A Single Active Site Mutation in the Pikromycin Thioesterase Generates a More Effective Macrocyclization Catalyst

Article abstract of DOI:10.1021/jacs.7b06436

Macrolactonization of natural product analogs presents a significant challenge to both biosynthetic assembly and synthetic chemistry. In the preceding paper, we identified a thioesterase (TE) domain catalytic bottleneck processing unnatural substrates in the pikromycin (Pik) system, preventing the formation of epimerized macrolactones. Here, we perform molecular dynamics simulations showing the epimerized hexaketide was accommodated within the Pik TE active site; however, intrinsic conformational preferences of the substrate resulted in predominately unproductive conformations, in agreement with the observed hydrolysis. Accordingly, we engineered the stereoselective Pik TE to yield a variant (TE_S148C) with improved reaction kinetics and gain-of-function processing of an unnatural, epimerized hexaketide. Quantum mechanical comparison of model TE_S148C and TE_WT reaction coordinate diagrams revealed a change in mechanism from a stepwise addition-elimination (TE_WT) to a lower energy concerted acyl substitution (TE_S148C), accounting for the gain-of-function and improved reaction kinetics. Finally, we introduced the S148C mutation into a polyketide synthase module (PikAIII-TE) to impart increased substrate flexibility, enabling the production of diastereomeric macrolactones.

Full text of DOI:10.1021/jacs.7b06436

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Article
A single active site mutation in the pikromycin thioesterase
generates a more effective macrocyclization catalyst
Aaron A. Koch, Douglas A. Hansen, Vikram V. Shende, Lawrence R.
Furan, Kendall N. Houk, Gonzalo Jiménez-Osés, and David H. Sherman
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b06436 • Publication Date (Web): 24 Aug 2017
Downloaded from http://pubs.acs.org on August 24, 2017
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Journal of the American Chemical Society
A single active site mutation in the pikromycin thioesterase generates
a more effective macrocyclization catalyst
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Aaron A. Kochα†, Douglas A. Hansen°†, Vikram V. Shendeε†, Lawrence R. Furan†, K. N. Houk*Ψ, Gonzalo Jiménez-Osés*Ф,
and David H. Sherman*α†π‡
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†Life Sciences Institute, α Cancer Biology Graduate Program, °Department of Medicinal Chemistry π, εProgram in Chemical
Biology, πDepartment of Chemistry, ‡Department of Microbiology & Immunology, University of Michigan, Ann Arbor, MI
48109, United States
ΨDepartment of Chemistry and Biochemistry, University of California, Los Angeles, 607 Charles E. Young Drive East, Los
Angeles, CA 90095, United States
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ФDepartamento de Química, Centro de Investigación en Síntesis Química, Universidad de La Rioja, 26006 Logroño, La
Rioja, Spain
Supporting Information Placeholder
ABSTRACT: Macrolactonization of natural product analogs presents a significant challenge to both biosynthetic assembly and
synthetic chemistry. In the preceding paper, we identified a thioesterase (TE) domain catalytic bottleneck processing unnatural
substrates in the pikromycin (Pik) system, preventing the formation of epimerized macrolactones. Here, we perform molecular
dynamics (MD) simulations showing the epimerized hexaketide was accommodated within the Pik TE active site; however, intrin-
sic conformational preferences of the substrate resulted in predominately unproductive conformations, in agreement with the ob-
served hydrolysis. Accordingly, we engineered the stereospecific Pik TE to yield a variant (TES148C) with improved reaction kinetics
and gain-of-function processing of an unnatural, epimerized hexaketide. Quantum mechanical (QM) comparison of model TES148C
and TEWT reaction coordinate diagrams revealed a change in mechanism from a stepwise addition-elimination (TEWT) to a lower
energy concerted acyl substitution (TES148C), accounting for the gain-of-function and improved reaction kinetics. Finally, we intro-
duced the S148C mutation into a polyketide synthase module (PikAIII-TE) to impart increased substrate flexibility, enabling the
production of diastereomeric macrolactones.
INTRODUCTION:
philic attack to release the product as a linear acid or macro-
lactone, respectively6 (Figure 1).
Macrocycles are a common motif amongst natural product
and natural product derived therapeutics, with >100 marketed
drugs possessing a macrocyclic core1. The conformational
preorganization of these large rings enables precise display of
functionality to engage challenging targets such as protein-
protein interactions, and, in the case of macrolides, ribosomal
Biochemical and structural studies of the Pik TE and related
DEBS TE (erythromycin pathway) have provided initial in-
sights into the mechanism of ring formation over hydrolysis7,8.
While it is generally accepted that modular type I TEs have
high substrate flexibility for the initial acylation step, the se-
cond, macrolactone forming release step is far more stringent6.
Since the formation of the macrocyclic core is essential for
downstream tailoring and biological activity of a natural prod-
uct, aberrant hydrolysis limits access to new macrocyclic
analogs.
machinery2-4
.
