A semipinacol rearrangement directed by an enzymatic system featuring dual-function FAD-dependent monooxygenase

Article abstract of DOI:10.1002/anie.201204138

The biosynthesis of aurachins includes the intriguing migration of the prenyl group by a pinacol-type rearrangement. In vitro analysis of AuaGH revealed that these enzymes catalyze epoxidation coupled with semipinacol rearrangement (see scheme) and ketoreduction. Thus, the AuaGH system was revealed to be a novel enzymatic system for establishing semipinacol rearrangements.

Full text of DOI:10.1002/anie.201204138

Angewandte
Chemie
DOI: 10.1002/anie.201204138
Biosynthesis
A Semipinacol Rearrangement Directed by an Enzymatic System
Featuring Dual-Function FAD-Dependent Monooxygenase**
Yohei Katsuyama, Kirsten Harmrolfs, Dominik Pistorius, Yanyan Li, and Rolf Mꢀller*
Nature has invented ingenious ways to biosynthesize biolog-
ically active small molecules that have been applied ever since
to benefit human life in various ways. During the underlying
biosynthetic processes, highly elaborate chemical reactions
are often catalyzed by enzymatic systems, thereby enabling
transformations under physiological conditions that would
require harsh conditions or are hardly possible without
enzymatic catalysis. Consequently, understanding novel bio-
chemical transformations is of importance to eventually apply
the knowledge gained to generate molecules of interest.
Aurachins are quinoline alkaloids isolated from the
myxobacterium Stigmatella aurantiaca Sg a15; they have
various biological activities, including antibacterial, antifun-
gal, antiplasmodial, and mitochondrial respiration inhibition
properties.[1–6] The biosynthesis of aurachin derivatives
includes several interesting features in which the most
intriguing reaction is the conversion of aurachin C (1) to B
(2). This step involves the migration of the prenyl group from
position C3 to C4, probably via a pinacol type rearrangement
(Scheme 1).[7,8] Pinacol rearrangements are proposed to occur
during the biosyntheses of various secondary metabolites,
Scheme 1. Biosynthetic pathway of aurachin B (2) from aurachin C (1).
including aflatoxin B1, (+)-liphagal, (+)-asteltoxin, brevia-
namides, paraherquamide B, verscicolamide B, and notoa-
mides.[9–11] However, no such biosynthetic hypotheses has
been biochemically proven, although the proposed pathways
in turn inspired biomimetic approaches for natural product
synthesis.[9] Therefore, an enzymatic system for pinacol-type
rearrangement remained to be discovered in the biosynthesis
of secondary metabolites. Even in primary metabolism there
are only two reported examples in course of the biosynthesis
of branched chain amino acids and 1-deoxy-d-xylulose-5-
phosphate.[12–17]
[*] Dr. Y. Katsuyama,[+] Dr. K. Harmrolfs,[$] Dr. D. Pistorius,[$] Dr. Y. Li,[#]
Prof. Dr. R. Mꢀller
Helmholtz Institute for Pharmaceutical Research, Helmholtz Center
for Infection Research and Department of Pharmaceutical Biotech-
nology, Saarland University
The biosynthetic conversion of aurachin C (1) to B (2) was
initially studied by feeding experiments carried out by Hçfle
and Kunze.[7] Importantly, they reported that the hydroxy
group of aurachin B (2) at C3 is derived from molecular
oxygen. Recent work by Pistorius et al.[8] discovered two gene
loci containing aurachin biosynthetic genes in addition to the
core biosynthetic gene cluster.[18–20] The authors speculated
that two enzymes encoded by auaG and auaH are responsible
for the migration of the farnesyl group from C3 to C4.
However, the detailed biosynthetic conversion still remains to
be solved, mainly because of the lack of detection of putative
intermediates in vivo in auaG and auaH mutants of S.
aurantiaca Sg a15.[8] To uncover the enzymatic chemistry
behind this intriguing rearrangement reaction, we here
describe in vitro experiments using recombinant AuaG and
AuaH proteins.
