Catalase Involved in Oxidative Cyclization of the Tetracyclic Ergoline of Fungal Ergot Alkaloids

Article abstract of DOI:10.1021/jacs.9b10217

A dedicated enzyme for the formation of the central C ring in the tetracyclic ergoline of clinically important ergot alkaloids has never been found. Herein, we report a dual role catalase (EasC), unexpectedly using O₂ as the oxidant, that catalyzes the oxidative cyclization of the central C ring from a 1,3-diene intermediate. Our study showcases how nature evolves the common catalase for enantioselective C-C bond construction of complex polycyclic scaffolds.

Full text of DOI:10.1021/jacs.9b10217

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Communication
A Catalase Involved in the Oxidative Cyclization of
the Tetracyclic Ergoline of Fungal Ergot Alkaloids
Yongpeng Yao, Chunyan An, Declan Evans, Weiwei Liu, Wei Wang,
Guangzheng Wei, Ning Ding, K. N. Houk, and Shu-Shan Gao
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b10217 • Publication Date (Web): 17 Oct 2019
Downloaded from pubs.acs.org on October 19, 2019
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A Catalase Involved in the Oxidative Cyclization of the Tetracyclic
Ergoline of Fungal Ergot Alkaloids
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Yongpeng Yao,†,§ Chunyan An,†,§ Declan Evans,‡ Weiwei Liu,† Wei Wang,†,# Guangzheng Wei,†,#
Ning Ding, K. N. Houk,*,‡ Shu-Shan Gao*,†
†State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, P.
R. China
‡Department of Chemistry and Biochemistry, University of California, Los Angeles, California 90095, United States.
#University of Chinese Academy of Sciences, Beijing 100049, P. R. China
School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122, P. R. China.
Supporting Information Placeholder
ABSTRACT: A dedicated enzyme for the formation of the central
C ring in the tetracyclic ergoline of clinically important ergot
alkaloids has never been found. Herein, we report a dual role
catalase (EasC), unexpectedly using O2 as the oxidant, catalyzes
the oxidative cyclization of the central C ring from a 1,3-diene
intermediate. Our study showcases how Nature evolves the
common catalase for enantioselective C-C bond construction of
complex polycyclic scaffolds.
Ergot alkaloids (EAs) are among the most important
pharmaceuticals and natural toxins. These bioactive compounds
were initially isolated from fungi in the genus Claviceps, a
group of parasitic fungi that infected rye and related crop
plants.1,2 The grains infected with Claviceps and subsequently
contaminated with EAs can cause ergotism (St. Anthony's fire)
in both human and livestock.3 In contrast to their toxicity to
human and livestock, both natural and semi-synthetic EAs
(Figure 1a and Figure S1), are used for the treatment of a wide
range of diseases and disorders.1 Until recently, more than a
hundred EAs have been identified from different fungal genera
including Aspergillus and Penicillium.4,5
EAs share a common indole-derived tetracyclic ergoline
moiety that can modulate the receptors of the central nervous
Figure 1. The chemical structures of ergot alkaloids (the central C
system, due to the structural similarities between the ergoline
ring is marked in red) and the catalytic cycle of a typical catalase.
moiety and neurotransmitters.6 Thus, the tetracyclic-ergoline
(a) The ergoline moiety is present in clinically used ergot alkaloids.
core structure is essential for the biological activity of EAs
(Figure 1a). Many biosynthetic steps of ergoline formation have
been established,1,2,7 yet the nature of the central C ring
formation, specifically the conversion of N-methyl-
dimethylallyltryptophan (N-Me-DMAT; 1) to chanoclavine-I 2
remains an enigma (Figure 1b). It is very intriguing that the
regio- and enantioselective cyclization of the central C ring was
enabled by creating a new C5-C10 single bond from two un-
activated sp3 carbons, despite the presence of functional groups
elsewhere in 1 (Figure 1b).
(b) EasC and EasE are essential for the formation of the central C
ring.1 (c) The catalytic cycle of a typical catalase.