The macrolactone core of macrolide5 natural products is
formed through two steps: first, the linear intermediate is
transferred from the upstream acyl carrier protein (ACP) to a
catalytic serine of the TE via a transesterification to form an
acyl-enzyme complex. Next, the acyl intermediate is offloaded
via intermolecular water hydrolysis or intramolecular nucleo-
In an accompanying report9, we employed a series of syn-
thetic Pik pentaketides to evaluate the substrate tolerance of
Figure 1. Mechanism of the TE-catalyzed macrolactonization or hydrolysis of an ACP-tethered polyketide intermediate.
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PikAIII-TE. Characterization of the in vitro reaction products tected hexaketides (Figure 2), using the conformation of the
derivatized products10 as the starting arrangement for the indi-
vidual substrates. MD simulations (see Computational Details
in the Supporting Information) were initiated with the hexake-
tide C-11 alcohol constrained in a reactive conformation by
imposing a maximum distance restraint of 2.3 Å between the
hexaketide nucleophilic hydroxyl hydrogen and the Nε nitro-
gen of His268. After 50 ns, the distance restraint was removed
and the simulations were allowed to continue for 500 ns.
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indicated a catalytic bottleneck localized to the Pik TE do-
main, preventing macrolactonization to form C-10 and C-11
epimers of 10-dml (1). Furthermore, this bottleneck was con-
firmed directly by incubation of 5 with Pik TE resulting exclu-
sively in hydrolysis9 (Scheme 1). Given the importance of the
TE domain in production of macrolactone analogs, we sought
to engineer the excised Pik TE domain to expand its substrate
scope and, potentially, improve reaction rates. In this study,
we report the identification of a single active site mutation
(S148C) in Pik TE that generates a more effective macrolac-
tonization catalyst by increasing both the substrate flexibility
and efficiency of the enzyme. Subsequent introduction of the
engineered TE into PikAIII-TE results in a polyketide syn-
thase with increased substrate flexibility capable of producing
epimeric macrolactones.
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Figure 2. Acyl-enzyme starting structures for the MD simula-
tions of 4 (Pik TE-4) and C-11-epimerized 5 (Pik TE-5).
Simulations of Pik TE-4 revealed that while the initial cata-
lytic restraint was in place, the native hexaketide readily
adopts two main conformations favorable towards macrolac-
tonization (denoted as I and II, Figure 3a). At the initial stages
of the simulation, conformation I predominates, accompanied
with a high level of shape complementarity with the TE active
site. The main interactions constituting conformation I are
hydrophobic contacts between the hexaketide and the side
chains of residues lining the active site. Additionally, a hydro-
gen bond between the hexaketide C-7 carbonyl and the side
chain of Thr77 facilitates stabilization of the cyclic, reactive
conformation (Figure S13). After ~30 ns the hexaketide
evolves to conformation II through a substrate tail rotation
that places the C-11 alcohol 0.2 Å and 0.7 Å closer to His268
and the C-1 carbonyl, respectively. This orientation resembles
the macrolactonization transition state (vide infra) even closer
than conformation I and is likely catalytically productive.
Scheme 1. Pik TE displays a high level of substrate stereo-
specificity. (a) Pik hexaketides used in this study to probe Pik
TE substrate flexibility. 3 is generated in situ by photolysis of
the 2-nitrobenzyloxymethyl ether (NBOM) protected native
hexaketide10. (b) Incubation of C-11-epimerized Pik hexake-
tide 5 results exclusively in hydrolysis9.
Following formation of conformation II, the hexaketide
displays a large amount of conformational freedom within the
TE active site. In the most prevalent conformation (III, Figure
3a) hydrophobic packing is largely reduced and non-
productive hydrogen bonds between the C-11 alcohol and
Tyr25/Leu193 are formed (Figure S13). Loss of the key hy-
drophobic interactions is detrimental to macrolactonization as
it increases the conformational space accessible to the hexake-
tide, which adopts a more linear, unreactive conformation.
Although conformation III accounts for the majority of the
Pik TE-4 simulation, conformation II developed again later in
the trajectory (406 to 443 ns, Figure 3c) suggesting that pro-
ductive conformations for macrocyclization are frequently
sampled despite the high substrate flexibility.