Saarbrꢀcken (Germany)
E-mail: rom@helmholtz-hzi.de
[+] Present address: Graduate School of Agricultural and Life Sciences,
The University of Tokyo
Tokyo (Japan)
[$] Present address: Natural Products Unit, Novartis Institutes for
BioMedical Research, Novartis Pharma AG
Novartis Campus, 4056 Basel (Switzerland)
[#] Present address: National Museum of Natural History, CNRS,
Laboratory of Communication Molecules and Adaptation of
Microorganisms
UMR 7245, 57 rue Cuvier, 75005 Paris (France)
[**] We thank Prof. Dr. G. Hçfle from HZI Braunschweig for authentic
reference material of aurachin B and C. Y.K. was supported by the
Humboldt foundation with a Humboldt Research Fellowship for
Postdoctoral Researchers. Research in the laboratory of R.M. was
funded by the Deutsche Forschungsgemeinschaft and the Bundes-
ministerium fꢀr Bildung und Forschung. FAD=flavin–adenine
dinucleotide.
AuaG belongs to the family of flavin-dependent mono-
oxygenases and appears to be relatively similar to PgaE
involved in angucycline biosynthesis according to results from
BLAST search and Phyre2 analysis, respectively.[21] The
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201204138.
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protein possesses conserved motifs, GxGxxG, DG and GD,
which are important for this class of enzymes.[22] AuaH seems
to possess a Rossman fold and therefore was speculated to be
a NAD(P)H-dependent reductase (NAD = nicotinamine–
adenine dinucleotide). Both the conserved TGxxxGxG
motif for NAD(P)H binding and the catalytic centers
important for ketoreduction activity (YxxxK motif and the
catalytic Ser residue) are present in AuaH.[23,24]
To analyze these enzymes in vitro, recombinant proteins
fused with a hexahistidine tag at the C-terminus of AuaG and
AuaH were expressed in Escherichia coli using the pET
(Novagen) and pCold (Takara) systems, respectively. The
recombinant proteins were purified using Ni2+ affinity
chromatography (Supporting Information, Figure S1). AuaG
was purified as a slightly yellow protein and the binding
cofactor was confirmed to be flavin–adenine dinucleotide
(FAD) by the method described previously (Figure 1),[25] thus
Figure 2. LC-MS analysis of the conversion of aurachin C (1) from
AuaGH in vitro assays. When AuaG and AuaH were incubated with
aurachin C (1) in the presence of NADH, synthesis of aurachin B (2)
and oxidized aurachin C (3) was observed (A). When inactive AuaG
and active AuaH was used, neither 3 nor aurachin B (2) was produced
(B). When AuaG and inactive AuaH were used, only production of 3
was observed (C). In case of inactivation of both enzymes, no reaction
took place (D).
Figure 1. Analysis of the co-factor bound to AuaG. AuaG solution was
mixed with equal volume of methanol and centrifuged to remove the
precipitated protein. The obtained solution was analyzed by LC-MS
analysis (A) and compared with authentic FAD (B) and flavin
mononucleotide (FMN;C).
oxidation and subsequent reduction. Both enzymes required
NADH or NADPH for the reaction, and they both showed
a preference for NADH over NADPH (Supporting Informa-
tion Tables S1,S2). Addition of FAD to the reaction mixture
did not significantly influence the reaction velocity of AuaG.
Based on the key amino acids that have been reported to
constitute the catalytic center of ketoreductases associated
with secondary metabolite biosynthetic pathways, S111A and
Y139F mutants of AuaH were constructed. Both mutated
enzymes failed to produce aurachin B (2) in vitro (Supporting
Information, Figure S3).[23,24]
Compound 3 seemed to be the main product of AuaG.
This compound was highly unstable and rapidly isomerized to
a second compound in 0.1m CHES (pH 9.5)/methanol (Sup-
porting Information, Figure S4). The resulting isomer dis-
played identical MS/MS fragments, suggesting that the two
compounds have similar core structures (Supporting Infor-
mation, Figure S2). However, it exhibited a UV spectrum
different from compound 3, probably because of a structural
variation in the configuration of the conjugated double bonds
(Supporting Information, Figure S2). Therefore we presume
that the isomer is an oxidized form of aurachin C, the farnesyl
side chain of which is found in a different position from
compound 3. Our efforts to stabilize the intermediate by
indicating that approximately 80% of AuaG did bind FAD.
