Controlling a structural branch point in ergot alkaloid biosynthesis

Article abstract of DOI:10.1021/ja105785p

The ergot alkaloids are a diverse class of fungal-derived indole alkaloid natural products with potent pharmacological activities. The biosynthetic intermediate chanoclavine-I aldehyde 1 represents a branch point in ergot biosynthesis. Ergot alkaloids festuclavine 2 and agroclavine 3 derive from alternate enzymatic pathways originating from the common biosynthetic precursor chanoclavine-I aldehyde 1. Here we show that while the Old Yellow Enzyme homologue EasA from the ergot biosynthetic gene cluster of Aspergillus fumigatus acts on chanoclavine-I aldehyde 1 to yield festuclavine 2, EasA from Neotyphodium lolii, in contrast, produces agroclavine 3. Mutational analysis suggests a mechanistic rationale for the switch in activity that controls this critical branch point of ergot alkaloid biosynthesis.

Full text of DOI:10.1021/ja105785p

Published on Web 08/25/2010
Controlling a Structural Branch Point in Ergot Alkaloid Biosynthesis
†
‡
,‡
,†
Johnathan Z. Cheng, Christine M. Coyle, Daniel G. Panaccione,* and Sarah E. O’Connor*
Massachusetts Institute of Technology, Department of Chemistry, 77 Massachusetts AVenue,
Cambridge, Massachusetts 02139, and West Virginia UniVersity, DiVision of Plant & Soil Sciences, Morgantown,
West Virginia 26506
Received June 30, 2010; E-mail: Dan.Panaccione@mail.wvu.edu; soc@mit.edu
a
Scheme 1
Abstract: The ergot alkaloids are a diverse class of fungal-
derived indole alkaloid natural products with potent pharmacologi-
cal activities. The biosynthetic intermediate chanoclavine-I alde-
hyde 1 represents a branch point in ergot biosynthesis. Ergot
alkaloids festuclavine 2 and agroclavine 3 derive from alternate
enzymatic pathways originating from the common biosynthetic
precursor chanoclavine-I aldehyde 1. Here we show that while
the Old Yellow Enzyme homologue EasA from the ergot biosyn-
thetic gene cluster of Aspergillus fumigatus acts on chanoclavine-I
aldehyde 1 to yield festuclavine 2, EasA from Neotyphodium lolii,
in contrast, produces agroclavine 3. Mutational analysis suggests
a mechanistic rationale for the switch in activity that controls this
critical branch point of ergot alkaloid biosynthesis.
a
(
A) Reduction of C8-C9 alkene by EasA_Af leads to festuclavine 2.
(
B) EasA_Nl leads to formation of agroclavine 3.
The ergot alkaloids are a diverse class of fungal-derived indole
alkaloid natural products with potent pharmacological activities. The
biosynthetic intermediate chanoclavine-I aldehyde 1 can be enzymati-
cally converted into festuclavine 2, which is further derivatized to form
1
isomerization step required for the conversion of chanoclavine-I
2,4
aldehyde 1 into agroclavine 3. Mutational analysis suggests a
mechanistic rationale for the switch in activity of EasA- from
reductase in A. fumigatus to isomerase in N. lolii- that controls this
critical branch point of ergot alkaloid biosynthesis.
alkaloids such as the fumigaclavines in Aspergillus fumigatus (Scheme
1
1
). Alternatively, in certain fungal species such as ClaViceps purpurea
3
Based on previous analyses and homology of EasA with Old
and Neotyphodium lolii, chanoclavine-I aldehyde 1 is converted to
Yellow Enzyme, a flavoenzyme that typically reduces the alkene
agroclavine 3, which goes on to form lysergic acid-derived ergot
5
1
of an R, ꢀ unsaturated carbonyl moiety, the role of EasA in fungal
