Dirigent proteins from cotton (Gossypium sp.) for the atropselective synthesis of gossypol

Article abstract of DOI:10.1002/anie.201507543

Gossypol is a defense compound in cotton plants for protection against pests and pathogens. Gossypol biosynthesis involves the oxidative coupling of hemigossypol and results in two atropisomers owing to hindered rotation around the central binaphthyl bond

Full text of DOI:10.1002/anie.201507543

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International Edition: DOI: 10.1002/anie.201507543
German Edition: DOI: 10.1002/ange.201507543
Biosynthesis
Dirigent Proteins from Cotton (Gossypium sp.) for the
Atropselective Synthesis of Gossypol
Isabelle Effenberger, Bin Zhang, Ling Li, Qiang Wang, Yuxiu Liu, Iris Klaiber,
Jens Pfannstiel, Qingmin Wang,* and Andreas Schaller*
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Abstract: Gossypol is a defense compound in cotton plants for
protection against pests and pathogens. Gossypol biosynthesis
involves the oxidative coupling of hemigossypol and results in
two atropisomers owing to hindered rotation around the
central binaphthyl bond. (+)-Gossypol predominates in vivo,
thus suggesting stereochemically controlled biosynthesis. The
aim was to identify the factors mediating (+)-gossypol
formation in cotton and to investigate their potential for
asymmetric biaryl synthesis. A dirigent protein from Gos-
sypium hirsutum (GhDIR4) was found to confer atropselec-
tivity to the coupling of hemigossypol in presence of laccase
streptomycetes, P450 enzymes are responsible for the regio-
[
5]
selective formation of biarylic pre-anthraquinones.
In
Daldinia eschscholzii, it is the conformational preference of
laccase for specific naphthol dimer radicals that leads to the
[
6]
enantiomeric excess of (À)-dalesconols. The second mech-
anism involves an unspecific oxidizing enzyme for radical
formation and so-called dirigent proteins (DIRs) that confer
regio- and enantioselectivity to the subsequent coupling
reaction. The discovery of DIRs in 1997 led to a new model
for phenoxy radical–radical coupling control in plant secon-
[
7]
dary metabolism. However, the concept is still a matter of
debate because DIRs are thus far only known for a single
reaction during lignan biosynthesis, in which DIRs control the
bimolecular coupling of coniferyl alcohol radicals to either
and O as an oxidizing agent. (+)-Gossypol was obtained in
2
greater than 80% enantiomeric excess compared to racemic
gossypol in the absence of GhDIR4. The identification of
GhDIR4 highlights a broader role for DIRs in plant secondary
metabolism and may eventually lead to the development of
DIRs as tools for the synthesis of axially chiral binaphthyls.
[
7,8]
(+)- or (À)-pinoresinol.
We show herein that DIRs are
also responsible for the atropselective formation of gossypol,
which supports a more general role for DIRs in plant
secondary metabolism.
A
xially chiral arene–arene linkages are frequent in biolog-
Gossypol (2, Scheme 1) is a natural product found in the
flowers, seeds, roots and foliage of cotton plants, where it
serves as a defense compound against insect pests and
pathogens. It has attracted a lot of interest for its multiple
pharmacological activities and because of its toxicity to
humans and non-ruminant animals, which limits the use of
ically active natural products, and such compounds are
rewarding targets for organic synthesis. Biosynthetic routes
to biaryls typically involve the oxidative coupling of phenols
or naphthols. While oxidative coupling is also the most direct
approach towards biaryl formation in organic synthesis, it
frequently suffers from poor regio- and enantioselectivity and
unwanted side reactions. Therefore, novel strategies for the
enantioselective formation of biaryl systems are urgently
[9]
[
1]
needed. In this respect, the biosynthetic repertoire evolved
by nature for the highly selective coupling of aromatic
compounds provides a largely untapped reservoir for bio-
technological approaches towards enantioselective biaryl
[
2]
synthesis.
There are at least two mechanisms that confer regio- and
enantioselectivity to oxidative coupling reactions in biological
systems. First, the oxidizing enzymes, cytochrome P450-de-
pendent enzymes or laccases, may provide the required
selectivity. The intramolecular coupling of (R)-reticuline
during morphine biosynthesis and of (S)-reticuline during
magnoflorine biosynthesis, for example, and the intermolec-
ular reaction for bisbenzylisoquinoline alkaloid formation are
catalyzed by highly specific cytochrome P450-dependent
Scheme 1. Oxidative coupling of 1 results in two atropisomers of 2.
[3]
oxidases in plants. In Aspergillus niger, a P450 enzyme
[10]
catalyzes the stereoselective coupling of dimethyl siderin as
cottonseed oil and protein for food and feed. The biosyn-
thesis of 2 involves the formation of 8-hydroxy-(+)-d-
cadinene from farnesyl diphosphate by cadinene synthase
[
4]
the penultimate step of (P)-(+)-kotanin biosynthesis, and in
[11]
and the P450 monooxygenase CYP706B1.
At least one
[
*] I. Effenberger, Prof. Dr. A. Schaller
more P450 enzyme is involved in the subsequent steps leading
Institut für Physiologie und Biotechnologie der Pflanzen
Universität Hohenheim (260), 70593 Stuttgart (Germany)
E-mail: andreas.schaller@uni-hohenheim.de
to hemigossypol (1), from which 2 is generated in a perox-
[12]
idase-catalyzed bimolecular radical coupling reaction. Two
atropisomers, (+)- and (À)-2, arise because of hindered
rotation around the central CÀC bond of the tetra-ortho-
I. Klaiber, Dr. J. Pfannstiel
Serviceeinheit Massenspektrometrie
Universität Hohenheim (690), 70593 Stuttgart (Germany)
substituted biaryl system. While organic synthesis typically