When these recombinant enzymes (2 mm each) were incu-
bated with 200 mm aurachin C (1) for 30 min at 308C in
a buffer containing 2 mm NADH and 0.1 mm of CHES
(pH 9.5), the formation of aurachin B (2) was detected by LC-
MS analysis (Figure 2A). The formation of aurachin B (2)
was confirmed by comparison with an authentic sample with
respect to retention time, UV, MS, and MS/MS spectra
(Supporting Information, Figure S2). This experiment also
revealed the production of unknown compounds. These
exhibited a molecular weight that is 16 Da heavier than
aurachin C (3) and were therefore speculated to be oxidized
forms. When heat-inactivated AuaG was added to the
reaction, neither aurachin B (2) nor oxidized aurachin C (1)
was detected (Figure 2B). Once AuaH was heat inactivated,
the formation of aurachin B (2) was eliminated but oxidized
aurachin C (3) was still produced (Figure 2C). Furthermore,
aurachin B (2) could also be formed by incubating AuaH with
NADH plus an ethyl acetate extract of AuaG reaction
products (Supporting Information, Figure S3). These results
clearly show that AuaG and AuaH act sequentially and that
conversion of aurachin C (1) into aurachin B (2) occurs by
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changing the pH from neu-
tral to basic led to various
ratios of compound 3, iso-
mers, and decomposition
products (Supporting Infor-
mation, Figures S4,S5). In
equilibrium state, the ratio
of isomer to compound 3
was approximately
9 at
pH 9.5 and 1.4 at pH 7.
Rapid degradation of com-
pound
3
was observed
under acidic conditions
(Supporting Information,
Figure S6).
To gain further insight
into the reaction catalyzed
by AuaGH we attempted to
characterize the oxidized
form of aurachin C (3), as
it seemed to be the major
product of the reaction cat-
alyzed by AuaG. Unfortu-
nately, isolation of com-
pound 3 failed owing to its
instability. Thus, we aimed
to characterize the com-
pound by direct analysis of
the enzymatic reaction by
NMR spectroscopy. The
AuaG reaction mixture
was
extracted
with
dichloromethane and the
obtained organic layer was
buffered with potassium
phosphate buffer (pH 7).
Scheme 2. Proposed reaction mechanism of AuaG reaction (A) and comparison with the reaction of
acetohydroxy acid isomeroreductase (B) and 1-deoxy-d-xylulose-5-phosphate reductoisomerase (C), which are
two enzymes that are known to catalyze the semipinacol rearrangement.
Dichloromethane
was
evaporated under cold N2-
stream and the residual crude product was immediately
dissolved in CD3CN for NMR analysis. Prior to NMR
analysis, the obtained sample was analyzed by LC-MS to
confirm that compound 3 was the main product (Supporting
Information, Figure S7). NMR analysis was performed by
combination of 1D and 2D experiments in CD3CN. Com-
pound 3 was found much more stable in organic solvent than
in water, although slight degradation of the compound after
NMR analysis was still observed. Detailed analysis of the
NMR data of the crude mixture revealed that the farnesyl
group in the intermediate (3) has already rearranged from C3
to C4. Evidence for this was obtained from the HMBC
spectra. HMBC correlations to a carbonyl group with
a chemical shift of d 195.3 could be detected for the methyl
group at d 2.23 (9-H) and a CH2 group at d 2.64 and 2.53(1’a,b-
H) respectively. Furthermore, the signals of 5-H and 1’a,b-H
showed HMBC correlations to a hydroxylated quartenary
carbon at d 77.5 (C4). These facts led us to conclude that the
farnesyl group is attached to C4 in 3, and 3 was thus identified
According to these results, we propose that AuaG
catalyzes the migration of the farnesyl chain by a semipinacol
rearrangement starting from aurachin C (Scheme 2).[9] First,
À
AuaG epoxidizes the double bond at C2 C3 position of the
quinoline core. After epoxidation, the N-hydroxy group is
likely to be deprotonated in conjunction with activation of the
carbonyl group by coordinative protonation. This type of
acid–base catalysis may induce the ring opening of the
epoxide followed by a semipinacol rearrangement, resulting
in migration of the farnesyl group from position C3 to C4 to
give rise to compound 3. Such proton shuttling is also
observed in other types of enzymes catalyzing semipinacol
rearrangements, which are discussed below.