A biosynthetic gene cluster (BGC) of fumigaclavine C 3 has
been identified in Aspergillus fumigatus (Figures 1b and S2).8
Previous studies have shown that 1 is produced by two upstream
genes dmaW and easF (Figure 2d),8-10 and dmaW, easC, easE,
and easF were sufficient for the production of 2.11-13 EasC was
demonstrated as a functional catalase14 and proposed to catalyze
the decarboxylation of 4 or its oxidative derivatives.15 By
knocking out either easE or easC, 1 was found to be the only
intermediate
accumulated.14,16
However,
the
direct
transformation of 1 to 2 under in vitro conditions remains
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Figure 2. Characterization of the biosynthesis of the central C ring (in red). (a) In vivo reconstitution of dmaW, easC, easE, and easF in A.
nidulans. (b) In vitro reconstitution of the activity of EasC. (c) Analysis of 18O-labelled 2 generated from EasC-reaction in 18O2 saturated
buffer. LC-HRMS data: 18O labeled chanoclavine-I 2 [Mꢀ+ꢀH]+ m/z: calculated 259.1696, observed 259.1691 (d) The complete biosynthetic
pathway from tryptophan to 5. See SI for reaction conditions.
elusive.7 Herein, we demonstrated that EasC, a catalase that
efficiently catalyzes the decomposition of H2O2 to generate O2
in vitro, unexpectedly uses O2 as the oxidant to catalyze the
oxidative cyclization of the central C ring via the construction
of a new C5-C10 single bond in an enantioselective fashion.
Firstly, we confirmed the production of 3 in Aspergillus
fumigatus (CGMGCC 3.772),17 following 3-days cultivation on
PDA medium (Figure S3, trace ii). The production of 3 was
abolished (Figure S3, trace vii) when dmaW, the
prenyltransferase gene responsible for the first biosynthetic
step,8 was knocked out in A. fumigatus (Figure S4). Next, 1 was
produced by co-expression of dmaW and easF genes in the
heterologous host Aspergillus nidulans (Figure 2a, trace i;
Figure 2d). 1 was purified from A. nidulans and fully
characterized by MS and NMR (Table S5, Figures S5, and S26-
27). Supplementation of 1 to the ΔdmaW mutant of A. fumigatus
could restore the production of fumigaclavine C (Figure S3,
trace vi), confirming it was an on-pathway intermediate.
Bioinformatic analysis suggested EasE is a FAD-linked
oxidoreductase (Figure S6). Enzymes in this family, such as the
well-characterized D-amino-acid dehydrogenase,18 catalyze
electron transfers using FAD as a cofactor. We hypothesized
that EasE was the immediate downstream enzyme after DmaW
and EasF. To test this hypothesis, easE was co-expressed with
the genes dmaW and easF in A. nidulans. Indeed, LCMS
analysis of the crude extract from A. nidulans showed the
emergence of a new peak (Figure 2a, trace ii). The new product
was purified and characterized as compound 4 with a 1,3-diene
moiety by MS and NMR (Table S5 and Figures 2d, S7, and S28-
S32). 4 has been previously synthesized by Floss and co-
workers and demonstrated to be an intermediate for ergoline
biosynthesis.19 We further confirmed 4 is an on-pathway
intermediate by feeding it to the ΔdmaW strain to yield 3
(Figure S3, trace v). 4 was potentially generated via base-
catalyzed removal of the C10 hydrogen and capture of the C7
hydrogen as a hydride by the FAD cofactor in EasE (Figure 2d).
Whole-cell biotransformation was performed to further
precluding the direct assay of this reaction using the purified
enzyme.20
EasC showed a high sequence identity with small subunit size
catalases (SSCs, Figures S9 and S25), with a conserved
NADPH-binding site (190H, 197S, 299W, 301L, Figure S10c)
and a heme-binding site (71H, 145N, 361Y, Figure S10b). SSCs
structurally bind NADPH, which is proposed to act as an
electron donor for preventing and reversing the formation of
Compound II.21 To evaluate its activity, EasC was purified to
homogeneity as N-His6–tagged protein in E. coli BL21 DE3
(Figure S11a). Purified EasC was dark red in color and
exhibited the characteristic heme absorbance at 406 nm in UV-
Vis spectra (Figure S12). HPLC analysis of the supernatant of
the denatured protein confirmed the presence of NADPH
(Figure S13). EasC was then incubated with H2O2 and the
disproportionation of H2O2 by different amount of EasC was
monitored by recording the absorbance of H2O2 at 240 nm. The
results show that H2O2 was reduced rapidly with EasC-
concentration dependencies (Figure S11b), confirming EasC is
an active catalase as previously described.14
Sequence identity alignment revealed that several widely
conserved amino acid residues of classic SSCs were different in
EasC and its homologues (Figure S9). In particular, 112T, 114
L, and 202M were observed in EasC, instead of the conserved
V, G, and P, as in classic SSCs. Phylogenetic analysis of EasC
and its homologues also led to a clear classification of them into
a separate clade, though it remains closely related to fungal
SSCs (clade III) (Figure 3). These indicate the function of EasC
could be different from the classic catalases. Indeed, co-
expression of easC with the genes dmaW, easF and easE led to
the detection of a new product by LCMS with m/z 257 [M+H]+
as expected for 2 (Figure 2a, trace iii, and Figure S14).