RESULTS:
Molecular dynamics (MD) simulations of Pik TE macro-
lactonization. Intrigued by the stark catalytic divergence from
macrolactonization to hydrolysis in Pik TE reactions contain-
ing hexaketide 5 bearing the epimerized C-11 hydroxyl group
(Scheme 1), we envisioned that comparing MD simulations of
Pik TEWT acylated with hexaketides 4 and 5 (denoted Pik TE-4
and Pik TE-5, respectively, Figure 2) could provide structural
insight into these experimental findings. Accordingly, we
performed MD simulations of each catalytic system modeled
as acyl-enzyme intermediates bound to the C-3 methyl pro-
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Figure 3. Comparison of the reactive conformations for each acyl-enzyme intermediate obtained from clustering analysis of MD
simulations with Pik TEWT. (a) Pik TE-4 and (b) Pik TE-5. Pik TE-4 conformations I and II contain a hexaketide orientation
most conducive to macrolactonization with the C-11 OH in close proximity to both His268 and the C-1 carbonyl. The corre-
sponding conformation (cluster I’) in the Pik TE-5 simulation likely represents a larger barrier to macrolactonization as the
distance between the C-11 OH and His268 has increased and the resulting geometry hinders deprotonation. The Pik TE-5 hex-
aketide continues to evolve towards a linear conformation till the final cluster III’ is reached which places the C-11 OH distal to
both His268 and the C-1 carbonyl and in an orientation susceptible to hydrolysis. The catalytic triad His268 and Asp176 resi-
dues are colored yellow. For each conformation the distance in angstroms from the nucleophilic hydroxyl oxygen to the Nε
nitrogen of His268 and the ester C-1 carbonyl is displayed above the dashed lines. Clusters containing catalytically productive
conformations contain red dashed lines. (c) The distance of the nucleophilic hydroxyl oxygen and the ester C-1 carbonyl plotted
for each frame of the MD simulation with each data point colored according to the corresponding clustered conformation. The
vertical dashed line at 50 ns indicates when the distance constraints were released.
The simulations of Pik TE-5 involving the C-11-epimerized
geometry impedes macrolactonization. Furthermore, His268
remains 4.0 Å away and positioned at an angle suboptimal for
deprotonation, making this conformation catalytically unpro-
ductive. After ~80 ns, unrestricted Pik TE-5 transitions to the
second most prevalent conformation that is primarily defined
by interruption of the intramolecular hydrogen-bond and rota-
tion of the hexaketide tail away from His268 resulting in poor
shape complementarity with the TE active site and a more
linear conformation of the hexaketide (II', Figure 3b). These
two unproductive conformations are alternately sampled for
hexaketide revealed a significantly different scenario. While
the initial distance restraint was in place, the hexaketide
adopted a cyclic conformation stabilized by hydrophobic
packing with the TE active site (I’, Figure 3b). However, in
contrast with the native hexaketide, the C-11 alcohol of the
epimerized hexaketide is prone to intramolecular hydrogen
bonding with the C-1 carbonyl oxygen. Despite maintaining
the substrate in a cyclic conformation with the nucleophilic
hydroxyl in close proximity to the C-1 carbonyl, the resulting
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Figure 4. Procatalytic sampling of Pik TE during MD simulations. (a,b) Low energy QM optimized transition states for macro-
lactonization of Pik hexaketides (a) Pik TE-4 and (b) Pik TE-5. Non-polar hydrogens have been removed for clarity. (c,d) Devia-
tions of the key catalytic distances (x axis) and angles (y axis) in the MD simulations of (c) Pik TE-4 and (d) Pik TE-5 from their
respective optimized transition structure (green square at the origin of coordinates). Each point represents a single frame from
the 550 ns simulation while the shaded rectangles represent frames from the MD that are likely in a catalytically productive
state.
the majority of the simulation (~470 ns). Afterwards, the hex-
aketide chain evolves towards even more extended confor-
mations (III', Figure 3b) characterized by a gradual loss of
hydrophobic interactions with the protein, and formation of
unproductive hydrogen bonds between the C-11 alcohol and
Glu85 (Figure S13). This completely impedes macrolactoniza-
tion and increases the likelihood competing hydrolysis of the
acyl-enzyme intermediate will occur.
with Pik TE-5, the energetic barrier to macrolactonization is
increased above that of competing hydrolysis.
Chemical lactonizations. As the MD simulations contain-
ing covalently bound hexaketides 4 and 5 revealed that both
substrates are accommodated by the Pik TE active site, we
next investigated if macrolactonization of the C-11-epimerized
5 suffered in bulk solution outside of enzymatic constraints.
Starting with conditions employed for similar seco-acids11,12
we screened contemporary lactonization methodologies capa-
ble of cyclizing 8 to 6. Optimization of Yamaguchi conditions
originally reported from the total synthesis of spinosyn ana-
logs13, enabled direct comparison of lactonization efficiency of
8 and 7. Employing identical reaction conditions resulted in
73% and 6% conversions to generate 6 and 9, respectively
(Scheme 2). This result confirmed the intrinsically poor reac-
tivity of 7 toward macrocyclization compared to 8, corroborat-
ing the MD findings that the lack of macrolactone formation
with the C-11-epimerized hexaketide is not due to steric clash-
es within the Pik TE active site.
We next compared the frequency of productive confor-
mations sampled in the MD simulations (Figure 4) by calculat-
ing quantum mechanically (QM) optimized macrolactoniza-
tion transition structures (TS) for each hexaketide (vide infra).
