Direct electrochemical regeneration of monooxygenase subunits for biocatalylic asymmetric epoxidation

Article abstract of DOI:10.1021/ja050997b

We report the first example of direct electrochemical regeneration of a flavin-dependent monooxygenase for asymmetric epoxidation catalysis. It is shown that electrochemical regeneration of the oxygenase subunit of the multicomponent styrene monooxygenase

Full text of DOI:10.1021/ja050997b

Published on Web 04/14/2005
Direct Electrochemical Regeneration of Monooxygenase Subunits for
Biocatalytic Asymmetric Epoxidation
Frank Hollmann,§,‡,† Karin Hofstetter,§,¶ Tilo Habicher,‡ Bernhard Hauer,‡ and Andreas Schmid*,§,¶
Institute for Biotechnology, Swiss Federal Institute of Technology, Zurich, Switzerland, BASF Aktiengesellschaft,
Ludwigshafen, Germany, and Department of Biochemical and Chemical Engineering, UniVersity of Dortmund,
Dortmund, Germany
Received February 16, 2005; E-mail: schmid@bci.uni-dortmund.de
Scheme 1. StyA-Catalyzed Epoxidation of Styrenes Following the
Native Cycle (A) and the Simplified Electroenzymatic Approach
(B)a
Enzymes are increasingly recognized as valuable tools for
asymmetric synthesis. The scope of enzyme catalysis remains
largely unexploited as the vast majority of synthetic biocatalytic
applications has been confined to simple, hydrolysis-based kinetic
resolution reactions.1 Monooxygenases catalyze the reductive
activation of molecular oxygen and its incorporation into a variety
of organic substrates, frequently with high regio- and stereoselec-
tivity and under environmentally benign reaction conditions.
However, monooxygenases are cofactor-dependent enzymes, which
have to be supplied with reducing equivalents derived from costly
and instable nicotinamide cofactors (NAD(P)H). The reducing
equivalents are often transferred to the terminal oxygenase via
complex multienzyme electron transport chains. Besides,
NAD(P)H-to-product stoichiometry is impaired by undesired oxida-
tive side-reactions.2 These impediments restrict the practical value
of monooxygenases as chiral catalysts.
A potential remedy for the mentioned challenges is the direct
introduction of reducing power into the catalytic cycle by cathodic
reduction. Research on electroenzymatic oxidations has been mainly
confined to P450-monooxygenase-catalyzed oxyfunctionaliza-
tions3,4 and flavin-dependent oxidases,5 whereas flavin-dependent
monooxygenases have been largely neglected. However, these
enzymes are versatile catalysts performing specific oxygenation
reactions, including hydroxylations, epoxidations, halogenations,
and heteroatom and Baeyer-Villiger oxidations.1
For example, styrene monooxygenase (StyAB) from Pseudomo-
nas sp. VLB120 catalyzes the specific S-epoxidation of styrene
derivatives.6 The enzyme is composed of an FADH2-dependent
oxygenase component (StyA), accomplishing the epoxidation
reaction and an NADH-dependent reductase component (StyB),
delivering reducing equivalents from NADH to StyA. Both
components possess their own catalytically active centers, and the
epoxidation capability of StyA is exclusively dependent on
FADH2.6a
a Holo-StyA binds reduced FAD for in situ activation of O2 followed
by stereospecific S-epoxidation of styrenes.6a
able when either StyA or FAD was omitted from the reaction
medium. Selected vinyl aromatic compounds could be transformed
into corresponding (S)-epoxides with high enantiomeric purity
(>98%) (Table 1). Apparently, the substrate spectrum, as well as
the stereodiscrimination of StyA, was not impaired under the
unnatural, highly simplified reaction conditions.
The rate of the electroenzymatic reaction did not meet the values
obtained with the native cycle. For example, a specific StyA activity
of 35.5 ( 2.1 U g-1 was calculated for the electroenzymatic
epoxidation of trans-â-methyl styrene,8 while specific StyA activi-
ties of up to 2100 U g-1 (corresponding to more than 1.6 catalytic
turnovers per second) have been observed in kinetic studies using
the native system.6 Apparently, the full catalytic potential of StyA
was by far not exploited yet.
In the present study, we establish direct electrochemical regen-
eration of FADH2 to substitute for the complex native regeneration
cycle including StyB and NADH (Scheme 1).
We assumed substrate limitation by FADH2 to account for the
low activity of the biocatalyst. Slow electrochemical reduction of
the flavin and fast aerobic reoxidation may explain the putatively
low in situ concentration of FADH2. No obvious change in the
spectroscopic properties of the reaction medium could be observed
throughout aerobic electrolyses (λ ) 450 nm), indicating a very
low steady-state concentration of reduced flavin. We therefore
investigated the influence of electrochemical parameters on the rate
of the epoxidation reaction. A shift of cathode potential to more
anodic values resulted in essentially identical reaction profiles,
indicating that electron transfer to FAD was not rate-limiting.7
However, the ratio of cathode surface to reaction volume had a
significant influence on the rate of the electroenzymatic epoxidation
Electrolyses were conducted potentiostatically in a temperature-
controlled stirred tank reactor (10 mL volume) with external
aeration. Cylindrical carbon felt served as cathode (working
electrode) and a Pt wire as anode (counter electrode).7 Application
of a cathode potential of -550 mV vs Ag/AgClsat. to the reaction
containing StyA, FAD, and catalase in phosphate buffer allowed
the conversion of trans-â-methyl styrene into practically enantiopure
(1S,2S)-1-phenylpropylene oxide. No product formation was detect-
† Current address: Max-Planck Institute for Coal Research, Mu¨lheim/Ruhr,
Germany.
§ Swiss Federal Institute of Technology.
‡ BASF.
¶ University of Dortmund.
9
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J. AM. CHEM. SOC. 2005, 127, 6540-6541
10.1021/ja050997b CCC: $30.25 © 2005 American Chemical Society

