A Polyketide Cyclase That Forms Medium-Ring Lactones

Article abstract of DOI:10.1021/jacs.0c11226

Medium-ring lactones are synthetically challenging due to unfavorable energetics involved in cyclization. We have discovered a thioesterase enzyme DcsB, from the decarestrictine C1 (1) biosynthetic pathway, that efficiently performs medium-ring lactonizations. DcsB shows broad substrate promiscuity toward linear substrates that vary in lengths and substituents, and is a potential biocatalyst for lactonization. X-ray crystal structure and computational analyses provide insights into the molecular basis of catalysis.

Full text of DOI:10.1021/jacs.0c11226

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A Polyketide Cyclase That Forms Medium-Ring Lactones
De-Wei Gao,∥ Cooper S. Jamieson,∥ Gaoqian Wang,∥ Yan Yan, Jiahai Zhou,* K. N. Houk,*
and Yi Tang*
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ABSTRACT: Medium-ring lactones are synthetically challenging due to unfavorable energetics involved in cyclization. We have
discovered a thioesterase enzyme DcsB, from the decarestrictine C1 (1) biosynthetic pathway, that efficiently performs medium-ring
lactonizations. DcsB shows broad substrate promiscuity toward linear substrates that vary in lengths and substituents, and is a
potential biocatalyst for lactonization. X-ray crystal structure and computational analyses provide insights into the molecular basis of
catalysis.
actones of all sizes are found widely in bioactive natural
Lproducts. Synthetically, the preparation of medium-ring
(8−11 membered) lactones is significantly more challenging
compared to small- and large-ring compounds.1 The reactivity
of intramolecular lactonization of medium-sized rings is
estimated to be nearly six-orders of magnitude slower than
that of a five-membered lactone.2 The steep increase in
activation energy arises from both entropic and enthalpic
penalties, with the latter playing a dominant role due to
transannular strain.2,3 Developing efficient strategies to
construct such rings systems by cyclization of hydroxyacids
has been an ongoing effort, anchored by methods such as
Corey−Nicolau,4 Yamaguchi,5 Mitsunobo,6 and others.7
Biocatalytically, enzymes responsible for forming small
lactones and macrocycles are found in biosynthetic pathways,8
Figure 1. Nonanolides and related natural products. (A) Compounds
with 10-membered lactones. (B) Compounds that are proposed to be
highlighted by the 12-membered and 14-membered forming
pikromycin (Pik)9 and erythromycin (DEBS)10 thioestereases
derived from 10-membered lactones.
(TEs), respectively. However, there is no example of a natural
enzyme that can promote lactonization to form 9- or 10-
membered lactone rings.11 Discovering and characterizing
enzymes that can catalyze such reactions can therefore bridge
this notable inventory and knowledge gap.
proposed TE therefore represents a potential medium-ring
lactonization biocatalyst.
Our search for the biosynthetic gene cluster (BGC) that
encodes the enzymes for 1 started with genome scanning of
Beauveria bassiana ARSEF 2860;18 a related fungus B. bassiana
EPF-5 was reported to produce pyridomacrolidin.14 Guided by
other fungal macrolide BGCs,17c,d we identified one BGC (dcs,
Figure 2A) that encodes an HRPKS (DcsA), a TE (DcsB), a
P450 (DcsC), a short-chain dehydrogenase/reductase (SDR,
DcsD), and a flavin-dependent monooxygenase (FMO, DcsE)
(Table S1). Close homologues of this five-gene cassette are
found in two other fungi, including Cordyceps species that are
known producers of nonanolides.19
Toward this objective, we focused on the biosynthesis of
nonanolides by filamentous fungi. Compounds in this family
contain a 10-membered lactone core (Figure 1A). Decar-
estrictine C1 (1) isolated from Penicillium simplicissimum and
other fungi12 is a representative molecule. Lactone 1 shows
inhibitory effects on cholesterol biosynthesis and contains an
(E)-alkene at C4 and C5 that is flanked by two allylic alcohols.
