Communications
DOI: 10.1002/anie.200904622
Enzyme Catalysis
An Enantiocomplementary Dirigent Protein for the Enantioselective
Laccase-Catalyzed Oxidative Coupling of Phenols**
Benjamin Pickel, Mihaela-Anca Constantin, Jens Pfannstiel, Jꢀrgen Conrad, Uwe Beifuss,* and
Andreas Schaller*
The oxidative coupling of phenols marks a key step in the
biosynthesis of lignans, flavonolignans, and alkaloids and
plays a central role in plant secondary metabolism.[1] The
dimerization of propenylphenol derivatives to form lignans
generally imparts high regio-, diastereo-, and enantioselec-
tivity in vivo,[2] whereas in vitro enantioselectivity is negli-
gible.[3,4] For example, after enzymatic oxidation of (E)-
coniferyl alcohol (1) in vitro coupling results in the formation
of only racemic pinoresinol ((Æ )-2).
Regio- and stereoselective phenol coupling is observed
not only in plant secondary metabolism but also in bacteria,
lichen, and fungi.[5] Examples include the regioselective
formation of the isomers vioxanthin in Penicillium citreoviride
and pigmentosin A in Hypotrachyna immaculate,[6] and the
formation of the enantiomeric perylene quinones, hypocrellin
and hypocrellin A, in Hypocrella bambusae and Shiraia
bambusicola, respectively.[7] The rationale for the dramati-
cally different routes taken during oxidative phenol coupling
in vivo and in vitro remains an open question.
In 1997, Lewis et al. showed that in the presence of a
dirigient protein (DP) from Forsythia intermedia (FiDIR1),
the oxidative coupling of 1 (Scheme 1) results in enantiomer-
Scheme 1. Oxidative coupling of 1 with FiDIR1.[8]
ically pure (+)-pinoresinol ((+)-2) as well as (Æ )-dehydro-
diconiferyl alcohol ((Æ )-3) and erythro/threo-(Æ )-guaiacyl-
glycerol 8-O-4’ coniferyl ether ((Æ )-4).[8,9] Additional DPs
were subsequently found in Thuja plicata. Like FiDIR1, the
T. plicata DPs lack catalytic activity but mediate the enantio-
selective formation of (+)-2 in the course of enzymatic
oxidation of 1.[2,10] There are also indications for a protein in
Linum usitatissimum that allows the preferential formation of
(À)-2.[11] The atropselective coupling of hemigossypol to (+)-
gossypol in Gossypium hirsutum further shows that enantio-
selective phenol coupling in plants is not restricted to the
formation of 2.[12]
Continuing studies of DPs and their effects on phenol
coupling selectivity are required to achieve a better under-
standing of lignan biosynthesis and for the development of
phenol coupling as a generally applicable tool in enantiose-
lective synthesis. The latter aspect is of particular importance,
since there is a high demand, but no general solution, for
enantioselective phenol coupling in organic synthesis, even
though some promising approaches exist.[13]
[*] M.-A. Constantin, Dr. J. Conrad, Prof. Dr. U. Beifuss
Institut fꢀr Chemie, Universitꢁt Hohenheim
Garbenstrasse 30, 70599 Stuttgart (Germany)
Fax: (+49)711-459-22951
E-mail: ubeifuss@uni-hohenheim.de
We present herein the cloning, expression, purification,
and functional characterization of a DP from Arabidopsis
thaliana, which mediates the laccase-catalyzed enantioselec-
tive oxidative phenol coupling of 1 to (À)-2. We further show
that the enantioselectivity of the newly characterized Arabi-
dopsis DP is opposed to that of the known Forsythia DP. In
analogy to enantiocomplementary enzymes[14] we would like
to propose the term enantiocomplementary dirigent protein
(EDP) to describe such proteins.
B. Pickel, Prof. Dr. A. Schaller
Institut fꢀr Physiologie und Biotechnologie der Pflanzen
Universitꢁt Hohenheim
Emil-Wolff-Strasse 25, 70599 Stuttgart (Germany)
Fax: (+49)711-459-23751
E-mail: schaller@uni-hohenheim.de
Dr. J. Pfannstiel
Life Science Center und Institut fꢀr Physiologie
Universitꢁt Hohenheim, August-von-Hartmann-Strasse 3
70593 Stuttgart (Deutschland)
Starting point in our search for EDPs was the recent
report on a pinoresinol reductase from Arabidopsis thaliana
specifically converting (À)-2 into (À)-lariciresinol.[15] This
finding suggested the existence of an EDP responsible for the
[**] Financial support from the German Research Foundation (DFG,
SFB 706) is gratefully acknowledged.
Supporting information for this article is available on the WWW
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Angew. Chem. Int. Ed. 2010, 49, 202 –204