Polyketide intermediate mimics as probes for revealing cryptic stereochemistry of ketoreductase domains

Article abstract of DOI:10.1021/cb5006883

Among natural product families, polyketides have shown the most promise for combinatorial biosynthesis of natural product-like libraries. Though recent research in the area has provided many mechanistic revelations, a basic-level understanding of kinetic and substrate tolerability is still needed before the full potential of combinatorial biosynthesis can be realized. We have developed a novel set of chemical probes for the study of ketoreductase domains of polyketide synthases. This chemical tool-based approach was validated using the ketoreductase of pikromycin module 2 (PikKR2) as a model system. Triketide substrate mimics 12 and 13 were designed to increase stability (incorporating a nonhydrolyzable thioether linkage) and minimize nonessential functionality (truncating the phosphopantetheinyl arm). PikKR2 reduction product identities as well as steady-state kinetic parameters were determined by a combination of LC-MS/MS analysis of synthetic standards and a NADPH consumption assay. The d-hydroxyl product is consistent with bioinformatic analysis and results from a complementary biochemical and molecular biological approach. When compared to widely employed substrates in previous studies, diketide 63 and trans-decalone 64, substrates 12 and 13 showed 2-10 fold lower K_M values (2.4 ± 0.8 and 7.8 ± 2.7 mM, respectively), indicating molecular recognition of intermediate-like substrates. Due to an abundance of the nonreducable enol-tautomer, the k_cat values were attenuated by as much as 15-336 fold relative to known substrates. This study reveals the high stereoselectivity of PikKR2 in the face of gross substrate permutation, highlighting the utility of a chemical probe-based approach in the study of polyketide ketoreductases.

Full text of DOI:10.1021/cb5006883

Articles
pubs.acs.org/acschemicalbiology
Polyketide Intermediate Mimics as Probes for Revealing Cryptic
Stereochemistry of Ketoreductase Domains
Yang Li, William D. Fiers, Steffen M. Bernard, Janet L. Smith, Courtney C. Aldrich,*,†
†
†
∥,§
‡,§
,†
and Robert A. Fecik*
†
Department of Medicinal Chemistry, College of Pharmacy, University of Minnesota, Minneapolis, Minnesota 55455, United States
∥
‡
§
Chemical Biology Program, Department of Biological Chemistry, and Life Sciences Institute, University of Michigan, Ann Arbor,
Michigan 48109, United States
*
S Supporting Information
ABSTRACT: Among natural product families, polyketides have
shown the most promise for combinatorial biosynthesis of natural
product-like libraries. Though recent research in the area has
provided many mechanistic revelations, a basic-level understanding
of kinetic and substrate tolerability is still needed before the full
potential of combinatorial biosynthesis can be realized. We have
developed a novel set of chemical probes for the study of
ketoreductase domains of polyketide synthases. This chemical tool-
based approach was validated using the ketoreductase of pikromycin
module 2 (PikKR2) as a model system. Triketide substrate mimics
12 and 13 were designed to increase stability (incorporating a
nonhydrolyzable thioether linkage) and minimize nonessential functionality (truncating the phosphopantetheinyl arm). PikKR2
reduction product identities as well as steady-state kinetic parameters were determined by a combination of LC-MS/MS analysis
of synthetic standards and a NADPH consumption assay. The D-hydroxyl product is consistent with bioinformatic analysis and
results from a complementary biochemical and molecular biological approach. When compared to widely employed substrates in
previous studies, diketide 63 and trans-decalone 64, substrates 12 and 13 showed 2−10 fold lower K values (2.4 ± 0.8 and 7.8
M
±
2.7 mM, respectively), indicating molecular recognition of intermediate-like substrates. Due to an abundance of the
nonreducable enol-tautomer, the kcat values were attenuated by as much as 15−336 fold relative to known substrates. This study
reveals the high stereoselectivity of PikKR2 in the face of gross substrate permutation, highlighting the utility of a chemical probe-
based approach in the study of polyketide ketoreductases.
acterial type I polyketide synthases (PKSs) are large,
multifunctional, modular proteins that biosynthesize a large
a thioesterase (TE) domain catalyzes intramolecular cyclization
or hydrolysis to release a macrolactone or carboxylic acid,
respectively. Bacterial type I PKSs such as 6-deoxyerythronolide
B synthase (DEBS), the biosynthetic pathway for erythromycins,
have been well-studied for their potential in drug discovery by
B
class of diverse, polyoxygenated secondary metabolites with
1
−3
important pharmacological and therapeutic activities.