Purification of the new product followed by NMR analysis
confirmed the structure indeed to be 2 (Table S5, and Figures
2d and S33-S38).
To further study the exact function of EasC, we conducted in
vitro reactions. In the enzymatic cycle of a heme catalase, H2O2
acts as the oxidizing substrate to generate the highly reactive
heme iron-oxo species Compound I (Figure 1c). In accordance
with this, a variety of catalases/peroxidases are known to use
H2O2 as the oxidant to catalyze redox reactions, including the
well-characterized HppE and SfmD.22,23 Thus, we proposed that
EasC also uses H2O2 as the oxidant to generate Compound I to
confirm the function of EasE. When
1 was fed to
Saccharomyces cerevisiae or A. nidulans expressing easE, a
30% and 10% conversion of 1 to 4 was demonstrated,
respectively (Figure S8). However, when we attempted to
express EasE as a soluble protein in Escherichia coli, S.
cerevisiae or Trichoderma reesei, it was not successful, thereby
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initiate the subsequent oxidative cyclization. To test this, 4 was
incubated with 10 μM EasC and 2 mM NADPH, and 1 mM
H2O2 in an anaerobic glove box, however, only 20% of 4 was
converted to 2 and an additional new product 5 in 4 hours
(Figure S15). The low in vitro peroxidase activity of EasC
prompted us to consider adding O2 instead of the H2O2 into the
reaction mixture. O2, the product of catalases (Figure 1c), has
rarely been characterized as the oxidant for catalase/peroxidase
in redox reactions.
chanoclavine-I aldehyde 5 by MS and NMR (Table S5, Figures
S20 and S39-S44), which was potentially produced via the
dehydrogenation of C7 hydroxyl in 2. However, no
interconversion between 2 and 5 could be observed under a
variety of in vitro conditions when 2 or 5 reacted with EasC
separately (Figure S21). This, combined with the following
results suggest that the radical species could be generated
during the reaction and be responsible for the formation of the
aldehyde 5: (i) in vivo co-expression only generated 2 without
the detection of 5 (Figure 2a, trace iii); (ii) a dedicated NAD+
dependent dehydrogenase, EasD, was already demonstrated to
convert 2 to 5 (Figure 2d),25 (iii) the radical species can catalyze
the oxidation of organic substrates;26 and (iv) catalase activity
is known to generate radical species, including hydroxyl
radicals, under various conditions.27 Indeed, the production of 5
could be clearly observed when 2 was incubated with a mixture
of H2O2, Fe2+ and ascorbic acid (Figure S22), which could
generate hydroxyl radicals through the Fenton reaction.28,29
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Figure 3. Phylogenetic analysis of EasC and its homologues and
previously categorized group I−III catalases.24 See SI for details.
Figure 4. The proposed catalytic mechanism for EasC (the central
C ring is marked in red).
Indeed, incubation of 4, 10 μM EasC and 2 mM NADPH in
the open air without the addition of H2O2 led to the gradual and
full conversion to 2 and the additional new product 5, with a
ratio of 1: 3, in only 1 hour (Figure 2b). In contrast,
deoxygenated reaction mixtures completely inhibited the
consumption of 4 by EasC (Figure S16, trace iii), indicating the
reaction was O2-dependent, and the peroxidase activity of EasC
in the anaerobic conditions (Figure S15) might actually have
resulted from the decomposition of H2O2 and utilization of the
resultant O2. As the test of this possibility, EasC reactions with
4 were carried out in the open air with the presence of varying
concentrations of H2O2, as shown in Figure S17, increasing the
concentration of H2O2 diminished the consumption of 4 and
reduced the yields of 2 and 5 with clear H2O2-concentration
dependences. This result suggests H2O2 competes with 4 and O2
for binding EasC, which results in reduced oxygenase activity.
Furthermore, we incubated 4 with EasC in the 18O2 saturated
A radical addition mechanism was proposed for EasC-
catalyzed cyclization by transforming 4 to 2 (Figure 4).
Compound I abstracts the hydrogen from 5-carboxylic acid to
generate the radical species 6. Delocalization of the radical at
C5 leads to release CO2 to yield 7, which can be stabilized via
the formation of an imine 8 with the adjacent secondary amine.
The subsequent radical addition of C5 to C10 yields the radical
9 that can be resonance delocalized at C7 of the terminal alkene
and the final hydroxyl rebound of Compound II yields 2.