We used these models to describe the precise angle of nucleo-
philic attack (O11-C1-O1) and distance (O11-C1) for macro-
lactonization of each hexaketide. The resulting geometric
values were then compared to those extracted from each frame
of the corresponding MD simulations and the entire trajecto-
ries were plotted according to their deviation from the ideal TS
values (Figure 4). Consistent with our clustering analysis of
the hexaketide conformations, comparison of the geometric
deviations from ideal within each simulation revealed that Pik
TE-4 contained more frames with the hexaketide in a position
favorable to macrolactonization (Figure 4c). In contrast, Pik
TE-5 presented a larger distribution of geometries with very
few catalytically productive structures (Figure 4d). Taken
together, the MD results on the covalent acyl-enzyme inter-
mediates indicate that hydrophobic interactions accompanying
a high level of substrate-TE shape complementarity are critical
for maintaining each hexaketide in a catalytically productive
conformation. These contacts are, to a lesser extent, main-
tained in simulations containing the C-11-epimerized hexake-
tide 5, indicating that the Pik TE active site has sufficient
flexibility to accommodate both epimers. The intrinsic con-
formational preferences of each hexaketide further influence
catalysis. The acyl intermediates must reach conformations
that are matched for deprotonation by His268 and subsequent
nucleophilic attack in order to achieve macrolactonization.
When these structural preferences are perturbed, as in the case
Scheme 2. Yamaguchi macrolactonization of methyl protect-
ed hexaketides. Conversion of 8 to 6 and 7 to 9 was monitored
by HPLC with data represented as the mean standard devia-
tion where n = 3.
Pik TES148C macrolactonization of epi-hexaketide 5. As
the strict substrate stereospecificity of Pik TEWT does not arise
from inherent steric clashes within the TE active site, we con-
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sidered the excised Pik TE domain to be a prime engineering Full-module processing. We next investigated whether the
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target to generate a catalyst capable of cyclizing 5. We envi-
sioned that altering substrate specificity would likely require a
protracted directed evolution campaign and, if successful,
would potentially yield a variant incapable of cyclizing the
starting, native substrate 3. Prior to library generation, we
performed preliminary site directed mutagenesis on select
residues in the active site, including a serine to cysteine muta-
tion of the serine-histidine-aspartate catalytic triad14,15. Re-
markably, this variant was able to efficiently cyclize 5 to 9
while also displaying improved native functionality (Scheme
3).
engineered Pik TES148C was able to improve substrate flexibil-
ity in the context of full-module catalysis. To accomplish this,
we generated PikAIII-TES148C and incubated it with an analo-
gous series of epimeric Pik pentaketides. LC-HRMS analysis
of reaction products revealed two new peaks in the chromato-
grams not observed in WT PikAIII-TE reactions (Figure S12),
with masses and retention time corresponding to 12-membered
ring macrolactones. As no authentic standards for the putative
novel products were available, a 0.2 millimole scale reaction
of 10 with PikAIII-TES148C was performed and the reaction
products were purified and characterized via MS and NMR.
Structural determination of the reaction products confirmed
that PikAIII-TES148C was indeed able to generate 11-epi-10-
dml 11 from 10, as well as 3-keto-11-epi-10-dml 12, due to
failed reduction of the β-keto intermediate by the PikAIII KR
domain prior to transfer to the terminal TE domain (Scheme
4).
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Diminished relative KR activity has been previously ob-
served in vitro by the production of both the predicted reduced
and 3-keto macrolactones from reactions containing Ery5-TE
with DEBS pentaketide.17 Additionally, PikAIII-PikAIV chi-
meras18 lacking a KR domain yielded exclusively 3-keto-10-
dml when incubated with Pik pentaketide in vivo, indicating
that the WT Pik TE domain is capable of cyclizing the C-3
keto intermediate. We next investigated if the KR-TE domain
competition observed in PikAIII-TES148C reactions with 10
also occurred with the native Pik pentaketide. Accordingly, we
performed analytical scale reactions containing PikAIII-
TES148C with its native substrate and analyzed the product
distribution using synthetic standards for each product. HPLC
quantification of the reaction products revealed the conversion
of native pentaketide to 3-keto-10-dml and 10-dml (1) to be
5.5% and 12.5%, respectively (see Supporting Information).
Generation of both the reduced and unreduced macrolactone
products indicates competition for the linear hexaketide inter-
mediate between the KR and TE domains even in the context
of the native substrate. Isolation of 11 and 12 at 12% yields
indicates that the identified thioesterase bottleneck had been
alleviated by a single amino acid change (S148C) enabling
substrate flux to generate novel epimerized macrolactones.
Scheme 3. Evaluation of Pik TES148C with methyl protected
hexaketides 4 and 5. Enzymatic reaction conditions: sodium
phosphate buffer (400 mM, pH 7.2), 1 mM hexaketide, 8 mM
2-vinylpyridine, purified Pik TE (10 ꢀM), 4 h, stationary, RT.
