The Mechanism of Dehydrating Bimodules in trans-Acyltransferase Polyketide Biosynthesis: A Showcase Study on Hepatoprotective Hangtaimycin

Article abstract of DOI:10.1002/anie.202106250

A bioassay-guided fractionation led to the isolation of hangtaimycin (HTM) from Streptomyces spectabilis CCTCC M2017417 and the discovery of its hepatoprotective properties. Structure elucidation by NMR suggested the need for a structural revision. A putative HTM degradation product was also isolated and its structure was confirmed by total synthesis. The biosynthetic gene cluster was identified and resembles a hybrid trans-AT PKS/NRPS biosynthetic machinery whose first PKS enzyme contains an internal dehydrating bimodule, which is usually found split in other trans-AT PKSs. The mechanisms of such dehydrating bimodules have often been proposed, but have never been deeply investigated. Here we present in vivo mutations and in vitro enzymatic experiments that give first and detailed mechanistic insights into catalysis by dehydrating bimodules.

Full text of DOI:10.1002/anie.202106250

Angewandte
Communications
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How to cite:
International Edition: doi.org/10.1002/anie.202106250
German Edition: doi.org/10.1002/ange.202106250
Natural Products
The Mechanism of Dehydrating Bimodules in trans-Acyltransferase
Polyketide Biosynthesis: A Showcase Study on Hepatoprotective
Hangtaimycin
+
+
Minghe Luo , Houchao Xu , Yulu Dong, Kun Shen, Junlei Lu, Zhiyong Yin, Miaomiao Qi,
Guo Sun, Lingjie Tang, Jin Xiang, Zixin Deng, Jeroen S. Dickschat,* and Yuhui Sun*
Abstract: A bioassay-guided fractionation led to the isolation
of hangtaimycin (HTM) from Streptomyces spectabilis
CCTCC M2017417 and the discovery of its hepatoprotective
properties. Structure elucidation by NMR suggested the need
for a structural revision. A putative HTM degradation product
was also isolated and its structure was confirmed by total
synthesis. The biosynthetic gene cluster was identified and
resembles a hybrid trans-AT PKS/NRPS biosynthetic machi-
nery whose first PKS enzyme contains an internal dehydrating
bimodule, which is usually found split in other trans-AT PKSs.
The mechanisms of such dehydrating bimodules have often
been proposed, but have never been deeply investigated. Here
we present in vivo mutations and in vitro enzymatic experi-
ments that give first and detailed mechanistic insights into
catalysis by dehydrating bimodules.
paracetamol overdosage and silymarin, a lipophilic extract
from milk thistle (Silybum marianum) seeds, is administered
in liver injuries by carbon tetrachloride, alcohol, in chronic
viral hepatitis or acute poisonings with Amanita mush-
[
4]
rooms. The limited availability of hepatoprotective drugs
raises an urgent need for the discovery of new compounds.
In the process to screen hepatoprotective drug leads,
crude culture extracts from Streptomyces spectabilis CCTCC
[
5]
M2017417 revealed an interesting activity by inhibiting the
increase of alanine (ALT) and aspartate aminotransferase
(AST) activities in the human hepatoma cell line HepG2
stimulated with CCl , which are indicators of liver over-
4
[
6]
function. A subsequent bioassay guided fractionation led to
the isolation of hangtaimycin (HTM, 1) as the active principle
[
7]
(Figure S1). This known compound was identified by ESI-
HRMS and NMR spectroscopy (Figures 1, S2–S9 and
Table S3). However, a detailed inspection of the NMR
F
or many years Actinomycetes have been a major source for
2
9,30
the discovery of bioactive natural products and the develop-
ment of new drugs such as the antibiotic erythromycin, the
immunosuppressant rapamycin, or the antitumor antibiotic
spectra indicated an E configured D
double bond instead
[
7]
of the published Z configuration, especially because of the
absence of a ROESY correlation between H29 and H30 in
conjunction with the signal multiplicity for H30 (dd, J = 15.0,
[
1,2]
bleomycin.