The 10-membered ring with multiple chiral substituents has
attracted several total synthesis efforts.13 Nonanolides such as
1 are also proposed to be biosynthetic precursors for
pyridomacrolidin14 and opaliferin15 (Figure 1B). Biosyntheti-
cally, the 10-membered ring in 1 is proposed to derive from a
polyketide pathway:16 one would expect a highly reducing
polyketide synthase (HRPKS) to iteratively assemble the
enzyme-bound linear polyketide, which is lactonized by a TE.17
Additional oxidoreductases complete the biosynthesis. The
Received: October 25, 2020
https://dx.doi.org/10.1021/jacs.0c11226
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Figure 2. Fungal nonanolide biosynthesis. (A) The dcs and homologous biosynthetic gene clusters. The percent sequence identities are shown in
parentheses. (B) Proposed biosynthetic pathway of 1. (C) LC-MS analysis of metabolites produced from expression of dcs genes in A. nidulans. (D)
Assaying activity of DcsB using small molecule thioesters.
The functions of the dcs enzymes were examined through
heterologous expression in Aspergillus nidulans A1145 (Figures
2 C and S1).20 Coexpression of DcsA and DcsB led to the
production of 2a (∼250 mg/L), which was confirmed to be the
10-membered lactone diplodialide-B (Table S5, Figures S50−
S54). This implies DcsA and DcsB together are sufficient to
synthesize the nonanolide. Coexpression of the P450 DcsC led
to disappearance of 2a, but no accumulation of new
metabolites. Additional coexpression of the SDR DcsD led to
formation of a new metabolite that has the same molecular
weight as 1. NMR and X-ray crystallography characterization
confirmed the compound to be identical to decarestricitine C1
(Figure 2B, Table S6, Figures S45−S49). As shown in Figure
2B, we propose DcsC oxidizes the allylic C6 position of 2a to
the ketone 3, which can be modified or metabolized in vivo as
a Michael acceptor. The SDR DcsD stereoselectively reduces
the ketone to the C6 alcohol to form 1. The FMO DcsE is not
required in the biosynthesis of 1, and its role is not evident
from heterologous expression.
We synthesized a panel of thioesters (4, Figure 2D) that
mimic the proposed HRPKS-tethered pentaketide, and assayed
with recombinant DcsB (Figure S2). Thioesters with the
natural acyl chain were prepared through hydrolysis of 2a
followed by reacting with different thiols (Supporting
Information Methods). As shown in Figure 2D and Table
S4, acyl-S-N-acetylcysteamine 4a-1 was a poor substrate and
gave a low yield of 2a. Using more lipophilic acyl thioesters,
such as acyl-S-MMP 4a-2,21 acyl-S-EMP 4a-3, and acyl-S-BMP
4a led to significant increases in yield and total turnover
numbers (TTNs). Lowering the enzyme loading to 0.5 μM led
to maximum observed TTN of 1258 (Table S4) with 4a. The
kinetic parameters of DcsB toward 4a were determined to be
kcat = 11.5 min−1 and KM = 298 μM (Figure S3). Sequence
comparison of DcsB to other TEs showed the presence of the
catalytic triad, S114, H276, and D247, that performs catalysis
in a well-established reaction mechanism (Figure S14).22
While the S114A mutant cannot be solubly obtained, both the
S114T and the S114C mutants retained <5% of the catalytic
activity using 4a as the substrate (Figure S2).
To examine how substitutions in the substrate affect
cyclization, we synthesized modified pentaketide-S-BMP
substrates 4b−4e by using semisynthetically modified 2a
(Supporting Information Methods). Products from the
enzymatic reactions were isolated and characterized by NMR
(Figures S55−S60). The C3-methoxy-containing substrate 4b
was efficiently converted to the lactone 2b with 53% isolated
yield (Figure 3A). The saturated pentaketide 4c was converted
to lactone 2c, indicating that the olefin is not essential. The C3
epimer of 4c, 4d, was cyclized to 2d with 43% isolated yield,
further indicating substitutions on the ring do not affect
catalysis. In contrast, DcsB failed to lactonize substrate 4e
containing an epimerized nucleophilic alcohol into 2e. Instead
only the hydrolyzed byproduct was observed by LCMS
analysis (Figure S5). This suggests that the proper spatial
orientation of the nucleophile is required for attacking the
Ser114-bound acyl chain in the lactonization reaction, in
contrast to the stereotolerance of other macrolactonizing TE
enzymes.17e
We next explored the substrate tolerance of DcsB toward
linear substrates of different sizes by using simplified acyl-S-
BMP compounds 4f−4l (Figure 3B). These compounds, once
cyclized, would afford lactones 2f−2l ranging in ring sizes from
7- to 13-membered. In cases where synthetic standards of the
lactones were available, such as in 2f−2h, the formation of
products and yields were measured from GCMS analysis
(Figure S4). In other cases, all putative lactone products were
purified and spectroscopically characterized (Figures S61−
S72). Gratifyingly, all of the substrates can be cyclized using
DcsB in yields ranging from 49% to 82%. Using DcsB, odd-
membered lactones, which are rarely found in nature, can be
readily accessed. These examples illustrate that DcsB has broad
substrate scope and is a biocatalyst for constructing lactones of
assorted ring-sizes.