alkaloids such as ergopeptines. Festuclavine 2 and agroclavine 3 differ
producers of festuclavine 2 is clear: reduction of the C8-C9 double
bond of chanoclavine-I aldehyde 1 (Scheme 1A). However, fungal
producers of agroclavine 3, which retains the C8-C9 alkene, also
only by the degree of unsaturation in the D ring (Scheme 1). Although
chanoclavine-I aldehyde 1 had been proposed as an ergot alkaloid
2
biosynthetic intermediate many years ago, the enzyme-catalyzed
1
contain an EasA homologue in the ergot gene clusters. EasA
mechanism of D ring formation has remained elusive. Recently, we
and others have reported that a homologue of Old Yellow Enzyme
from A. fumigatus, EasA_Af, reduces the alkene of the chanoclavine-I
therefore likely plays a different role in this biosynthetic pathway.
Notably, an A. fumigatus (a festuclavine 2 producer) ∆easA
disruption strain transformed with easA from C. purpurea (an
3
aldehyde R, ꢀ unsaturated carbonyl moiety (C8-C9, Scheme 1A).
6
agroclavine 3 producer) yielded agroclavine 3 derived alkaloids.
This reduction facilitates an intramolecular reaction between the
aldehyde and the amine moieties to allow formation of the D ring of
festuclavine 2 (Scheme 1A). Yet it is still unclear how chanoclavine-I
aldehyde 1 is converted into agroclavine 3, which retains the C8-C9
double bond of the starting precursor, but with this alkene in the
opposite geometrical configuration. Formation of agroclavine 3 from
chanoclavine-I aldehyde 1 was investigated previously by Floss and
This experiment strongly suggests that EasA is the checkpoint that
controls whether chanoclavine-I aldehyde 1 is converted to festu-
clavine 2 or agroclavine 3. To gather mechanistic data to test this
hypothesis, an in Vitro assay for agroclavine 3 producing EasA
enzymes is required. Unfortunately, when EasA from C. purpurea
was heterologously expressed in E. coli, the resulting protein was
catalytically inactive with chanoclavine-I aldehyde 1 and lacked
the expected flavin cofactor as evidenced by UV-vis spectroscopy.
However, EasA from another agroclavine 3 producer N. lolii
2,4
colleagues in whole-cell precursor feeding studies. Results of these
studies not only clearly demonstrated precursor-product relationships
but also raised unanswered questions about the enzymes and mech-
anisms involved. One particularly puzzling step implicated a cis-trans
isomerization involving intermolecular transfer of a hydrogen between
successive substrate molecules. Here we show that an EasA homologue
from the ergot biosynthetic gene cluster of N. lolii, EasA_Nl acts on
chanoclavine-I aldehyde 1 to yield agroclavine 3 rather than festucla-
vine 2, therefore supporting the previously proposed cis-trans
(
EasA_Nl) could be expressed as an active, holo-enzyme from E.
coli (Supporting Information, SI).
We compared the products of EasA_Af and EasA_Nl when
7a
incubated with chanoclavine-I aldehyde 1 substrate in the presence
of NADPH-dependent oxidoreductase EasG (A. fumigatus). Recent
work by Wallwey et al. has revealed that EasG reduces the cyclic
iminium product generated by EasA_Af to form festuclavine 2
3
b
†
(Scheme 1A). EasG, which is present in all ergot biosynthetic
Massachusetts Institute of Technology.
West Virginia University.
‡
gene clusters, likely plays a similar role in agroclavine 3 biosyn-
10.1021/ja105785p  2010 American Chemical Society
J. AM. CHEM. SOC. 2010, 132, 12835–12837 9 12835