As an important “detoxification” organ the
[
3]
1
liver plays an important role in human metabolism. Liver
injury may cause fatigue, coagulopathy, encephalopathy and
liver failure, which are threating peopleꢀs lives, but only a few
hepatoprotective drugs are clinically available for specific
applications, for example, acetylcysteine is used to treat
10.2 Hz). The H-NMR signals for H29 and H31 are over-
lapping with other signals, which prevents a reverse assign-
ment of coupling constants, but J = 15.0 Hz must be relevant
for the coupling over an olefinic double bond (between H29
and H30, thus revealing its E configuration), while J = 10.2 Hz
must be assigned to the coupling between H30 and H31 over
a formal single bond. Besides HTM also a putative degrada-
[
+]
+
[
*] M. Luo, Y. Dong, Dr. K. Shen, J. Lu, M. Qi, G. Sun, L. Tang,
Dr. J. Xiang, Prof. Dr. Z. Deng, Prof. Dr. Y. Sun
Key Laboratory of Combinatorial Biosynthesis and Drug Discovery,
Ministry of Education, and School of Pharmaceutical Sciences,
Wuhan University
tion product was detected by LC-ESI-HRMS ([M+H] m/z
+
2
23.14349, calcd for C H O N m/z 223.14410), which
1
2
18
2
2
corresponds to the hemiaminal cleavage product HTM222
index 222 referring to its molecular mass, Scheme S1a,
(
Figure 1). This compound was synthesized from l-alanine
(Scheme S1b, Figures S10–S48), and the natural product and
the synthetic material showed identical chromatographic
behaviour and mass spectra (Figure S49). These findings
also confirmed the assigned 2Z,4E configuration in the sorbic
acid amide portion of HTM. HTM222 has been isolated before
No. 185 East Lake Road, Wuhan 430071 (People’s Republic of China)
E-mail: yhsun@whu.edu.cn
[
+]
H. Xu, Z. Yin, Prof. Dr. J. S. Dickschat
Kekulꢀ-Institute for Organic Chemistry and Biochemistry, University
of Bonn
Gerhard-Domagk-Straße 1, 53121 Bonn (Germany)
E-mail: dickschat@uni-bonn.de
[8]
from Streptomyces sp. SBI108 and named sarmentosamide
which may also arise by degradation of HTM or a similar
+
[
] These authors contributed equally to this work.
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
https://doi.org/10.1002/anie.202106250.
metabolite in this organism. The optical rotation of the
25
synthetic compound, [a] = À194 (c 0.28, MeOH), shows the
D
2
D
8
same sign as the natural product, [a] = À141 (c 0.05,
ꢁ
2021 The Authors. Angewandte Chemie International Edition
[8]
MeOH).
published by Wiley-VCH GmbH. This is an open access article under
the terms of the Creative Commons Attribution License, which
permits use, distribution and reproduction in any medium, provided
the original work is properly cited.
HTM is the product of a hybrid polyketide synthase/
nonribosomal peptide synthetase (PKS/NRPS). According to
the classical assembly line paradigm of PKS/NRPS, for the
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Figure 1. Structure of hangtaimycin (1) and its biosynthetic gene cluster. Proposed functions for individual ORFs are summarized in Table S2.
modular type of these enzymes usually a colinearity logic
exists with one module being responsible for the incorpo-
tion are missing KR and DH domains in modules 11–13 that
would be needed to explain the HTM structure. Here also KR
and/or DH domains may act in trans. In-frame deletion of
htmA7 completely abolished HTM production, verifying its
essential role as an in trans acting AT for HTM biosynthesis
(Figure S51).
[
9]
ration of one unit, while the domain organization with acyl
carrier proteins (ACP) for binding of intermediates, acyl
transferase (AT) for upload of an extender unit, ketosyn-
thases (KS) for catalysis of chain elongations by decarbox-
ylative Claisen condensations, and optional ketoreductase
More strikingly, an unusual modular organization was
found for HtmA1, in which—according to the structure of
HTM and against classical PKS colinearity—modules 1–4 are
responsible for the elongation of the acetyl-CoA starter with
only two malonyl-CoA units. Modules 2 and 3 together show
a domain organization that closely resembles that of a type A
dehydrating bimodule. Such bimodules are usually split after
(
KR), dehydratase (DH) and enoyl reductase (ER) domains
for b-keto group processing allows for detailed functional
[
9]
predictions. In contrast, trans-AT PKSs contain a much
more complex and less understood domain architecture, often
with split modules, unfunctional or repetitive domains or even
modules, and domains with special functions only seen in one
[
10,11]
0
or few cases.
the second non-elongating KS domain and generally show
0
Bioinformatic analysis of the genome sequence of S.
spectabilis CCTCC M2017417 resulted in the discovery of
a putative biosynthetic gene cluster with 74.4% G + C
content that could explain the structure of HTM (Table S4).