B
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αL2, αL3) (Figures 4A and S8). Although DcsB has less than
10% sequence identity to the well-characterized DEBS TE10
and Pik TE9a domains, the core regions have structural
similarities with an RMSD of 2.5 and 2.7 Å, respectively
(Figure S9). The catalytic triad is located in the loops of the
core domain, and adopts the same relative conformations as in
other TEs (Figure S9).
We then attempted crystallization of DcsB with the substrate
4a-2. Unexpectedly, we obtained a 2.11 Å resolution structure
of DcsB complexed with the substrate analogue 4a-2′, the 3R-
epimer of 4a-2 (PDB ID: 7D79; Figures 4A and S10).
Epimerization of the allylic and β-alcohol appears to take place
nonenzymatically under crystallization conditions. The overall
structure of the DcsB-4a-2′ complex is essentially identical to
the DcsB structure, with a RMSD of 0.146 Å (Figure S10A).
However, the conformation of 4a-2′ is a nonproductive one, as
the nucleophilic alcohol that undergoes lactonization is
hydrogen bonded to the catalytic Ser114. This pushes the
thioester 7.9 Å away from the catalytic triad (Figure S10C) and
gives a conformation that is unproductive toward lactonization.
Nevertheless, the presence of 4a-2′ allowed us to visualize the
active site cavity, which is an ∼151 Å3 hydrophobic chamber
calculated using POCASA.23 The active site is significantly
different from those of the DEBS10 and Pik9a TEs, both of
which have a substrate channel passing through the entire
dimer (Figure S11). In these structures, the catalytic triad is
located in the middle of channel to catalyze cyclization of the
substrate that enters from one side and exits from the other
side. The reaction chamber of DcsB is only open on one side,
because the other side is blocked by the residues of I139 and
F142 located in the loop between β6-αL1 connecting the lid
and the TE core (Figure S11).
Figure 3. Assaying the substrate promiscuity of DcsB. (A) Analogues
4b−4e used to probe functional group requirements. (B) Simple
alcohol-terminated substrates 4f−4l. aIsolated yield. bThe cyclized
product was not formed and only substrate hydrolysis was observed.
cYield determined by GC-MS analysis. Reaction conditions: 1 mM
substrate, 0.5 μM DcsB except for 2k in which 2.4 μM DcsB was used.
We determined the crystal structure of DcsB by single
anomalous diffraction at 1.56 Å resolution (PDB ID: 7D78;
Figure S6 and Table S7). DcsB exists as a homodimer in the
asymmetric unit, consistent with sedimentation velocity
experiment results (Figure S7). DcsB possesses a canonical
α/β-hydrolase fold with an eight-stranded β-sheet and a lid
region inserted between β6 and β7 with three helices (αL1,
We computationally removed the 4a-2′ ligand and docked
4a-2 into the crystal structure (Figure 4B). 4a-2 docks into the
enzyme in a conformation more consistent with catalysis. Now,
the secondary alcohol is positioned at the entrance of active
site and hydrogen bonds with Ser281. This conformation
places the thioester 5.6 Å from the catalytic triad. We then
Figure 4. Crystal structures of DcsB with bound and modeled substrate complexes. (A) Overall structure of DcsB-substrate analogue 4a-2′
complex. The active site catalytic triad residues (S114, H276, and D247) are shown in blue sticks while the substrate analogue 4a-2′ is shown in
yellow. The β-hydroxyl group in 4a-2′ is epimerized compared to the natural substrate 4a-2 and is bound in an unproductive conformation. (B)
Active site of DcsB with docked substrate 4a-2 that is shown in gray sticks. The thioester is 5.6 Å from the catalytic S114 residue. (C) Covalent
docking of the Ser114-bound acyl-intermediate of 4a-2 shown in teal.
C
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performed covalent docking of the Ser114-bound acyl-
intermediate (Figure 4C). The Ser114-bound acyl-intermedi-
ate adopts a folded conformation with the secondary alcohol
∼4 Å from the catalytic His276 and the oxyester carbonyl
where cyclization occurs. The backbone amides of F40 and
F115 hydrogen bond to the alcohol and organize the substrate
into a folded conformation ready for cyclization. This suggests
that if the acyl-intermediate is formed, it will readily cyclize.