All
modular type I PKSs contain a loading module and several
extension modules, each responsible for one round of polyketide
chain elongation and β-carbon processing, organized in an
assembly line fashion. Each extension module features a minimal
set of three domains responsible for a two-carbon extension of
the chain elongation intermediate: acyl carrier protein (ACP),
acyltransferase (AT), and ketosynthase (KS) domains. All chain
elongation intermediates are bound to the noncatalytic ACP
domains through thioester bonds with the phosphopantetheinyl
arm of the ACP domain. After extension, the newly formed β-
keto thioester intermediate is frequently followed by sequential
β-carbon processing by ketoreductase (KR), dehydratase (DH),
and enoylreductase (ER) domains. The KR domain is the most
abundant β-carbon processing domain throughout all PKSs. It
stereoselectively reduces the newly formed β-ketone to an
alcohol with NADPH and can also control the stereochemistry of
9
combinatorial biosynthesis and metabolic engineering. We, and
others, have studied the pikromycin (Pik) PKS of Streptomyces
venezuelae (Figure 1) for these purposes based upon its unique
ability to produce both 12- and 14-membered macrolactones,
including 10-deoxymethynolide (1), narbonolide (2), methy-
10−15
mycin (3), and pikromycin (4).
Each catalytic domain in a PKS conducts its reaction in a highly
stereoselective manner. Generally, the stereochemical outcome
of the β-carbon processing domains can be readily deduced by
examining the absolute stereochemistry of the natural product.
For instance, the initial KR domain of the Pik PKS (KR1)
reduces the β-ketone to an alcohol, which establishes the
configuration at C-11 of methymycin and C-13 of pikromycin
Received: May 16, 2014
Accepted: October 9, 2014
Published: October 9, 2014
4
−8
the α-substituent through enzymatic epimerization.
Follow-
ing multiple rounds of chain elongation and β-carbon processing,
©
2014 American Chemical Society
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Figure 1. Pikromycin and methymycin biosynthetic pathway in Streptomyces venezuelae ATCC 15439.
(Figure 1). However, this observational method cannot be
utilized for modules that contain a DH domain since the resulting
dehydration eliminates the stereogenic center as seen in modules
2
and 4 of the Pik PKS. This type of “hidden” stereoselectivity,
when referral to the mature polyketide gives no implication of the
stereochemical outcome of the KR domain of interest, has been
termed cryptic stereochemistry.
Biochemical and molecular biological approaches have been
widely used to provide valuable insights into the mechanism as
well as substrate and stereoselectivity of PKS domains, especially
1
6−19
KRs.
Cane and co-workers established the stereochemical
outcome of PikKR2 by engineering Pik module 2 with PikTE and
inactivation of PikDH2 by site-directed mutagenesis (Figure
1
6
2
a). Incubation of diketide thioester 5, methylmalonyl-CoA,
and NADPH with this construct produced triketide lactone 6
exclusively, establishing the D-configuration of the C3-alcohol. As
a general approach, this method may require extensive work on
protein expression, mutant construction, and product detection.
More importantly, most of the substrates used in this and other
previous studies are simple diketide analogs, which likely show
low affinity and poor stereoselectivity for the study of domains in
later, downstream modules due to poor structural similarity to
Figure 2. (a) Previous approach to determining the cryptic stereo-
chemistry in PikKR2 using an engineered Pik module 2. (b) Design of
PikKR2 substrate mimics 10−13 for a KR-based assay to determine
cryptic stereochemistry.