C O M M U N I C A T I O N S
reaction,6 depends directly on the oxygen concentration. Increased
supply of O2 increases the rate of the enzymatic reaction, while
the rate of the uncoupling reaction remains largely unaltered.
Stripping out of the reactants and StyA inactivation correlate
with the vigorousness of aeration under the experimental conditions
applied in this study. The low stability of StyA currently represents
the major impediment to preparative applications of the electro-
enzymatic epoxidation reaction. We are evaluating bubble-free
aeration techniques, such as hollow-fiber modules or anodic water
oxidation, that will allow efficient aeration under mild reaction
conditions.7
Table 1. Electroenzymatic Epoxidation of Substituted Styrene
Derivativesa
In conclusion, we have demonstrated the feasibility of direct
electrochemical regeneration of a flavin-dependent monooxygenase
for catalysis. Driven only by electrical power, optically pure
epoxides were synthesized from corresponding vinyl aromatic
compounds. The complicated native enzyme system consisting of
three enzymes (StyA, StyB, and an NADH regenerating enzyme)
and two cofactors (NADH and FAD) was minimized to the
oxygenase component and its flavin prosthetic group. In principle,
this approach is applicable to any enzymatic reaction involving
reduced flavins within the catalytic cycle.4,11 Optimization of the
bottlenecks identified in this study is underway and is expected to
result in a practical route to enantiopure epoxides, thus adding
electroenzymatic oxyfunctionalization to the toolbox of asymmetric
synthesis.
a General conditions: 10 mL of potassium phosphate buffer (50 mM,
pH 7.5), T ) 30 °C, c(StyA) ) 2.13 µM, c(FAD) ) 300 µM, c(catalase)
) 480 U mL-1, c(substrate) ) 2 mM, cathode area ) 14 cm2. Due to the
volatility of the reactants, no yields are given. b Values in parentheses
originate from whole-cell biotransformations.6b
Table 2. StyA Activity Depending on Cathode Surface and
Aerationa
area/volume quotient
aeration
(cm3 min-
StyA activity
1
b
1
1
c
(cm-
)
)
[U g-1] (min-
)
Acknowledgment. Financial support by the BASF Aktieng-
esellschaft, Ludwigshafen, Germany and the European Commission
(EC Enzymes, QLRT-1999-00439) is gratefully acknowledged.
1
2
3
4
5
6
0.21
0.74
2.12
2.12
2.12
0.94
7
7
7
0
6
15.1 (0.71)
34.5 (1.62)
74.5 (3.50)
26.6 (1.25)
64.5 (3.03)
178.7 (8.40)
Supporting Information Available: Further experimental data and
materials and methods. This material is available free of charge via
the Internet at http://pubs.acs.org.
40
a General conditions: phosphate buffer (50 mM, pH 7.5), T ) 30 °C,
c(StyA) ) 4.9-5.1 µM, c(FAD) ) 300 µM, c(catalase) ) 480 U mL-1
,
References
c(substrate) ) 2 mM. b Macroscopic cathode surface (cm2) divided by
medium volume (cm3). b Product formation rates may be normalized to the
biocatalyst concentration since this linearly correlates to the product
formation rate.7
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reaction (Table 2, entries 1-3), which can be ascribed to the
heterogeneous character of the electrochemical regeneration reac-
tion.
Since reduced flavins are subject to aerobic reoxidation,9 O2 may
further reduce the in situ concentration of FADH2 and negatively
influence the enzymatic epoxidation rate (KM,StyA,FADH > 10 µM10).
2
Similarly, electroenzymatic reactions with P450-monooxygenases
are hampered by undesired oxidative quenching of the regeneration
reaction.3,4 We found a positive correlation between aeration rate
and epoxide formation (Table 2, entries 4-6). The specific StyA
activity could be increased more than 5-fold compared to initial
values, corresponding to over 170 U g(StyA)-1 by increasing the
rate of external aeration. Apparently, the enzymatic epoxidation
rate is more oxygen sensitive than the oxidative quenching reaction
rate. The proposed autocatalytic FADH2 oxidation mechanism might
explain this observation.7,9 According to the mechanism, formation
of a flavin-semiquinone would be rate-limiting, and aerobic
FADH2 reoxidation should be largely independent from the
concentration of oxygen. Adversely, formation of the catalytically
active 4a-peroxoflavin, the rate-limiting step of the enzymatic
(8) Massey, V. J. Biol. Chem. 1994, 269, 22459-22462.
(9) One international unit (U) is defined as the biocatalyst amount forming
one micromole of product per minute. In the case of StyA (MW ) 47 ×
103 g mol-1), a specific enzyme activity of 1 U g-1 translates into a
catalytic performance (turnover frequency, TF) of 0.047 min-1
.
(10) KM value ) Michaelis-Menten constant, the concentration of (co)substrate
at which the catalyst exhibits half-maximal activity: Otto, K.; Schmid,
A. Unpublished results.
(11) Various P450-monooxygenases rely on FAD and/or FMN as electron
shuttles.
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