C O M M U N I C A T I O N S
thesis (Scheme 1A); EasG from N. lolii exhibits 65% amino acid
similarity to EasG from A. fumigatus (SI). As expected, when
EasA_Af and EasG were incubated with chanoclavine-I aldehyde
the substitution to Phe, which cannot serve as a proton donor, would
allow enolization of the substrate following donation of a hydride
from the flavin. Subsequent rotation around the C8-C9 bond,
tautomerization, and transfer of the hydride back to the flavin lead
to the Z alkene geometry to allow intramolecular iminium ion
formation (Scheme 2A). Notably, our data and interpretation provide
an explanation for observations documented by Floss and co-
workers in labeled precursor feeding experiments. These investiga-
tors performed numerous labeling experiments supporting a mech-
anism that involves an intermolecular transfer of the hydrogen at
1
and NADPH, a product with a mass and retention time identical
to those of an authentic standard of festuclavine 2 was observed
7
b
(
Figure 1A,E, SI). Gratifyingly, in contrast, EasA_Nl and EasG
yielded a product with a mass and retention time identical to those of
7b
agroclavine 3 (Figure 1B,D, SI). Therefore, EasA_Nl exhibits the
biochemical activity predicted by the alkaloid production profile of
the producing organism, validating that EasA controls formation of
festuclavine 2 vs agroclavine 3 from chanoclavine-I aldehyde 1.
Although the C8-C9 double bond is retained in both chanocla-
vine-I aldehyde 1 and agroclavine 3, the alkene geometry switches
1
c,2,4e
C9.
The most conclusive of these experiments showed that
2
13
separately labeled H and C chanoclavine-I 4 (the direct precursor
to chanoclavine-I aldehyde 1) are converted by ClaViceps to doubly
labeled agroclavine 3 derived products (Scheme 2B) (see Table 5
of ref 2). Doubly labeled product indicates that the hydrogen at C9
undergoes an intermolecular transfer from one substrate to another.
Floss proposed that this transfer may occur by addition of a
hydrogen at C9, followed by removal of a hydrogen, and that one
4
from E in 1 to Z in the product 3. This switch from E to Z is
required to allow intramolecular iminium formation (Scheme 1B).
EasA_Nl therefore displays an isomerase rather than the reductase
function of EasA_Af. This switch in enzymatic function clearly
explains how the biosynthetic intermediate 1 with the alkene E
configuration is converted into product 3 with Z configuration.
4
e
of these steps may occur in a nonstereoselective fashion. Our
results suggest that this intermolecular H transfer likely results from
hydrogen transfer between flavin and substrate as depicted in
Scheme 2A. Additionally, Floss proposed that the transferred
1c,4f
hydrogen could be either a proton or a hydride;
the involvement
of a flavin-containing enzyme suggests that the hydrogen transfer
at C9 likely involves a hydride. More detailed studies are required
to further explore this aspect of the enzyme mechanism and to
clarify whether these hydrogen transfer steps are nonstereoselective.
b
Scheme 2
b
(
A) Proposed isomerization reaction catalyzed by EasA_Nl. (B) The
products of whole cell labeling experiments, which derive from a mixture
of unlabeled, single, and double labeled 3 shown above, reported in ref 2
suggest that H of C9 undergoes intermolecular transfer.
Figure 1. LCMS chromatograms (selected ion monitoring at m/z 239 and
2
41) showing product formation in EasA assays. (A) EasA_Af + EasG.
(
B) EasA_Nl + EasG. (C) EasA_Nl Phe176Tyr + EasG. (D) Agroclavine
To validate the hypothesis that the Tyr to Phe mutation is
responsible for this switch in activity from a reductase (as found
in A. fumigatus) to an isomerase (as found in N. lolii), we made
the EasA_Nl Phe176Tyr and the EasA_Af Tyr178Phe mutations.
Gratifyingly, EasA_Nl Phe176Tyr produced a compound with a
mass and retention time identical to those of festuclavine 2 when
incubated with chanoclavine-I aldehyde 1, EasG, and NADPH
(Figure 1C). A compound with a mass and retention time matching
those of the wild type product, agroclavine 3, was also observed,
suggesting that while the Phe176Tyr mutation is critical for
reduction of the alkene substrate, other residues also contribute to
a complete switch in enzymatic activity. Interestingly, the activity
of the EasA_Af Tyr178Phe mutant was not qualitatively different
from wild type EasA_Af; both the mutant and wild type EasA_Af
enzymes appeared to produce only festuclavine 2 with no agro-
3
standard m/z 239. (E) Festuclavine 2 standard m/z 241.
To explore the mechanistic basis for the catalytic differences
between these two EasA homologues, we compared the sequences
of EasA_Nl and EasA_Af (76% amino acid similarity), along with
the sequences of other EasA homologues from both festuclavine 2
and agroclavine 3 producing fungal species (SI). The most intriguing
difference between the two sets of producers was substitution of a
phenylalanine for a tyrosine residue in the sequences of fungi that
produce agroclavine and are thus expected to have EasA with
isomerase activity. Previous mechanistic work on Old Yellow
Enzyme from Saccharomyces carlsbergensis suggests that this
tyrosine residue acts as a general acid that protonates the R carbon
of the alkene substrate after a hydride from the flavin is added to
5a
the ꢀ carbon. We therefore speculate that, in isomerase EasA_Nl,
1
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C O M M U N I C A T I O N S
clavine 3 observed (SI). More extensive mutations may be required
to engineer an isomerase-type enzyme. The active site must
rigorously exclude all other proton sources such as water that could
protonate C8 after hydride addition to C9. Additionally, it is possible
that the residues of EasA_Nl make the enolate shown in Scheme
Supporting Information Available: Experimental procedures,
sequence alignments, and additional experimental data. This material
is available free of charge via the Internet at http://pubs.acs.org.
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2
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(
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2
and agroclavine 3 are reported in ref 3a.
JA105785P
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