Conversion of 4 to 6 and 5 to 9 was monitored (HPLC) with
data represented as the mean standard deviation where n =
3.
Kinetic analysis of Pik TE WT and S148C. In addition to
the gain-of-function macrolactonization of 5, we also exam-
ined the kinetic effect of the S148C mutation on cyclization
(Table 1). Steady state kinetic analysis of Pik TEWT and
TES148C was performed using both methyl protected Pik hex-
aketides 4 and 5, as well as the previously reported NBOM
protected10 native hexaketide 3, following a similar procedure
used previously16. Kinetic analysis revealed that the serine to
cysteine substitution afforded a superior cyclization catalyst
for each substrate tested. Pik TES148C displayed a 4.3 and 12-
fold increase in the kcat/Km for macrolactonization of both the
native (3) and C-3 methoxy hexaketide 4 compared to Pik
TEWT, respectively. In reactions containing methyl protected
epimerized 5, not only did Pik TES148C retain the ability to
catalyze macrolactonization of the linear substrate, but notably
the kcat/Km was 5.5-fold higher than Pik TEWT catalyzed hy-
drolysis. In addition to increased substrate flexibility of Pik
TES148C, the gain-of-function mutation provided a 2.6-fold rate
enhancement with the native hexaketide 3 over wild type.
These kinetic parameters demonstrate that the S148C mutation
of Pik TE produces a catalyst with both expanded substrate
scope and increased catalytic efficiency.
Scheme 4. Reaction of PikAIII-TES148C with C-9-epimerized
pentaketide 10. Enzymatic reaction conditions: 1 mM Pik
pentaketide, 20 mM (20 equiv) MM-NAC, 8 mM (8 equiv) 2-
vinylpyridine, 0.5 mM (50 mol %) NADP+, 2.5 mM (2.5
equiv) glucose-6-phosphate, glucose-6-phosphate dehydro-
genase (2 units/mL), 3 ꢀM (0.3 mol %) PikAIII-TE, 8 h,
stationary, RT.
Quantum mechanical (QM) modelling of Pik TE
macrolactonization. Since MD simulations revealed the Pik
TEWT active site to be competent in binding the unnatural
substrate 5, we hypothesized the catalytic advantages imparted
by the S148C mutation were kinetic in nature as opposed to
structural. To further our understanding of the energetics of
these processes and how Pik TES148C is able to overcome the
Table 1. Steady state kinetic values for Pik TEWT and TES148C
.
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Figure 5. Reaction coordinate diagram representing the relative free energies for Pik TE catalyzed macrolactonization of hex-
aketides 13 and 14. Calculations were performed at the PCM/M06-2X/6-31+G(d,p) level using reduced models that define the
enzymatic active site (theozymes19). Relative free energies are in kcal mol-1. Only the lowest energy conformers are represented;
see Supporting Information for details and all the calculated structures. *This intermediate is higher in energy than its preceding
TS due to conformational differences between both stationary points.
barrier to macrolactone formation with 5, we turned to QM
modeling of the catalytic steps comprising macrolactonization
(see Computational Details in the Supporting Information)
after formation of the acyl-enzyme intermediate. To accom-
plish this, we generated abbreviated active site models (theo-
zymes19) for Pik TEWT and Pik TES148C containing the native C-
3-unprotected hexaketide (Pik TEWT-13 and Pik TES148C-13)
mation of the acyl-enzyme intermediate is faster in Pik TES148C
compared to TEWT
The calculated increase of 4.2 kcal mol-1 in the activation
barrier of the macrolactonization of Pik TEWT-13 vs Pik TEWT
.
-
14, agrees well with the experimentally observed lack of epi-
merized-macrolactone formation. This higher macrolactoniza-
tion energy barrier in the TEWT system increases the difficulty
in proceeding from the acyl-enzyme intermediate, which is
vulnerable to water hydrolysis6. While we did not observe
significant water hydrolysis in reactions containing Pik TEWT
and the native substrate, incubation with epimerized methyl
protected 5 resulted exclusively in hydrolyzed product, the
result of an inability to form the epimerized macrolactone 9. In
contrast, the 6.4 kcal mol-1 decrease in activation barrier for
Pik TES148C-14 macrolactonization is consistent with our ex-
perimentally observed product formation.
and the C-11-epi hexaketide (Pik TEWT-14 and Pik TES148C
-
14). Analysis of the resulting free energy landscapes revealed
that macrolactonization of the linear hexaketide intermediates
is an exergonic process in all four systems after product re-
lease (Figure 5). Cyclization of the native hexaketide was
more thermodynamically favorable compared to C-11-
epimerized hexaketide, particularly with the wild-type en-
zyme. Moreover, the macrolactonization mechanism with each
TE was calculated to change from a stepwise addition-
elimination with existence of a tetrahedral intermediate in Pik
TEWT to a concerted acyl substitution20 upon the S148C muta-
tion. This change in mechanism was evidenced by the flat
potential energy surface which precludes the formation of a
tetrahedral intermediate.
