On-Line Polyketide Cyclization into Diverse Medium-Sized Lactones by a Specialized Ketosynthase Domain

Article abstract of DOI:10.1002/anie.201804991

Ketosynthase (KS) domains of modular type I polyketide synthases (PKSs) typically catalyze the Claisen condensation of acyl and malonyl units to form linear chains. In stark contrast, the KS of the rhizoxin PKS branching module mediates a Michael addition, which sets the basis for a pharmacophoric δ-lactone moiety. The precise role of the KS was evaluated by site-directed mutagenesis, chemical probes, and biotransformations. Biochemical and kinetic analyses helped to dissect branching and lactonization reactions and unequivocally assign the entire sequence to the KS. Probing the range of accepted substrates with diverse synthetic surrogates in vitro, we found that the KS tolerates defined acyl chain lengths to produce five- to seven-membered lactones. These results show that the KS is multifunctional, as it catalyzes β-branching and lactonization. Information on the increased product portfolio of the unusual, TE-independent on-line cyclization is relevant for synthetic biology approaches.

Full text of DOI:10.1002/anie.201804991

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Accepted Article
Title: On-line Polyketide Cyclization into Diverse Medium-Sized
Lactones by a Specialized Ketosynthase Domain
Authors: Srividhya Sundaram, Hak Joong Kim, Ruth Bauer, Tawatchai
Thongkongkaew, Daniel Heine, and Christian Hertweck
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10.1002/anie.201804991
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On-line Polyketide Cyclization into Diverse Medium-Sized
Lactones by a Specialized Ketosynthase Domain
Srividhya Sundaram, Hak Joong Kim, Ruth Bauer, Tawatchai Thongkongkaew, Daniel Heine and
Christian Hertweck*
Abstract: Ketosynthase (KS) domains of modular type I polyketide
synthases (PKSs) typically catalyze Claisen condensation of acyl
and malonyl units to form linear chains. In stark contrast, the KS of
the rhizoxin PKS branching module mediates a Michael addition,
which sets the basis for a pharmacophoric δ-lactone moiety. The
precise role of the KS was evaluated by site-directed mutagenesis,
chemical probes, and biotransformations. Biochemical and kinetic
analyses helped to dissect branching and lactonization reactions,
and unequivocally assign the entire sequence to the KS. Probing the
range of accepted substrates with diverse synthetic surrogates in
vitro, we found that the KS tolerates defined acyl chain lengths to
produce 5- to 7-membered lactones. These results show that the KS
is multifunctional as it catalyzes β-branching and lactonization.
Information on the increased product portfolio of the unusual, TE-
independent on-line cyclization is relevant for synthetic biology
approaches.
lactonizations are generally catalyzed by TE domains, which
promote an intramolecular attack of an alcohol residue onto the
ACP-thioester.[8] In contrast to this scheme, the formation of the
δ-lactone residue of rhizoxin does not involve any designated TE
domain. Studies at the genetic, biochemical and chemical levels
have revealed that the side chain is introduced by a non-
canonical PKS module consisting of KS, branching (B), and acyl
carrier protein (ACP) domains.[9] Unlike its homologues found in
regular PKS modules, this KS domain catalyzes the vinylogous
addition of ACP-bound malonyl units to an α,β-unsaturated
thioester (Figure 1).[10] The branched chain can only propagate
after cleavage of the KS-bound thioester, which yields the δ-
lactone. By analogy, δ-lactam and glutarimide moieties are
formed when the hydroxy nucleophile is replaced with amine
and carboxamide nucleophiles, respectively.[11]
Domain substitution experiments have indicated that the B
domain maintains the structural integrity of the module but does
not have any significant impact on the choice of substrates used
for heterocyclizations.[12] The biochemical basis for the
cyclization reaction and its scope, however, has remained
obscure. One may conceive that the lactonization is independent
of enzyme catalysis. In lovastatin biosynthesis, for example, the
hydroxy acid released by the TE cyclizes spontaneously under
an acidic condition.[13] Here we show by in vitro assays,
mutational studies and kinetic analyses that the lactonization is
promoted by the branching KS domain and that the scope of
cyclizations is broader than previously believed.