Deletion of the entire cluster from htmA4 to htmK resulted in
an abolished HTM production, confirming its function as
HTM biosynthetic gene cluster (Figure S50). The central part
encodes five multidomain enzymes comprising a trans-
the domain organization KS-KR-ACP-KS // DH-ACP and
[12]
are believed to catalyze the installation of a Z double bond.
Type B dehydrating bimodules are also split (KS-KR-ACP-
KS// DH-ACP-KR), but here both KS domains are elongating
and the DH catalyzes two eliminations of water with
[
13]
formation of a 2E,4Z diene. Dehydrating bimodules are
a special feature of trans-AT PKSs and occur inter alia in the
[
14]
[15]
biosynthetic machineries for bacillaene, difficidin, batu-
[
18,19]
[16]
[17]
[18]
AT
PKS/NRPS hybrid machinery (Table S4) with an
min,
kirromycin
and gladiolin.
For the dehydrating
integral flanking subdomain of 90–110 amino acids located
immediately after each KS except the loading module and
module 3 to which the discrete duplicate AT encoded by
hmtA7 might dock (Figure 1). Another interesting observa-
bimodule in the gladiolin PKS the importance of a docking
domain for protein-protein interaction and efficient acyl
group transfer has been demonstrated in vitro, but further
aspects of catalysis by dehydrating bimodules have not been
[
19]
2
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studied so far. This prompted us to aim at a deep investigation
function of module 1 could still not completely be ruled out,
of the hypothetical integral dehydrating bimodule of HtmA1.
Alternative biosynthetic hypotheses for the biosynthesis
of the (2Z,4E)-sorbic acid portion made up by C51–C56 in
HTM are shown in Figure 2. The starter unit is likely malonyl-
CoA that is uploaded to the loading module with decarbox-
ylation by the GNAT domain. This step must be followed
by two chain extensions with double bond formation, but the
only module in HtmA1 that is fully equipped for double bond
installation is module 1. In order to test whether module
because it is possible that downstream modules do not take
over such hydroxyl- or ketoacyl intermediates from ACP , but
1
this hypothesis also seems unlikely, because one and the same
set of KR and DH domains would need to be responsible for
the installation of one E and one Z double bond.
[
20]
Similar to the situation in type A dehydrating bimodules,
HtmA1 KS is predicted to be inactive with respect to chain
3
elongation based on the observed Cys-Asn-His motif instead
[21]
of the usual highly conserved Cys-His-His catalytic triad,
1
functions iteratively to perform two rounds of chain
but because of the retained Cys in this motif KS could still act
3
extension (Figure 2a), individual in vivo inactivations of
DH and KR (the index refers to the module number) by
as a transacylase for passing on the substrate to a downstream
ACP (Figure S54). Instead of an iteration of module 1 and as
suggested for (split) dehydrating bimodules, modules 2 and 3
could cooperate in one chain elongation with formation of a Z
olefin. This may proceed with attachment of the triketide
intermediate solely to ACP , i. e. with a silent ACP and KR
1
1
mutating active site and NADPH binding sites, respectively
Figures S52 and S53). LC-ESI-HRMS analysis of culture
extracts from the DH (H26A) and KR (G10Q, G12L, G15A)
(
1
1
mutants showed a disrupted HTM production, but also no
other product such as a derivative with hydroxyl or keto
groups at C53 and C55 was observed. At this stage an iterative
3
2
2
acting on the ACP bound intermediate (Figure 2b). Alter-
3
natively, the triketide intermediate may still be tethered on
ACP after ketoreduction by KR and dehydration by DH
3
2
2
“in-trans-like” acting on the ACP2 bound intermediate,
followed by module 3 skipping (Figure 2c). Finally, chain
elongation to the triketide and ketoreduction could be
performed at ACP , followed by KS -mediated transacylation
2
3
to ACP3 and dehydration by DH3 (Figure 2d), as it is
generally proposed for type A dehydrating bimodules, but
has never been verified experimentally.