DISCUSSION
TE substrate tolerance. In vivo engineering of the DEBS
biosynthetic pathway has shown that DEBS TE possesses
some tolerance to modifications in both the length and the
functionality of the linear polyketide substrate. Modifications
in the DEBS pathway have yielded a large number of 6-dEB
analogs from differential extender unit incorporation and
reductive processing21-28 as well as macrolactones ranging in
size from 6- to 16-membered rings29-32. However, the titers of
these unnatural products are greatly diminished compared to
wild type production levels6, and the inherent complexity of in
vivo biosynthesis has prevented identification of pathway
bottlenecks, such as TE domains.
Figure 6 shows the lowest energy transition structures and
associated activation barriers (ꢁG‡) for the macrolactonization
of the acyl intermediates of both the natural 13 and C-11-
epimerized 14 substrates with the Pik TEWT and TES148C pro-
tein models. The rate-limiting activation barriers for the
TES148C catalyzed reactions are significantly lower than those
for the TEWT reactions, which correspond to the final Ser148-
acyl cleavage. Thus, Pik TES148C performs the macrolactoniza-
tion of the native and C-11-epimerized hexaketides with ꢁG‡
values of 14.8 and 16.0 kcal mol-1, respectively, while Pik
TEWT displays ꢁG‡ values of 18.2 and 22.4 kcal mol-1 for the
same substrates (Figure 6). These values predict that the rate
of reactions containing the native hexaketide after the for-
Initial in vitro biochemical characterization of the DEBS
TE33,34 provided further evidence for the relatively high sub-
strate tolerance of PKS TEs for acylation and hydrolysis, as
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Figure 6. Lowest energy rate-limiting transition structures calculated with PCM/M06-2X/6-31+G(d,p) for the abbreviated active
site models (theozymes19) of the Pik TEWT (left) and Pik TES148C (right) catalyzed macrolactonization of native hexaketide 13
(top, in blue) and C-11-epi hexaketide 14 (bottom, in red). Activation free energies (ꢁG‡) calculated form the corresponding Pik
TE hexaketides are given in kcal mol-1 and distances in angstroms. Relevant breaking/forming C–O and C–S bonds are shown in
boldface. Non-polar hydrogens have been removed for clarity. See Supporting Information for all calculated structures along the
reaction pathway.
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terminally (omega) hydroxylated fatty acids and substrates
Direct comparison of PikAIII-TEWT to PikAIII-TES148C with
a panel of stereoisomer Pik pentaketides demonstrated the
success of this approach as a 0.2 millimole scale reaction with
10 resulted in the production of two new macrolactone prod-
ucts, 11-epi-10-dml 11 and 3-keto-11-epi-10-dml 12. The 1:1
isolation of 11 and 12 from reactions of 10 with PikAIII-
TES148C indicates that the TE was indeed responsible for the
failed catalysis using WT PikAIII-TE, as the KR domain re-
tained a sufficient level of activity towards the unnatural in-
termediate. This demonstration of TE engineering offers a
potential means for increasing the substrate flexibility of bio-
synthetic pathways for the production of natural product ana-
logs.
resembling simplified DEBS heptaketides were all hydrolyzed
by DEBS TE. However, the ability of PKS TEs to cyclize
substrates other than their native linear intermediates has
proved to be much more limited. In fact, TE mediated macro-
lactonization has only been observed in a select few
studies.10,16,35-42
In addition to their native substrates, Pik TE has been shown
to catalyze macrolactonization of C-3 methyl 4 and NBOM
protected derivatives10, though not C-7 reduced analogs16,36
.
DEBS TE has been shown to catalyze macrolactonization of
unnatural mimics of the DEBS heptaketide39,42. However,
there are no reports of either the Pik or DEBS TEs catalyzing
macrolactonization of a substrate containing a nucleophilic
hydroxyl group with an unnatural, epimerized (S)-
configuration. When probed for the ability to form an epimeric
heptaketide mimic of 6-dEB , the DEBS TE displayed a high
level of stereoselectivity for the natural (R)-configuration, and
exclusively hydrolyzed the unnatural (S)-stereoisomer42, add-
ing to the observations in this study for strict stereospecificity
in TE catalyzed macrolactonization.
In a previous report we assayed a series of different thio-
and oxoester protected hexaketides against the terminal Pik
module (PikAIV) as well as the excised TE domain10. We
found that thio- or oxoester choice determined the catalytic
route of the hexaketide in the presence of PikAIV in vitro,
with thiophenol and N-acetylcysteamine thioesters having a
10:1 preference for either full module processing or direct
cyclization, respectively. Moreover, previous studies have
demonstrated that TE domains can function as hydrolases with
a large degree of substrate flexibility16, 17. Thus, we reasoned
that the low yields from the PikAIII-TES148C reactions with 10
are likely due to direct loading of the S148C TE domain and
subsequent hydrolysis. Indeed, control reactions (see Support-
ing Information) confirmed (i) the S148C mutation overrides
the KS preference previously enjoyed by thiophenol thioesters
(ii) enzymatic reactions performed without extender unit or
PKS catalysis with engineered TE domains. In an accom-
panying article, we describe the identification of the TE do-
main as a catalytic bottleneck in the processing of unnatural
substrates. Accordingly, we have engineered the TE domain
for increased substrate flexibility and introduced it into
PikAIII-TE to yield the engineered variant PikAIII-TES148C
.