Modular polyketide synthases (PKS) are efficient
biosynthetic machineries that generate highly diverse natural
products with a broad range of biological activities. The motors
of these molecular assembly lines are ketosynthase (KS)
domains that promote sequential Claisen condensations to
generate nascent polyketide chains tethered to acyl carrier
protein (ACP) domains.[1] As soon as the polyketide has reached
its final length, it is generally off-loaded by a thioesterase (TE)
domain located at the N-terminus of the enzymatic processing
line. Post-PKS enzymes optionally tailor the primary product to
further functionalize the scaffolds.[2] There is growing evidence,
however, that structural modifications of the polyketide
backbone may be introduced while the intermediates are still
tethered to the assembly line.[3] Among these on-line
modifications, cyclization reactions are highly prolific as they
alter the overall structure and add rigidity to the polyketide
backbone.[4] This is well illustrated by the cycloether moieties
found in a range of polyketides, which result from vinylogous
cycloadditions catalyzed by pyran synthase (PS) domains.[5]
Another important example of
a
pharmacophoric
polyketide-derived heterocycle is the δ-lactone residue of
rhizoxin (1, Figure 1), a powerful phytotoxin produced by the
bacterial endosymbiont of the rice seedling blight fungus
Rhizopus microsporus.[6] Owing to its potent antiproliferative
activity, this unusual bicyclic polyketide has been considered as
an antitumoral therapeutic. In light of the complex overall
structure of the macrolactone it is remarkable that the small
carbon chain branch is totally essential for the binding of the
toxin to the β-tubulin subunit and thus for antimitotic activity.[7]
According to text-book knowledge, polyketide (macro)
Figure 1. Model of on-line branching and lactonization in rhizoxin (1)
biosynthesis. KS, ketosynthase; AT, acyl transferase; B, branching domain;
ACP, acyl carrier protein.
To evaluate the role of the chain-branching KS domain in
the formation of the δ-lactone ring and to dissect branching and
cyclization, we examined the primary sequence of the KS
domain by bioinformatics. A phylogenetic analysis revealed that
the KS domain of the rhizoxin PKS branching module (Rhi-
KS14) falls into clade IX that corresponds to canonical KS
domains utilizing olefinic substrates.[14] By multisequence
alignments we compared the amino acid sequence of Rhi-KS14
with other KSs (both branching and elongating) from clade IX.
Excluding the conserved catalytic residues, ten positions in the
family of branching KSs differ considerably when compared
against classical KSs mediating Claisen condensations (Table
S1 and Figure S1). Conserved residues of the Rhi-KS14 (PDB
code: 4kc5) were mapped and superimposed on the bacillaene
KS5 structure (PDB code: 5erb, Figure 2A).[15] These residues
[*]
Dr. S. Sundaram, Dr. H. J. Kim, R. Bauer, T.
Thongkongkaew, Dr. D. Heine, Prof. Dr. C. Hertweck
Department of Biomolecular Chemistry
Leibniz Institute for Natural Product Research and Infection
Biology – Hans Knöll Institute
Beutenbergstr. 11a, 07745 Jena, Germany
E-mail: christian.hertweck@leibniz-hki.de
S.S. and H.J.K. contributed equally to this work.
Prof. Dr. C. Hertweck
Chair for Natural Product Chemistry
Friedrich Schiller University, Jena, Germany
Supporting information for this article is given via a link at
the end of the document.
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were mutated to the corresponding residues represented in the
canonical KSs. In the case of S3232, it was changed to Leu as
well as to Ala. In total, eleven single mutations were introduced.
To understand the cumulative effect of all selected amino acids,
we designed a synthetic gene coding for KS’-B, in which all nine
residues (except T3290) were mutated. All mutant KS domains
were heterologously produced in E. coli BL21 (DE3) (Figure S2).
Except for KS’-B, of which soluble protein could only be obtained
in minute amounts, CD spectra showed no significant
differences between the secondary structures of all recombinant
proteins (Figure S3).
(Figure 3A, iii) indicates that this residue is involved in the C-C
bond formation.
We next tested the impact of these residues on the
Michael addition and the subsequent lactonization. To identify
residues that are involved in the Michael addition we probed all
mutants in vitro with the coenzyme
A
mimicking N-
acetylcysteamine (SNAC) thioester 2.[16] Since this polyketide
surrogate lacks the δ-hydroxy group, the branching reaction is
arrested after the initial C–C bond formation.[10a] The trapped
intermediate (3), which covalently links the KS-B with the ACP,
can be visualized on the SDS gel (Figure 2B). All mutants,
except C3228A, T3290Y and the KS’-B, retained the vinylogous
C-C bond-forming activity (Figure 2C).
Figure 3. (A) LC-HRMS analysis of mutants showing extracted ion
chromatograms of lactone 5 [M+H]+) 173.0808, (C8H13O4). (B) Rate of lactone
(5) formation by wild-type KS-B and mutants. Data represent mean ± s.d from
three independent experiments.
