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Biocatalysis
Back-to-back cycloadditions in nature
Richiro Ushimaru & Ikuro Abe
Tandem cycloaddition reactions have significant
applications in organic synthetic chemistry.
Now, two enzymes are shown to catalyse tandem
hetero-Diels–Alder reactions with a synergistic
interplay between a calcium cofactor and
N-glycan post-translational modifications during
the biosynthesis of bistropolone-sesquiterpene
secondary metabolites.
The Diels–Alder (DA) reaction is a [4 + 2] cycloaddition reaction between
a conjugated diene and a dienophile, enabling the rapid construction of
complex molecular frameworks in a single step. Owing to the versatility
and predictable regio- and stereoselectively of the DA reaction, it is one
of the most powerful and widely utilized chemical transformations in
organic synthesis of natural products and pharmaceuticals1. Conse-
quently, the synthetic routes of target molecules containing complex
polycyclic ring systems include tandem DA reactions as key steps. Now,
writing in Nature Chemistry, Hu, Zhou, Houk and co-workers demon-
strate that such tandem DA reactions can be catalysed by enzymes
called Diels–Alderases (DAases) during the biosynthesis of fungal
bistropolone-sesquiterpenes, eupenifeldin (1) and pycnidione (1′)
(ref. 2). They also uncover the detailed molecular mechanism by which
the DAases selectively facilitate the tandem polycyclic ring formation,
which involves a synergy of calcium binding and glycan decoration.
Over the past dozen years, numerous enzymes that catalyse peri­
cyclic reactions such as DA reactions in natural product biosynthesis have
been reported3. Although DAases are powerful biocatalysts for producing
structurally complex natural products, most of them are limited to cata-
lysing single-step DA reactions. Enzymatic catalysis of sequential DA reac-
tions by a single enzyme would require accurate recognition of multiple
substrates at each step during the cascade process, which is non-trivial.
Eupenifeldin4 and pycnidione5 are stereoisomeric bistropolone-
sesquiterpene natural products produced by fungi that share an
identical two-dimensional carbon skeleton, wherein the central
sesquiterpene-derived 11-membered macrocycles with distinct stereo­
configurations are flanked by two dihydropyrans, each conjugated with
a tropolone moiety (Fig. 1a). The presence of two identical tropolone
fragments in their molecular architectures led the researchers
to hypothesize that the biosynthetic pathways of 1 and 1′ involve a
two-fold conjugation event between the sesquiterpene core with the
tropolone moiety. Previous studies on the biosynthetic gene clusters
(eupf from Penicillium janthinellum and pyc from Leptobacillium sp.
CF-236968) (refs. 6,7) responsible for generating these natural products
have shown that EupfF catalyses a hetero-Diels–Alder (hDA) reaction
between modified sesquiterpene 4 and tropolone 7 to form a mono-
cycloaddition product, neosetophomone B (2). However, when the
eupf gene cluster was heterologously expressed in the fungal host
Aspergillus nidulans, compound 1 containing two tropolone units
was produced. This led to the hypothesis that EupfF is the enzyme
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responsible for catalysing the tandem hDA reactions to form com-
pound 1 (or respectively the homologous protein PycR1 for the bio-
synthesis of 1′).
To test these hypotheses, precursors 5 and 4 were fed to an
A. nidulans transformant expressing eupfE and eupfF. Production of 1
as a metabolite implied that EupfF is responsible for the tandem
intermolecular hDA reaction of 7 and 4, resulting in the formation of 1
because the short-chain dehydrogenase/reductase EupfE, encoded
by eupfE, catalyses the reduction of compound 5 to 6. Furthermore,
in vitro enzyme analysis of EupfE and PycR1 purified from A. nidulans
verified their roles as tandem DAases with distinct substrate specifici-
ties. Specifically, EupfE and PycR1 selectively accept 4 and 4′ as their
respective dienophiles.
Hu, Zhou, Houk, and co-workers also solved the crystal structures
of EupfE and PycR1. Both EupfE and PycR1 exhibit a propeller-like
overall folding and are post-translationally modified by N-glycans at
multiple asparagine residues (N45, N68, N213 and N300 in EupfF, and
N43 and N211 in PycR1) (Fig. 1b). They also both contained a catalytic
Ca2+ ion. The crystal structure and mutagenesis experiments indicated
that the N-glycan modification at N211 in PycR1, which is located at the
dimer interface, played a crucial role in both dimer formation and Ca2+
ion binding. This finding was consistent with the dramatic reduction
in the activity of a glycan-free PycR1 purified from Escherichia coli.
Subsequent molecular dynamics (MD) simulations and ONIOM (short
for 'our own n-layered integrated molecular orbital and molecular
mechanics') calculations suggested that the Ca2+ ion, acting as a Lewis
acid, is critical for the dehydration of compound 6 to enone 7, which is
the immediate precursor preceding the hDA reaction with 4 (Fig. 1a).
The proposed molecular catalytic mechanism of PycR1 and EupfF
suggests that substrate 6 undergoes dehydration to form enone 7 via
the Lewis acidity of the Ca2+ ion. Enone 7 then reacts with 4 (or 4′),
leading to the formation of the first product, 2 (or 2′), which is either
released from the enzyme cavity or translocated to another region
within the cavity. Upon the arrival of another molecule of 7, the second
hDA reaction takes place (Fig. 1a). MD simulations provided insights
into the stereoselectivities of the first and second cycloadditions,
which are primarily controlled by the main chains of Y223 and V225,
respectively (Fig. 1c). Sequence alignment and mutagenesis experi-
ments also revealed that N341 and S242 in PycR1, as well as N340 and
Q239 in EupfF, play crucial roles in the second hDA reaction because
substituting these residues with the corresponding amino acids found
in a one-step DAase AsR5 resulted in the abolishment of the second
hDA reaction. Additionally, it was found that the S242Q variant of
PycR1 accepted 6, while the Q239S variant of EupfF accepted 6.
Based on MD simulations, these critical residues likely affect the
hydrogen-bonding network and conformation of active-site residues
such as Y223 and V225, thereby leading to altered substrate preferences.
The work by Hu, Zhou, Houk, and colleagues provides new biosyn-
thetic insights into the assembly of natural products containing polycyclic
systems by a single enzyme. The detailed structural and computational
analysis of EupfF and PycR1 highlights the cycloaddition mechanism,
in which Ca2+ binding and N-glycan post-translation modification
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News & views