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reductive cofactors showed a much higher hydrolysis of 5 with QM calculations provides a plausible explanation for im-
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PikAIII-TES148C relative to wild type.
proved substrate flexibility and catalytic efficiency of Pik
TES148C. According to the results from our MD simulations,
epimerization of the hexaketide C-11 stereocenter generates a
substrate with a reduced propensity for acquiring a catalytical-
ly competent conformation within the TE active site. Howev-
er, the ability of the hexaketides to reach a conformation via-
ble for catalysis is not the only factor affecting macrocyliciza-
tion, especially if this step is not rate-limiting44. Hence, even if
a substrate has a poor propensity to arrange in a productive
conformation and/or lactonization is structurally hindered (as
with the C-11-epimerized hexaketide) a significant accelera-
tion through a key single mutation can overcome these struc-
tural limitations, boosting reactivity even with unnatural sub-
strates.
Recently, Schaffer et al. reported a TE domain with a native
cysteine nucleophile critical to the cyclization of a strained β-
lactone ring in the obafluorin (Obi) nonribosomal peptide
pathway.43 Of note, an analogous cysteine to serine mutation
in the Obi TE resulted solely in the hydrolysis product, further
implicating the catalytic advantage of a cysteine TE in the
cyclization of strained ring systems. Examples of modular
type I polyketide TE domains containing cysteine active site
nucleophiles are notably rare6. Further studies will focus on
delineating the biosynthetic parameters that select for cysteine
or serine active site residues.
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Computational investigation of the TE domain catalysis.
Our investigation revealed mutual recognition, TE substrate
shape complementarity, and intrinsic substrate structural pref-
erences to be critical for the hexaketide to reach a confor-
mation productive towards macrolactonization. MD simula-
tions revealed hydrophobic interactions between the substrate
and TE residues lining the active site as being vital for guiding
the hexaketide to a catalytically competent pro-cyclic confor-
mation. These findings are consistent with mutational analysis
of DEBS TE where exchange of potential hydrogen-bonding
residues did not substantively affect the specificity constant
for hydrolysis of four unnatural thioester substrates. In this
case, Wang and Boddy suggested that hydrophobic interac-
tions between the active site and substrate are the main driving
force of substrate specificity37. Additionally, our results are in
agreement with the available crystal structures of Pik TE
bound with phosphonate substrate mimics, which displayed a
lack of specific TE-substrate polar contacts7,8.
Recently, Chen et al.44 reported theoretical investigations of
the thioesterase domain from the erythromycin biosynthetic
pathway (DEBS TE) to describe the mechanistic parameters
that determine the catalytic partitioning of substrates to either
macrocyclic or linear hydrolysis products. MD simulations
coupled with QM calculations were performed on systems of
DEBS TE modeled with the native DEBS heptaketide and Pik
hexaketide, which both lead to macrolactonization, as well as
two diastereomers of a reduced C-7 hydroxyl analog of Pik
hexaketide that result exclusively in substrate hydrolysis.
Analysis of the resulting MD simulations provided findings
consistent with those in this report, particularly highlighting
the importance of the formation of a substrate pre-reaction
state through induced-fit mutual recognition between the en-
zyme and the substrate for macrolactonization to occur. Con-
sistent with the present study, the authors found a hydrogen
bond between the lactonizing hydroxyl group of the substrate
and the catalytic histidine as well as hydrophobic interactions
to be critical for formation of a catalytically competent pre-
reaction state.
CONCLUSION
Based on the TE catalytic stringency observed in the pro-
cessing of unnatural substrates9, we focused the current study
on investigating full length substrate analogs as probes for the
Pik TE domain and describe the identification of a single
active site mutation that generates a more effective macrolac-
tonization catalyst. Remarkably, this single S148C mutation
provided a Pik TE variant with increases in both substrate
scope and catalytic efficiency. Notably, Pik TES148C was able
to catalyze the cyclization of a hexaketide with an epimerized
nucleophilic hydroxyl to generate a novel epimerized macro-
lactone, while TEWT catalyzed complete hydrolysis of the
same substrate due to inefficient macrocyclization. Further-
more, application of the engineered TE through reactions
containing PikAIII-TES148C allowed for the full-module pro-
cessing of a C-9-epimerized Pik pentaketide 10 to the corre-
sponding epimeric macrolactones 11 and 12. Computational
investigation of both variants revealed that a single mutation
greatly lowers the activation barriers to macrolactonization; as
a consequence, the catalytic process occurs faster and the
substrate specificity previously dictated by shape complemen-
tarity is kinetically overcome, in line with the observed in-
crease in kcat.