When testing mutants H3364A and H3404A, increasing
the enzyme concentration and the duration of the assay also
enlarged the amount of product formed (Figure 3A, iv and v). It
is possible that both His residues complement each other in the
branching reaction. A report on bacterial fatty acid synthases
(FabF) suggested that the H3364 counterpart is not an absolute
necessity for the C–C bond forming reaction, but enhances the
overall rate of the reaction.[17] Analogous mutations of the
catalytic His residues of the KS of 6-deoxyerythronolide B
synthase (DEBS) module 1 and E. coli FabB resulted in
Figure 2. (A) Comparison of active-site residues of KS-B (orange) (PDB code
4kc5) with bacillaene KS5 (green) (PDB code 5erb). Residues chosen for
mutational analysis are colored black. Residues in blue constitute catalytic
triad. Numbers correspond to residues of Rhi–KS14 (PDB code 4kc5). (B)
Chain-branching assay setup with deoxy variant of SNAC thioester substrate 2.
(C) SDS-PAGE analysis of the reaction mixture with 2. The 100 kDa band
corresponds to the KS-B didomain, upper band corresponds to fused KS-B
and ACP domains (intermediate 3). All proteins were verified by tryptic mass
fingerprinting followed by MALDI analysis. M, protein molecular weight marker.
moderate to very low turnover of the substrate, too.[17-18]
A
double mutant (H3364A and H3404A) stalled the formation of 5,
proving that these residues are indispensable for the Michael
addition. According to functional studies with homologous KS
domains,[17-19] H3404 activates the malonyl carboxylate and
stabilizes the enolate. Reduction of the rates in the carbon-
carbon bond formation as well as lactonization (Figure 2C and
3A, v) indicates that H3404 plays a major role in all steps of the
reaction. To complement the LC-MS analysis, LC-ESI-MS of the
intact ACP-bound proteins was also performed for KS-B,
H3364A and H3404A and analyzed by the PPant-ejection
method.[20] The lactone-bound PPant arm was observed in all
the samples tested. However, compared to the wild-type KS-B
(Figure S5), the intensities of the mass fragments were
approximately 10-fold lower in H3364A and H3404A (Figure S6
and S7). Nonetheless, all mutants retained malonyl-CoA
decarboxylation activity.
To investigate the role of the selected residues in the
lactonization reaction, we performed in vitro assays with 4 to
monitor the formation of lactone 5 (Figure 3A and 4A). Mutants
H3097A, S3117G, S3141D, S3232L, S3232A, C3234A, T3267L,
L3406E, S3409A and I3478F did not markedly hamper the
cyclization activity (Figure S4). In all cases, 5 was observed in
comparable amounts to KS-B (Figure 3A, i). Corroborating the
SDS gel-shift experiments, mutants C3228A, T3290Y and KS’-B
did not result in 5, even when the corresponding enzymes were
tested at 10 times higher concentrations for prolonged reaction
times (16 h) (Figure 3A, ii, iii and vi). The inactivity of C3228A
mutation is not surprising (Figure 3A, ii) because the
nucleophilic Cys residue is needed for binding the polyketide
intermediate. However, the completely inactive T3290Y mutant
To identify other residues that could affect the reaction rate,
the mutants (except for the inactive C3228A and T3290Y) were
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further analyzed by kinetic experiments at saturating
concentration of 4 (1 mM) and increased enzyme concentrations
of KS-B and ACP, respectively. Combined kinetic data from
three independent experiments showed that the mutants can be
grouped into three types (Figure 3B). The largest group of
mutants (represented by black curves) reached product
saturation after approximately 45 min, thus showing that the
lactonization is not impaired. In contrast, the rate of lactonization
was substantially decreased in the S3141D, I3478F and S3409A
mutants (Figure 3B and Figure S8). The H3364A, H3404A and
KS’-B mutants exhibited only negligible activities within the
specified time limit (75 min). The impact of the S3141 residue on
the reaction is remarkable as it is distant from the active center
and thus likely not directly involved in the branching/lactonization
reaction. The mutation of I3478 to a bulkier phenyl alanine
residue may have caused perturbations in the β-sheet where it is
located.[10a] Phenyl alanine residues close to the active site are
termed as gatekeeper residues that dictate the order of the
reaction.[17] Apart from the active site mutants, the only mutant
that showed attenuated reaction rate is S3409A. Since this
serine moiety is located in the active site, we concluded that this
residue is an important determinant for lactonization. However, it
is conceivable that additional residues contribute to the
lactonization reaction.
addition is working, but the subsequent lactonization is impaired
for the secondary alcohol.