To further examine the above hypotheses, site-directed
mutations were introduced into the active sites of KR and
2
DH , yielding the mutant strains KR (Y168A) and DH -
3
2
3
(H25A) (Figures S55 and S56). Both mutants neither pro-
duced HTM nor a new derivative with a hydroxyl or keto
group at C53, revealing the involvement of KR and DH in
2
3
HTM biosynthesis and thus ultimately ruling out the possi-
bility of an iterative usage of module 1 (Figure 2a). To
distinguish between models 2, 3 and 4, site-directed mutation
was performed at ACP2 and ACP , yielding the in vivo
3
mutants ACP (S40A, I41F) and ACP (D43L, S44A), respec-
2
3
tively (Figures S57 and S58). LC-ESI-HRMS analysis of
culture extracts showed an abolished HTM biosynthesis in
both cases, indicating the involvement of ACP and ACP in
2
3
HTM biosynthesis. These results disfavor models 1–3 and
support model 4. Furthermore, also the site-directed mutation
of the transacylation site of KS (KS (C155A), Figure S59)
3
3
abolished HTM production, which further supports model 4.
In particular, the negative HTM biosynthesis by KS (C155A)
3
suggests that KS3 plays a role as a “springboard” in the
intermediate transfer from ACP to ACP .
2
3
A detailed mechanistic hypothesis for the cooperativity of
modules 2 and 3 is shown in Figure S60. This mechanistic
model was further investigated by in vitro enzymatic reac-
tions. Modules 1, 2 and 3 of HtmA1 were expressed as
recombinant multidomain proteins in Escherichia coli BAP1
and purified (Figure S61). This strain contains the sfp gene
under control of the T7 promoter leading to efficient
Figure 2. Alternative models for triketide biosynthesis by HtmA1.
a) Module 1 iteratively catalyzes two elongations with skipping of
modules 2 and 3. b) Modules 2 and 3 represent an active dehydrating
bimodule with processing of the acyl group solely bound to ACP3
[22]
phosphopantetheinylation of carrier proteins (holo-CPs).
For loading of the malonyl-CoA extender unit, HtmA7 was
(
ACP is skipped), c) solely bound to ACP (ACP is skipped), or
2 2 3
also expressed in E. coli BL21(DE3) containing plasmid
pGro7 with GroEL/ES chaperone (Figure S61). As a sub-
d) bound to ACP for chain extension and ketoreduction and to ACP
for dehydration.
2
3
[
23]
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strate surrogate the N-acetylcysteamine thioester (SNAC
ester) of crotonic acid, (E)-2-butenoyl-SNAC (2), was syn-
thesized (Figures S62–S64) and incubated with holo-module
absolute configurations were independently confirmed by
chemical correlation with (S)- and (R)-3-hydroxyhexanoic
[
26,27]
acid by catalytic hydrogenation.
1
in the presence of malonyl-CoA and NADPH, followed by
When the same reaction was done in the presence of holo-
module 2 plus holo-module 3, a major compound identical to
authentic (2Z,4E)-hexa-2,4-dienoic acid (4) was produced,
confirming chain elongation with ensuing ketoreduction and
dehydration (Figure 3d). The retention time of the product
(16.46 min) closely matched that of synthetic 4 (16.49 min,
Figure 3 f), while a small, but noticeable difference was
observed for the retention time of commercial (2E,4E)-
hexa-2,4-dienoic acid (5, 16.78 min, Figure 3g), clearly prov-
ing Z configuration of the newly installed double bond in the
triketide, as observed in HTM and HTM222. Product 4 was not
detected with holo-module 3 alone (Figure 3c), showing that
module 3 can take over the intermediate polyketide chain
from module 2 and subsequently dehydrate the hydroxyl
group, but cannot condense and extend the carbon chain.
Consistent with the corresponding in vivo results, the
combination of wild-type holo-module 2 and the holo-
[
17,24]
alkaline hydrolysis of the thioester-bound products.
chain elongation product (3) was detected (Figure 3a),
confirming again in vitro that module 1 is not used iteratively.
