions. Using 10 mm
[
**] This work was supported by the Biotechnology Research and
Development Corporation (Peoria, IL) and an NSF Graduate
Research Fellowship awarded to P.C.C. The authors thank Ulrich
Schwaneberg for guidance and Denis Shcherbakov for laboratory
assistance.
H O , the initial rate of lauric acid hydroxylation, estimated
from the amount of product formed in the first minute of
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
2
2
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DOI: 10.1002/anie.200351434
ꢀ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
3299

Communications
summarizes the activities of 21B3 compared to HF87A for the
various substrates tested.
The major limitation of this system is rapid enzyme
inactivation as a result of peroxide-mediated heme degrada-
tion, as indicated by a decrease in the heme absorbance peak
in the presence of H O (not shown). Figure 3 shows the time-
2
2
Figure 2. Chromatograms of the trimethylsilyl-derivatized hydroxylated
products from H O -driven reactions of P450 BMP variants HF87A and
2
2
2
1B3 with lauric acid and myristic acid. The reactions contained puri-
fied enzyme (1–4 mm) and 1–2 mm substrate in 500 mL 100 mm Tris-
HCl, pH 8.2. The reactions were initiated by the addition of 5 mm or
Figure 3. Time-courses of pNP formation for reactions catalyzed by
P450 BMP variant 21B3 with 12-pNCA in 1, 5, and 10 mm H O .
Increasing peroxide concentration increases the peroxygenase activity
as well as the rate of enzyme inactivation. The reactions were per-
formed at room temperature and contained 250 mm 12-pNCA, 6.3%
2
2
1
0 mm H O , carried out at room temperature, and stopped by the
2 2
[
12]
addition of 7.5 mL 6m HCl, as described. I.S. is the internal standard
10-hydroxydecanoic acid), which was added to all samples in an equal
(
amount (30 nmoles) at the end of each reaction so that products
could be quantified relative to the area of the I.S. peak.
DMSO, and either 0.94 mm F87A or 0.15 mm 21B3 in 100 mm Tris-HCl,
À1
pH 8.2. Turnover is expressed in (mol pNP formed)(mol P450)
.
reaction (see ref. [12] or Supporting Information), is about
courses of pNP formation from reactions of 21B3 on 12-
À1
À1
5
0 mol(mol P450) min for 21B3. The TON for lauric acid
pNCA in 1 mm, 5 mm, and 10 mm H O . The enzyme is
2
2
in 5 mm H O is about 280. Similar results are observed with
essentially inactive within 5 minutes in 10 mm H O . Inacti-
2
2
2
2
decanoic (capric)acid.
vation appears to be turnover-dependent (similar TONs are
Oxidation of styrene to styrene oxide is also significantly
improved with 21B3. (Relaxed substrate specificity was
expected since specificity was not among the selection criteria
during screening.)The initial rate of styrene oxide production
reached from reactions in 1 mm, 5 mm, and 10 mm H O ), and
2
2
the ratio of peroxygenase turnovers to heme-degrading
turnovers increased with each generation of evolution (half-
lives in a given concentration of peroxide did not increase).
Sequence analysis of 21B3 reveals thirteen nucleotide
substitutions relative to the HF87A sequence, nine of which
result in amino acid substitutions. The BMP crystal struc-
À1
À1
by 21B3 is 55 mol(mol P450) min in 10 mm H O (esti-
2
2
mated from the amount of product formed in the first minute
of reaction), with a TON of about 240 in 5 mm H O . In
2
2
[
18]
contrast, the initial rate for HF87A in 10 mm H O is
ture shows that the mutations are dispersed throughout the
protein scaffold. No mutations appear in the active site,
substrate binding channel, or F and G helices of the heme
domain. There are also no mutations in any b-sheets of the b-
sheet-rich region of the structure. Four mutations lie on the
protein surface. A list of the amino acid substitutions and
information on the positions of these mutations are available
in the Supporting Information.
2
2
À1
À1
approximately 2.4 mol(mol P450) min , with a TON of
only about 10. For comparison, the NADPH-driven styrene
oxidation activity of wild-type BM-3 (for which styrene is a
À1
À1
poor substrate)is about 30 mol(mol P450) min . Table 1
Table 1: Summary of peroxygenase activities of P450 BMP variants
HF87A and 21B3.
The fungal heme enzyme chloroperoxidase (CPO)has
received much attention for its ability to catalyze the H O -
Substrate
Measurement
HF87A
21B3
2
2
[
a]
[b]
[19]
1
1
2-pNCA
initial rate
TON
23
90
10
70
2.4
10
430
980
50
280
54
driven, enantioselective epoxidation of alkenes. CPO does
[
a]
[c]
2-pNCA
[20]
not efficiently hydroxylate unactivated CÀH bonds, which
lauric acid
lauric acid
initial rate
TON
initial rate
TON
is the hallmark of P450 chemistry. Furthermore, protein
engineering on CPO has been extremely limited, as a result of
its lack of functional expression in bacteria or yeast. The
P450s SPa (CYP152B1)and BS b (CYP152A1)are two
recently discovered H O -driven fatty acid a- and b-hydrox-
[
d]
styrene
styrene
[
d]
240
[a] Activity on 12-pNCA was measured in the presence of 6% DMSO (see
2
2
ref. [16]). [b] Initial rates were determined in 10 mm H O and are
reported as (mol product)(mol P450) min . [c] Total enzyme turn-
2
2
[
21–23]
[24]
À1
À1
ylases.
Their highly specialized catalytic mechanisms
limits the substrate range and therefore the applications of
these P450s for biotransformations.
overs. TON values were determined in 5 mm H O . [d] Styrene oxide is
the only product.
2
2
3
300
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Angew. Chem. Int. Ed. 2003, 42, 3299 – 3301

Angewandte
Chemie
Cytochrome P450 BM-3 is a versatile hydroxylase whose
[15] U. Schwaneberg, C. Schmidt-Dannert, J. Schmitt, R. D. Schmid,
[
25]
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“
designability” has been demonstrated and whose biotech-
[
26]
[16] Screening was performed in the presence of DMSO (6.3%)to
help solubilize 12-pNCA (see Supporting Information). The
initial rate of HF87A with 12-pNCA in 6% DMSO
nological relevance has been established. Directed evolu-
tion allows us to engineer into this enzyme functions not
required or permitted in its natural biological context. For
example, a P450 BM-3 variant which efficiently hydroxylates
À1
À1
(
1
23 (mol product)(mol P450) min )is nearly half of that in
À1
À1
% DMSO (40 (mol product)(mol P450) min ), while similar
[
27]
alkanes was recently described. An efficient H O -driven
TONs are reached. As a result of evolution in DMSO, 21B3
activity is not inhibited by DMSO until concentrations exceed
2
2
BMP variant allows us to exploit this powerful and versatile
hydroxylase in a cell-free reaction system that requires
neither NADPH nor reductase. This catalyst is easy to
synthesize (high expression levels are achieved in E. coli),
10%.
[
[
17] Both HF87A and 21B3 are approximately 100% selective for
the C-12 position of 12-pNCA, and result in nearly complete
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28]
requires minimal preparation,
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conditions (namely, 258C, 1 atm). The self-sufficiency of the
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catalytic system.
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probe substrate (trans-2-phenyl-1-methylcyclopropane)with a
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Toy, M. Newcomb, L. P. Hager, Chem. Res. Toxicol. 1998, 11, 816.
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oxidation · peroxygenase
2
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Angew. Chem. Int. Ed. 2003, 42, 3299 – 3301
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ꢀ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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