Both epoxidation and rearrangement are likely to be
catalyzed by AuaG for the following reasons: First, the
reaction seems to be selective for compound 3 under
optimized conditions (Supporting Information, Figure S7).
Second, compound 3 was rapidly converted into its isomer
under basic conditions (Supporting Information, Figure S4).
This result suggests that the intermediate 3 is not the favored
state of the semipinacol rearrangement equilibrium and
as
4-hydroxy-2-methyl-3-oxo-4-farnesyl-3,4-dihydroquino-
line-1-oxide.
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therefore the reaction is likely to be driven enzymatically. The
rearrangement appears to take place in an inverse direction
under neutral and basic conditions, which is probably driven
by the deprotonation of the hydroxy group at position C4,
thus providing electrons to push back the farnesyl group to
the C3 position. The forward rearrangement may be stimu-
lated by a similar mechanism, but the “inverse isomer” is
likely to be more favored because C3 has a stronger electro-
philic property that is probably enhanced by the N-oxide at N-
1. Strong support for this proposal comes from the description
of 2-benzoyl-3-isonitrosobutan-2-ol, which was prepared
under strong acidic conditions.[26] The equilibrium state
described above and the instability of compound 3 suggested
the requirement of immediate reduction catalyzed by AuaH
to fix the farnesyl group at the position C4 and prevent the
degradation of this intermediate.
To the best of our knowledge, there are only two examples
of enzymes which catalyze semipinacol rearrangements
(acyloin rearrangements):[12–17] Acetohydroxy acid isomer-
oreductase and 1-deoxy-d-xylulose-5-phosphate reductoiso-
merase are involved in valine/isoleucine biosynthesis[12–14] and
2-C-methyl-d-erythritol 4-phosphate (MEP) pathway, respec-
tively.[15–17] (Scheme 2B,C). Both enzymes exhibit dual func-
tionality by catalyzing isomerization followed by reduction at
a single active site and require divalent metal ions for
isomerization in addition to NAD(P)H. As proposed by Tyagi
et al., the reduction is important for overcoming the unfav-
orable equilibrium state of the isomerization. In contrast,
AuaG does not require divalent metal ions and catalyzes
isomerization coupled with epoxidation, not with reduction.
AuaG probably establishes a selective isomerization by
catalyzing a rearrangement coupled with the ring opening of
an epoxide. The main product of AuaG appears to be an
energetically less-favored isomer that is immediately stabi-
lized by reduction and subsequent aromatization. The latter
step is catalyzed by AuaH, which is thus required to complete
the rearrangement.
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In summary, we have reported a novel enzymatic system
that establishes prenyl migration by employing a novel FAD-
dependent monooxygenase (AuaG), which catalyzes epox-
idation coupled with semipinacol rearrangement, and a keto-
reductase (AuaH). This discovery provides important infor-
mation regarding enzymatic rearrangements in natural prod-
uct biosynthesis and triggers efforts to achieve biomimetic
semipinacol rearrangements in organic synthesis.
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Received: May 28, 2012
Published online: && &&, &&&&
[26] G. Wittig, H. Kleiner, H. J. Conrad, Justus Liebigs Ann. Chem.
1929, 469, 1 – 16.
Keywords: aurachins · biosynthesis · enzymes · epoxidation ·
.
rearrangements
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Communications
Biosynthesis
Y. Katsuyama, K. Harmrolfs, D. Pistorius,
Y. Li, R. Mꢁller*
&&&& — &&&&
A Semipinacol Rearrangement Directed
by an Enzymatic System Featuring Dual-
Function FAD-Dependent
Monooxygenase
The biosynthesis of aurachins includes
the intriguing migration of the prenyl
group by a pinacol-type rearrangement. In
vitro analysis of AuaGH revealed that
these enzymes catalyze epoxidation cou-
pled with semipinacol rearrangement
(see scheme) and ketoreduction. Thus,
AuaGH system was revealed to be a novel
enzymatic system for establishing semi-
pinacol rearrangements.
Angew. Chem. Int. Ed. 2012, 51, 1 – 5
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5
These are not the final page numbers!