DOI: 10.1002/anie.201005597
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Enzymatic C H Activation
A Regioselective Biocatalyst for Alkane Activation under Mild
Conditions**
Mꢀlanie Bordeaux, Anne Galarneau, Franꢁois Fajula, and Jullien Drone*
The activation of alkanes to provide compounds of high value
is an important scientific challenge.[1] Increasing environ-
mental concerns call for the design of alternative and efficient
oxidation routes under mild conditions. Such benign methods
should enable the problems associated with the use of toxic
acids, solvents, and metals to be circumvented, and should
enable reactions to be carried out in an aqueous medium at
low temperature with a clean oxidant, such as oxygen.[2] The
Scheme 1. Oxidation of a substrate (RH) under the catalysis of a
cytochrome P450 hydroxylase (CYP) coupled to redox protein part-
ner(s) (RED). One molecule of the cofactor (NADPH) and one
molecule of molecular oxygen are consumed to yield one molecule of
use of biological catalysts instead of chemical catalysts can be
advantageous, because difficult reactions can often be per-
formed under mild conditions. Indeed, alkanes are partic-
ularly inert molecules, and their selective transformation
remains an important problem.[3]
the product (ROH).
Cytochromes P450 (CYP) form a superfamily of enzymes
widely distributed from prokaryotes to superior eukaryotes.[4]
They are known to catalyze the monooxygenation of a large
variety of hydrophobic chemical compounds, such as alkanes,
xenobiotics, steroids, and prostaglandins.[4c,5] An important
element for P450 catalysis is the nicotinamide adenine
dinucleotide cofactor, which, in its reduced form (NADH or
NADPH), provides the electrons necessary for the catalytic
cycle. As the electrons cannot be transferred directly to the
heme prosthetic group, redox protein partner(s) (P450
reductase) should be present to shuttle the reducing equiv-
alents from NAD(P)H to the hydroxylase domain[6]
(Scheme 1). Slow or uncoupled electron transfer currently
limits the catalytic efficiency of P450s.
It is possible to overcome this limitation by artificially
fusing a P450 hydroxylase to its natural reductase partner(s)[7]
(homologous fusion). Interestingly, functional enzymes can
also be obtained by fusion with a reductase from another
organism[7] (heterologous fusion). This strategy is of great
interest because many P450s catalyze interesting reactions
but have no identified reductase. The resulting biosynthetic
single-component cytochromes P450 are self-sufficient: the
different partners involved in the reaction remain together,
and the coupling efficiency and reaction rate are significantly
increased. The cytochrome CYP102A1 from Bacillus mega-
terium (P450 BM3) was the first natural self-sufficient P450
discovered[8,9] and remains one of the most efficient cyto-
chromes to date.
Alkane hydroxylases of the CYP153 family (AH153) are
monomeric and soluble proteins that convert medium-chain
alkanes (C5 to C12) into their corresponding alcohols.[10–12] This
process is thought to take place in the first step of alkane
metabolism, whereby the oxidation of the terminal and/or
subterminal carbon atoms leads to 1- and/or 2-alcohols. Such
a reaction at room temperature and atmospheric pressure is
of great interest to synthetic organic chemists, because the
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activation of inert C H bonds remains difficult by classical
chemical methods.[2] Pioneering studies on AH153 by van
Beilen and co-workers have produced a significant amount of
in vitro data for several members of this family.[12] Biocon-
versions were also conducted by van Beilen et al., who used
Pseudomonas putida whole cells expressing CYP153A6 from
Mycobacterium sp. HXN-1500 to produce perillyl alcohol
from limonene.[13] In another study, Fujii et al. used Escher-
ichia coli to successfully express CYP153A1 from Acineto-
bacter sp. EB104. They produced primary alcohols and a,g-
alkanediols by bioconversion. Although CYP153A1 was
correctly folded, functional in vivo, and present in the soluble
fraction of cell extracts, its purification and in vitro character-
ization were not reported.[14] Kubota et al. reported the
cloning of large genes encoding several CYP153 hydroxylases
fused to an FMN/Fe2S2-containing reductase (RhFred, earlier
found in the self-sufficient P450RhF from Rhodococcus sp.
NCIMB 9784[15]) in Escherichia coli (FMN = flavin mononu-
cleotide). For experiments with CYP153A13a from Alcani-
vorax borkumensis SK2, the P450 hydroxylase domain was
correctly folded, and its in vivo hydroxylation activity enabled
the production of primary alcohols by resting cells.[16] How-
[*] M. Bordeaux, Dr. A. Galarneau, Dr. F. Fajula, Dr. J. Drone
Institut Charles Gerhardt Montpellier
UMR 5253 CNRS/ENSCM/UM2/UM1
8, rue de l’Ecole Normale, 34296 Montpellier Cedex 5 (France)
Fax: (+33)4-6716-3470
E-mail: jullien.drone@enscm.fr
[**] We are grateful to Philippe Gonzales for GC/MS instrumentation, to
Irina Randrianjatovo, Emeline Vernhes, and Carl Ghaleb for their
excellent technical help, and to Dr. N. Ramsahye for proofreading
this article. This research was supported by the French Ministry of
Education (M.B.), the CNRS, the Graduate School of Chemistry of
Montpellier (ENSCM), and the University of Montpellier 2. We
thank Dr. D. Sherman (University of Michigan (USA)) for providing
us with a genetic sample of RhFred.
Supporting information for this article is available on the WWW
Angew. Chem. Int. Ed. 2011, 50, 2075 –2079
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
2075