Our mutational and kinetic analyses demonstrated that the
lactonization of the branched polyketide chain is catalyzed by
the KS. To probe the flexibility of this multifunctional KS with
respect to the ring size, we synthesized a range of SNAC
thioester analogues. By metathesis we prepared substrates with
γ-hydroxy groups (6 and 7, Figure 4B), which would give rise to
ring-contracted, five-membered lactones. The substrate
analogues were tested in vitro using the native KS-B didomain,
ACP, AT and malonyl-CoA. The biotransformation was
monitored by MALDI and LCMS to detect ACP-bound and
hydrolyzed products. Analysis of the reaction mixture with 6
resulted in a new species with m/z of 12705.7, whereas the
reaction with 7 resulted in a species with m/z 12722.5. These
m/z values correspond to the expected ACP-bound five-
membered lactones with methyl and ethyl side chains,
respectively (Figure S9, lactone 5 and lactone 5*). High-
resolution mass spectrometric analysis of the hydrolyzed
products confirmed the formation of new compounds, 8, [M+H]+
159.0652 (C7H11O4) and 9, [M+H]+ 173.0808 (C8H13O4) (Figure
4D, ii and iii). To unequivocally prove their structures we
prepared synthetic references and used these authentic
compounds for HPLC-MS comparisons. In both cases, the
resulting double peaks indicated the formation of the expected
syn and anti diastereomers.[11]
To test whether the branching module can also produce
larger rings such as ε-caprolactones, SNAC thioesters 10 and
11 were used as probes. With 10 as the substrate, MALDI
analysis of the reaction mixture revealed the formation of the 7-
membered lactone bound to the ACP (Figure S9, lactone 7*).
Analysis of the biotransformation products indicated the
formation of a new compound 12, with m/z [M+H]+ 173.0808
(C8H13O4) (Figure 4D, iv). In contrast to 10, compound 11 could
not be transformed into the corresponding ε-substituted
caprolactone. Even after prolonged reaction time (16 h at 23 °C)
no ACP-bound intermediate could be detected. However,
analysis of the hydrolyzed reaction mixture revealed a new
compound (13) with m/z [M+H]+ 205.1065 (C9H17O5) (Figure 4D,
v). According to the deviations in molecular masses and HPLC
retention times, 13 differed from the expected 7-membered
lactone 14. The structure of 13 was elucidated as the
dicarboxylic acid derivative by comparison with a synthetic
reference (see SI). This finding showed that the initial Michael
Figure 4. In vitro evaluation of KS-catalyzed branching/lactonization with
synthetic surrogates. (A) Set-up of assay leading to δ-lactone. (B) Synthesis of
substrate mimics (C) Schematic of observed biotransformation products. (D)
LC-HRMS analysis of biotransformation assays. Solid lines, assay; dashed
lines, synthetic references for lactones; dotted lines synthetic references for
dicarboxylic acid derivatives; dash-dot lines, negative control. Negative control
refers to the assays performed with heat-inactivated enzyme.
To corroborate this result, we synthesized and tested
SNAC thioesters with extended carbon backbones (15, 16 and
17). In all cases, the corresponding dicarboxylic acids, 18 (m/z
[M+H]+ 219.1219 (C10H19O5), 19 m/z [M+H]+ 233.1380
(C11H21O5) and 20 m/z [M+H]+ 247.1541 (C12H23O5), were
formed, as proven by comparison with synthetic references
(Figure 4D, vi-viii). Notably, when the assays were performed
with heat-inactivated KS-B (negative control), no products were
observed, thus proving that the vinylogous carbon bond
formation is always enzyme catalyzed. Since the OH group is
necessary to form the lactone, we proposed that the farther the
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In conclusion, we have shed light on the versatility of the
KS domain that catalyzes the vinylogous Michael addition and
lactonization. Using a series of mutational and kinetic analyses,
we showed the interplay of certain amino acid residues in
catalyzing the enzymatic steps. Moreover, through the synthesis
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Keywords: Lactones • Macrolides • Michael addition • Natural
Products • Polyketide Synthases
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Refer Supplementary Information.
Acknowledgements
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Entry for the Table of Contents
COMMUNICATION
Srividhya Sundaram, Hak Joong
Kim, Ruth Bauer, Tawatchai
Thongkongkaew, Daniel Heine and
Christian Hertweck
Stepping out of line: Site-specific
mutagenesis and kinetics of the
versatile rhizoxin PKS branching
module identified critical conserved
residues and show that the KS
domain promotes both Michael
Page No. – Page No.
addition
and
lactonization.
Moreover, the branching module is
tolerant towards substrates with
varied acyl chain lengths to form
various medium-sized lactones.
On-line Polyketide Cyclization
into Diverse Medium-Sized
Lactones by a Specialized
Ketosynthase Domain
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