Quantum mechanical calculations on cyclization transition
states with “theozyme” models of the active site further
corroborated this mechanism; the computed transition states
are very similar to the docked acyl-intermediate (Figure S12).
The hydrogen bonding interactions between the substrate and
F40 and F115 backbone amides distort the reactant toward a
transition state-like geometry. Also of note is that the alkene
and allylic alcohol do not form any stabilizing interactions with
nearby residues, which is consistent with the fact that these
substituents are not required for catalysis.
AUTHOR INFORMATION
■
Corresponding Authors
Yi Tang − Department of Chemical and Biomolecular
Engineering and Department of Chemistry and Biochemistry,
University of California Los Angeles, Los Angeles, California
90095, United States; orcid.org/0000-0003-1597-0141;
Email: yitang@ucla.edu
K. N. Houk − Department of Chemical and Biomolecular
Engineering and Department of Chemistry and Biochemistry,
University of California Los Angeles, Los Angeles, California
90095, United States; orcid.org/0000-0002-8387-5261;
Email: houk@chem.ucla.edu
Jiahai Zhou − State Key Laboratory of Bio-organic and
Natural Products Chemistry, Center for Excellence in
Molecular Synthesis, Shanghai Institute of Organic
Chemistry, University of Chinese Academy of Sciences,
Shanghai, China; orcid.org/0000-0001-5795-3804;
Email: jiahai@sioc.ac.cn
We performed covalent docking on Ser114-bound inter-
mediates from the substrate 4f−4l and found that many
structures produced a folded conformation as the best
predicted docked pose (Figure S13). All structures except 4g
produced folded conformations in the docked ensembles, but
the lowest energy docked poses for substrates 4f, g, h, and i (n
= 1−4) have extended conformations. The transannular strain
associated with cyclizing these small to medium-ring substrates
is unavoidable, but in the enzyme pocket these substrates can
overcome this barrier by forming stabilized folded conforma-
tions. We propose that the broad substrate scope arises from
the nature of the active site. One end of the substrate is bound
to Ser114, and the other end hydrogen bonds with the
backbone amides of F40 and F115. Positioning the termini in
close proximity to each other with strong electrostatic
interactions counteracts entropic and enthalpic barriers to
cyclization. The active site residues that hug the alkyl chain are
flexible methionines, bulky nonpolar leucines, and a phenyl-
alanine that afford hydrophobic binding. These residues make
up a large nonpolar cavity with the Ser114-bound acyl-
intermediate 4a-2. As the alkyl chain increases in length, the
nonpolar cavity can flex and accommodate the extra carbons.
This general arrangement, in which the tails of the substrate
are anchored by covalent modification and hydrogen bonds
within a hydrophobic pocket, facilitates the promiscuity of
DcsB.
Authors
De-Wei Gao − Department of Chemical and Biomolecular
Engineering, University of California Los Angeles, Los
Angeles, California 90095, United States
Cooper S. Jamieson − Department of Chemistry and
Biochemistry, University of California Los Angeles, Los
Angeles, California 90095, United States; orcid.org/
0000-0002-6076-6230
Gaoqian Wang − State Key Laboratory of Bio-organic and
Natural Products Chemistry, Center for Excellence in
Molecular Synthesis, Shanghai Institute of Organic
Chemistry, University of Chinese Academy of Sciences,
Shanghai, China
Yan Yan − Department of Chemical and Biomolecular
Engineering, University of California Los Angeles, Los
Angeles, California 90095, United States
Complete contact information is available at:
https://pubs.acs.org/10.1021/jacs.0c11226
Author Contributions
∥D.-W.G., C.S.J., and G.W. contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the NIH 1R35GM118056 to Y.T.
and GM124480 to K.N.H. We thank Dr. Yang Hai, Zhuan
Zhang, and Masao Ohashi for helpful discussion.
In conclusion, we have identified the enzymes involved in
forming the 10-membered lactone 1. DcsB is shown to have
broad substrate promiscuity to form medium-ring lactones that
are challenging to prepare chemically. DcsB adds to the
collection of thioesterases discovered from biosynthetic
pathways that are useful in the chemoenzymatic preparation
of lactones.
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ASSOCIATED CONTENT
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* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/jacs.0c11226.
Experimental procedures and spectroscopic data (PDF)
Crystallographic data (CIF)
D
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