Incubation of holo-module
No
2 with malonyl-CoA and
NADPH, alkaline workup and LC-ESI-HRMS analysis
revealed formation of a single product that showed an
identical chromatographic behaviour and mass spectrum as
authentic (E)-3-hydroxyhex-4-enoic acid (3), demonstrating
that module 2 catalyzes one chain elongation step with
ketoreduction (Figures 3b,h). The absolute configuration of 3
was determined by synthesis of (rac)-3 and enantiomerically
enriched (R)-3 by kinetic resolution through Sharpless
epoxidation of ethyl (rac)-3-hydroxyhex-4-enoate and sapo-
nification (Scheme S2, Figures S65–S67). HPLC analysis of
the synthetic materials and the enzyme product using a chiral
stationary phase demonstrated R configuration for enzymati-
cally generated 3 (Figure S68). In a second approach,
enantiomerically pure (R)- and (S)-3 were prepared by
[25]
module 3-KS (C155A) enzyme variant obtained by site-
3
directed mutation made the in vitro extension of module 3
impossible (Figure 3e, Figure S59), which excludes model 3
(Figure 2).
[26]
using the Evans oxazolidinone method (Scheme S3). As
the assignment for the absolute configurations to the enan-
tiomers of 3 is not very well explained in the literature, their
Taken together, HTM biosynthesis is achieved by a hybrid
trans-AT PKS-NRPS and starts with the formation of
a triketide by HtmA1 with involvement of an internal type
A dehydrating bimodule. Such dehydrating bimodules are
frequently observed in trans-AT PKSs but are usually split
over two proteins after the second KS domain. Apart from the
recently reported importance of a docking domain for
[
19]
efficient cooperation of these split didomains
not much
was known about the mechanism of dehydrating bimodules,
although their function has often been hypothesized in
previous reports. We here show by in vivo mutations and in
vitro enzyme reactions how they act: The first module (KS-
KR-ACP) takes over the acyl group from the preceding
module with its KS, followed by decarboxylative Claisen
condensation with an ACP-bound malonate unit and ketor-
0
0
eduction. The KS of the second module, composed of KS -
DH-ACP, is a non-elongating transacylase and passes on the
acyl group to the ACP, followed by dehydration. Dehydrating
[
28]
bimodules usually contain A-type ketoreductases,
which
introduce R configured hydroxyl groups. In cooperation with
dehydratases these systems are generally involved in the
[
12,13,29]
biosynthesis of polyketides with Z double bonds.
Acknowledgements
This work was supported by National Key R&D Program of
China (2018YFA0903200), the Open Funding Project from
State Key Laboratory of Microbial Metabolism (MMLKF18-
Figure 3. In vitro reconstitution of triketide biosynthesis by HtmA1.
The SNAC ester 2 serves as a substrate mimic of module 2 and is
condensed with one unit of malonyl-CoA. The extension products were
released from HtmA1 by alkaline hydrolysis and monitored by LC-ESI-
1
2
1) and the German Research Foundation DFG (SFB TRR
61). Open access funding enabled and organized by Projekt
HRMS. The KS domain that is unable to catalyze chain extension and
3
DEAL.
only acts as a transacylase is shown in black. The KS domain fully
3
inactivated by site-directed mutation on the transacylation active site
(
C155A) is indicated by an asterisk. Synthetic 3 and 4 and commercial
5
were used as standards for compound identification.
4
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Conflict of Interest
1
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Communications
Natural Products
Hangtaimycin biosynthesis proceeds
through a hybrid trans-AT PKS/NRPS
biosynthetic machinery that contains
a distinctive dehydrating bimodules.
Here we present detailed mechanistic
insights into catalysis by dehydrating
bimodules that are generally involved in
the biosynthesis of polyketides with Z
double bonds. This work lays the foun-
dations for future structural modifica-
tions by protein engineering.
M. Luo, H. Xu, Y. Dong, K. Shen, J. Lu,
Z. Yin, M. Qi, G. Sun, L. Tang, J. Xiang,
Z. Deng, J. S. Dickschat,*
Y. Sun*
&&&& — &&&&
The Mechanism of Dehydrating
Bimodules in trans-Acyltransferase
Polyketide Biosynthesis: A Showcase
Study on Hepatoprotective Hangtaimycin
6
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ꢁ 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH
Angew. Chem. Int. Ed. 2021, 60, 1 – 6
These are not the final page numbers!