Notably, in the context of our system, the KR domain of
PikAIII-TES148C displayed diminished relative activity for both
the native Pik pentaketide as well as 10 with an epimerized
distal hydroxyl stereocenter. The product distribution indicates
competition between the KR mediated β-keto reduction and
TE cyclization of the linear hexaketide intermediate. This
competition is remarkable since the KR domain is fully com-
petent toward processing the native substrate and no unre-
duced products are observed in reactions containing PikAIII-
TEWT, suggesting that the engineered TE domain is able to
outcompete the native catalytic sequence45,46. While this do-
main competition for the hexaketide intermediate diminished
the product yields for 11-epi-10-dml 11, it provides insight
into the effects of engineered domains on the sequence of
catalytic events in PKS catalysis.
To understand the energetic consequences of the S148C
mutation during the macrolactonization process, we performed
DFT analysis of Pik TEWT and TES148C modeled as acyl-
enzyme intermediates with the native and C-11-epimerized
hexaketides. The results of our QM calculations revealed a
significant kinetic advantage in the reactions catalyzed by Pik
TES148C. The S148C mutation provides a mechanistic change
during the macrolactonization step from a two-step transesteri-
fication in the TEWT reaction (i.e. addition-elimination) to a
lower energy single concerted step in the TES148C pathway.
While additional biochemical studies of excised domains
and full-modules with full-length analogs of native substrates
are necessary to support and expand our understanding of PKS
function, it is becoming apparent that efficient production of
specific, designer macrolide analogs will require significant
pathway engineering. However, production of a desired natu-
ral product analog should be obtainable through the following
workflow: (i) a targeted domain is engineered to perform an
unnatural function (ii) downstream modules are biochemically
Overall, our combined computational method for investigat-
ing Pik TE catalysis using MD simulations in concert with
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characterized in vitro with the resulting unnatural polyketide
(8)
Giraldes, J. W.; Akey, D. L.; Kittendorf, J. D.;
1
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in order to identify bottlenecks (iii) catalytically inefficient
domain(s) are engineered with the goal of restoring effective
processing to generate unnatural products. Indeed, as noted in
a recent review by Weissman47, it is perhaps more realistic to
envision the future application of PKS engineering as a syn-
thetic biology tool for producing specific, high-value natural
product derivatives through targeted reprogramming of modu-
lar type I polyketide pathways than generation of natural
product libraries.
Sherman, D. H.; Smith, J. L.; Fecik, R. A. Nat. Chem. Biol.
2006, 2, 531.
(9)
probing of PikAIII-TE with a series of pentaketide analogs
and reveal a thioesterase domain bottleneck in the processing
of unnatural substrates.
(10) Hansen, D. A.; Koch, A. A.; Sherman, D. H. J. Am.
Chem. Soc. 2015, 137, 3735.
In an accompanying manuscript, we report the
(11) Stang, E. M.; White, M. C. Angew. Chem. Int. Ed.
2011, 50, 2094.
9
The results described herein lay the groundwork for future
engineering of PKS TE domains in order to generate more
flexible catalysts for the production of novel natural product
analogs.
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ASSOCIATED CONTENT
Supporting Information. Full experimental details and the spec-
troscopic data is available free of charge via the Internet at
http://pubs.acs.org.”
AUTHOR INFORMATION
Corresponding Authors
(17) Mortison, J. D.; Kittendorf, J. D.; Sherman, D. H. J.
Am. Chem. Soc. 2009, 131, 15784.
(18) Chemler, J. A.; Tripathi, A.; Hansen, D. A.; O'Neil-
Johnson, M.; Williams, R. B.; Starks, C.; Park, S. R.;
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*davidhs@umich.edu
*gonzalo.jimenez@unirioja.es
*houk@chem.ucla.edu
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
This research was supported by Rackham Merit Predoctoral Fel-
lowships (D.A.H. and A.A.K.), American Foundation for Phar-
maceutical Education Predoctoral Fellowship (D.A.H.), and NIH
T-32-CA009676 (A.A.K.). Support for L.F. was provided through
a grant from the National Science Foundation REU Program (NSF
DBI #1263079). NIH Grants R35 GM118101, GM076477, the
Hans W. Vahlteich Professorship (to D.H.S.) and
MINECO/FEDER (grants CTQ2015-70524-R and RYC-2013-
14706 to G.J.O.) are gratefully acknowledged. Calculations were
performed using the Extreme Science and Engineering Discovery
Environment (XSEDE), which is funded by the NSF (OCI-
1053575), UR (Beronia cluster), and the UCLA Institute of Digi-
tal Research and Education (IDRE). We are grateful to Dr. J. W.
Kampf for small molecule X-ray crystallography.
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