Density functional theory (DFT) calculation results
demonstrate that 6 undergoes barrierless release of CO2 to form
the radical
7 following the EasC-catalyzed hydrogen
abstraction of 4 (Figure S23). The C ring is formed by the
radical addition of C5 to C10 in a low energy transition state
(ΔG‡ = 6.1 kcal/mol) to form the final intermediate 9 (Figure
S23). Several radial inhibitors30,31 including DMPO (5,5-
Dimethyl-1-Pyrroline N-oxide), 5-HTP (5-hydroxytryptophan)
and L-AA (L-ascorbic acid), were added into the EasC reaction
mixture, respectively, which all led to significantly reduced
consumption of 4 (Figure S24) , supporting the proposed radical
mechanism for EasC (Figure 4).
In conclusion, we demonstrated the biosynthetic basis for the
formation of the central C ring in the tetracyclic ergoline
moiety, a decades-old problem since initial studies began in the
1950s.32 Our research opens the door to utilize this untapped
repertoire of eukaryotic catalase-monooxygenase catalysts to
generate more polycyclic indole-alkaloids, which have largely
been neglected in previous studies.
Tris-HCl buffer. The LC-HRMS indicated
a 2 units
enhancement in the molecular weight of 2 (Figure 2c). In
contrast, no incorporation of 18O into 2 was observed when 4
was incubated with EasC in the buffer made from H218O without
18O2 (Figure S18). When the reaction was performed without
the addition of NADPH, an only trace amount of 2 and 5 was
observed in a 2-hour reaction (Figure S19), which suggests the
reaction was also NADPH dependent, and the remaining
activity was proposed due to enzyme-bound NADPH in
purified EasC (Figures S10 and S13).
Considering the additional new product 5 was not detected
from in vivo co-expression conditions (Figure 2a, trace iii), we
opted to scale up the in vitro reaction using purified EasC.
Surprisingly, 5 was determined to be the known product
ASSOCIATED CONTENT
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O'Connor, S. E.; Naesby, M. The important ergot alkaloid intermediate
Supporting Information
The Supporting Information is available free of charge on the ACS
Publications website at DOI:
chanoclavine-I produced in the yeast Saccharomyces cerevisiae by the
combined action of EasC and EasE from Aspergillus japonicus. Microbial
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pathway-enzymatic formation of a cyclopropyl group. Angew. Chem.
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Experimental details; spectroscopic and computational data; Tables
S1−S6 and Figures S1−S44
AUTHOR INFORMATION
Corresponding Author
*houk@chem.ucla.edu
*gaoss@im.ac.cn
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ORCID
Kendall Houk: 0000-0002-8387-5261
Shu-Shan Gao: 0000-0001-6335-2052
Chun-Yan An: 0000-0001-9918-5903
Yongpeng Yao: 0000-0002-6084-1683
Weiwei Liu: 0000-0002-5059-9416
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gene ccsA of the ergot fungus Claviceps purpurea encodes chanoclavine I
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mediating the transformation of N-methyl-dimethylallyltryptophan to
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Physiological functions of D-amino acid oxidases: from yeast to humans.
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H. G. Probing ergot alkaloid biosynthesis: intermediates in the formation of
ring C. J. Am. Chem. Soc. 1993, 115, 2482.
(20) Walsh, C. T.; Wencewicz, T. A. Flavoenzymes: versatile catalysts in
biosynthetic pathways. Nat. Prod. Rep. 2013, 30, 175.
(21) Kirkman, H. N.; Galiano, S.; Gaetani, G. F. The function of catalase-
bound NADPH. J. Biol. Chem. 1987, 262, 660.
(22) Wang, C.; Chang, W.-C.; Guo, Y.; Huang, H.; C Peck, S.; Pandelia,
M.-E.; Lin, G.-M.; Liu, H.-W.; Krebs, C.; Martin Bollinger, J. Evidence that
the fosfomycin-producing epoxidase, HppE, is a non-heme-iron peroxidase.
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Author Contributions
§These authors contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
This study was supported by the National Key Research and
Development Program of China 2018YFA0901600, by the
Strategic Priority Research Program, CAS, under grant No.
XDA22050401, by the National Science Foundation of China
under grant No. 31872614 and 31600270, by the Youth Innovation
Promotion Association, CAS, under the grant No. Y92R011CX2,
by the National Institutes of General Medical Sciences, NIH, under
grant No. GM124480, by the UCLA Chemistry-Biology Interface
training program, NIH, under grant No. T32GM008496.
methyltyrosine to 3-hydroxy-5-methyltyrosine in saframycin
A
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