a
EupfF
OH OH O
or
HO O HO OH HO
EupfE PycR1

O O O

5 6 H2O
7

7 7 H
cis
O
O
Z
H
Z EupfF H EupfF HO O
HO HO
HO
HO
O O
4 O
OH
Neosetophomone B (2) Eupenifeldin (1)

7 7 H
O trans
E
O
E H
PycR1 PycR1 HO HO O
H OH
HO
OH O O
O OH
4′
Epolone B (2′) Pycnidione (1′)

b c

Y223

N211

Y176

2
V225

N43 7 R94
N293
G320
S63
Ca2+

Fig. 1 | Tandem Diels–Alder reactions catalysed by EupfF and PycR1. a, Schemes respectively. The calcium ion is shown as a green sphere. Sites of the glycosylation
for the EupfF and PycR1 reactions2. b, Overall structure of PycR1 in complex with are also indicated. c, Active site of PycR1. Panels b and c were generated using the
7 and 2 (PDB ID: 7X2S). N-glycans, 7, and 2 are shown in magenta, yellow and cyan, PyMOL Molecular Graphics System, Version 2.4.1, Schrödinger, LLC.

cooperatively fine-tune the tandem DA reactions. The presence of genes Published online: 24 July 2023
encoding EupfF homologues in fungal genomes implies that such tandem
cycloaddition reactions are more relevant and operative in natural product References
biosynthesis than previously recognized. Lastly, the successful engineer- 1. Nicolaou, K. C., Synder, S. A., Montagnon, T. & Vassilikogiannakis, G. Angew. Chem. Int. Ed.
41, 1668–1698 (2002).
ing of PycR1 and EupfF, guided by the knowledge of evolutionary conserva- 2. Liu, J. et al. Nat. Chem. https://doi.org/10.1038/s41557-023-01260-8 (2023).
tion, represents an important milestone in the development of practical 3. Jeon, B.-S., Wang, S.-A., Ruszczycky, M. W. & Liu, H.-W. Chem. Rev. 117, 5367–5388
designer biocatalysts for the rapid assembly of polycyclic compounds. (2017).
4. Mayerl, F. et al. J. Antibiot. 46, 1082–1088 (1993).
5. Harris, G. H. et al. Tetrahedron 49, 2139–2144 (1993).
Richiro Ushimaru      & Ikuro Abe      6. Chen, Q. et al. J. Am. Chem. Soc. 141, 14052–14056 (2019).
Graduate School of Pharmaceutical Sciences and Collaborative 7. Schotte, C., Li, L., Wibberg, D., Kalinowski, J. & Cox, R. J. Angew. Chem. Int. Ed. 59,
23870–23878 (2020).
Research Institute for Innovative Microbiology, The University of Tokyo,
Tokyo, Japan. Competing interests
 e-mail: ushimaru@mol.f.u-tokyo.ac.jp; abei@mol.f.u-tokyo.ac.jp The authors declare no competing interests.

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