yields racemic mixtures of the two isomers (with the notable
exception of (+)-2 synthesis by asymmetric Ullmann coupling
Dr. B. Zhang, Dr. L. Li, Q. Wang, Prof. Dr. Y. Liu, Prof. Dr. Q. Wang
State Key Laboratory of Elemento-Organic Chemistry, Collaborative
Innovation Center of Chemical Science and Engineering (Tianjin)
Nankai University, Tianjin 300071 (P.R. China)
[13]
of chiral oxazoline-activated naphthyls), biosynthesis of 2
appears to be under stereochemical control. DIRs have been
suggested to be involved in the process, and DIR activity for
E-mail: wangqm@nankai.edu.cn
Supporting information and ORCID(s) from the author(s) for this
article are available on the WWW under http://dx.doi.org/10.1002/
anie.201507543.
(
+)-2 formation has in fact been detected in protein extracts
[12c,14]
from cotton embryos and flower petals.
However, the
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proteins responsible for atropselective biosynthesis of 2 have
not been identified yet, and this was the goal of the present
study.
In a candidate gene approach, two DIR-related cDNAs
reported by Zhu et al. from sea island cotton (G. barbadense;
GbDIR1 and GbDIR2) were considered to be involved in
gossypol biosynthesis because they are pathogen-inducible
and gossypol formation is known to be upregulated in cotton
[9,15]
plants as part of the pathogen defense response.
The
closest homologue of GbDIR1 and 2, GhDIR4, was cloned
from G. hirsutum var. marie-galante, a variety of upland
cotton containing (+)-2 in greater than 90% enantiomeric
excess (ee). The open reading frame of GhDIR4 was extended
with a hexa-histidine tag and expressed in a transgenic plant
cell culture system. Recombinant GhDIR4 was purified to
homogeneity from cell-wall extracts (Figure S18a in the
Supporting Information). The N terminus of the purified
protein was identified by mass spectrometry, which indicated
that the signal peptide is cleaved between Ser22 and Gln23
(
Figure S19). The apparent molecular weight of GhDIR4
differed under denaturing and native conditions (31 kDa for
SDS-PAGE, 125 kDa for gel filtration; Figure S18a,b), thus
suggesting a homotetramer as the native state of the protein.
However, considering the recently solved crystal structure of
a trimeric (+)-pinoresinol-forming DIR from pea plants, the
[16]
possibility of a homotrimer should not be dismissed.
To investigate potential “dirigent” activity, the product
spectrum generated during oxidative coupling of 1 was
compared in the presence and absence of GhDIR4. One-
electron oxidation of 1 was catalyzed by laccase from
Trametes versicolor with O as the oxidizing agent, which
2
results in (rac)-2 when no DIR is present. Preferential
formation of (+)-2 was observed upon addition of GhDIR4
(
Figure 1a). The enantiomeric excess of (+)-2 increased to
more than 80% with increasing concentrations of GhDIR4
Figure 1b). Only (rac)-2 was observed when GhDIR4 was
(
Figure 1. Demonstration of dirigent activity. The enantiomeric compo-
sition of 2 resulting from oxidative coupling of 1 was analyzed by
derivatization with a chiral amine and separation of the Schiff-base
adducts by reversed-phase HPLC. a) HPLC profiles are shown for the
heat-inactivated or substituted with DIRs known to be
involved in lignan formation (Figure S20b). GhDIR4, on the
other hand, was inactive with coniferylalcohol as the substrate
substrate (1), the reaction with laccase/O (1+L), the reaction with
2
(
Figure S20c). The data indicate that GhDIR4 is a novel
laccase/O in presence of GhDIR4 (1+L+DIR), and a (+)-2 standard.
2
dirigent protein that confers atropselectivity to the oxidative
coupling of 1. Its specificity for (+)-2 is consistent with (+)-2
being the predominant isomer in G. hirsutum var. marie-
galante.
An enantiocomplementary (À)-2-forming DIR might be
expected in plants that contain an excess of this particular
isomer. However, most cotton plants produce (+)- and (À)-2
at a 3:2 ratio. G. barbadense is a rare exception, with a 30–
b) Enantiomeric excess of (+)-2 with increasing amounts of GhDIR4.
gossypol biosynthesis and may act on different substrates. The
(À)-2-forming DIR thus remains elusive. Plants showing
a more pronounced ee of (À)-2, Thespesia danis, for example,
[18]
a tree from eastern Africa,
sources for such activity.
ought to be considered as
3
5% excess of (À)-2 in at least some of its organs, thus
We present herein the cloning and characterization of
cotton DIRs that mediate the laccase-catalyzed atropselective
coupling of 1 to (+)-2. The results highlight the importance of
DIRs for stereochemical control in plant secondary metab-
olism. The road from the discovery of (+)-2-forming DIRs
towards the development of DIRs as tools for the synthesis of
axially chiral binaphthols will certainly be long. Important
first steps have been made with the development of efficient
suggesting the presence of a (À)-2-forming DIR in this
[
17]
species. We thus cloned the pathogen-inducible GbDIR1
from G. barbadense, along with GaDIR1 from G. arboreum,
a species with the more typical 3:2 excess of (+)-2. The two
novel DIRs were expressed (Figure 2a) and their activities
were compared with that of GhDIR4 (Figure 2b). GaDIR1
also directed (+)-2 formation, which is consistent with (+)-2
being the predominant isomer in G. arboreum (Figure 2b).
No activity was detected for GbDIR1 during the coupling of
[19]
expression systems for DIR production, and the application
of targeted mutagenesis for the engineering of DIR selectiv-
[
20]
1, thus suggesting that this protein does not contribute to
ity. With respect to the use of cotton seeds as a source of
1
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Received: August 12, 2015
Published online: October 13, 2015
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