19
11
the native substrates. For example, in a recent study with KR
domains from the amphotericin, pikromycin, and spinosyn PKSs
(
AmpKR10, PikKR5, and SpnKR3), multiple diastereomeric
biochemical and molecular biological workload in studying
cryptic stereochemistry since it requires only a single domain
versus an entire multimodular PKS protein. Additionally, the
substrate is not tethered to the ACP domain; thus neither a TE
domain nor a complicated workup procedure is required to
release the enzymatic product. Second, this intermolecular assay
design allows for interrogation of the steady-state kinetic
parameters of the individual excised domains. Testing of non-
natural substrates enables investigation of substrate−protein
interactions. It would also be possible to investigate protein−
protein interactions if the probes are linked to the ACP domain.
products resulted from use of the simple (2RS)-methyl-3-
oxobutane-SNAC (N-acetylcysteamine) thioester as the sub-
strate.
We describe here an alternative, chemical probe-based
approach to understand the structural and mechanistic features
of KR domains. This method entails incubation of synthetic
substrate mimics with excised KR domains and observation of
enzymatic products by LC-MS/MS (Figure 2b). Compared to
previous approaches, we envision our new method offers unique
insights in three aspects. First, this method reduces the
2
0
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Third, compatibility of substrates is guaranteed. Given that the
substrate is specifically designed on the basis of the natural
substrate, substrate and stereoselectivity is ensured due to nearly
the C1-epimeric product mixture was deemed sufficient for these
studies.
Synthesis of Substrate Mimics. Synthesis of secondary and
2
1,22
identical steric properties.
tertiary amide substrate analogs 10 and 11 commenced with the
3
4,35
Recent research has shown a strong correlation between the
amino acid sequence of KR domains and the stereochemistry of
the KR products. In KRs, the fingerprint LDD motif predicts a D-
alcohol product, while KRs with a W motif produce an L-
acetate aldol reaction of thiazolidinethione 26
27
with aldehyde
3
6,37
to give alcohol 28 as the major diastereomer (Scheme 1).
Scheme 1. Synthesis of Amide Substrate Analogs 10 and 11
2
3
alcohol. Moreover, absolute configuration of an α-substituent
2
4
can be determined through additional motif indicators.
Although the amino acid sequence can provide insight into the
stereochemistry of uncharacterized KRs, current work has
revealed several exceptions. For instance, rifamycin (Rif) KR7
and RifKR10 do not possess the signature W motif, yet they still
25
yield L-alcohol products. Due to the empirical nature of the
fingerprint method, the predicted stereochemical outcome of
cryptic domains is not absolutely infallible. The geometry of the
olefin created by the DH domain may also indicate the
stereochemistry of the alcohol precursor, as trans-olefins arise
2
2,23
from D-alcohols.
However, the olefin geometry may be
reduced by the following ER domain or isomerized later in the
biosynthetic pathway. This highlights the need for novel
experimental methods to complement signature sequence
methods to decipher cryptic stereochemistry.
RESULTS AND DISCUSSION
■
In light of previous research (vide supra), we chose PikKR2 to
validate this chemical probe-based approach. Both PikKR2
substrate and product mimics were designed and synthesized
Direct aminolysis of the chiral auxiliary with amine 29 or N-
methylamine 30 (synthesized in three steps from N-(2-
aminoethyl)acetamide, Supporting Information Scheme 1)
yielded amide 31 and N-methylamide 32, respectively. TPAP/
NMO oxidation and deprotection afforded amide substrate
mimics 10 and 11. The stereochemistry of aldol product 28 was
established by deprotection of amide 31 and conversion to its
(
Figure 2b). The substrate mimic designs were based on the
natural substrate of PikKR2 (7) with two modifications. First,
truncation of the ACP-phosphopantetheinyl arm to a NAC-
based substrate was planned to ease synthetic access to the target
molecules. Since the ACP-phosphopantetheinyl arm does not
affect the stereocontrol of KRs, NAC thioester derivatives have
acetonide, which was subject to Rychnovsky’s acetonide analysis
38,39
(
Supporting Information Scheme 2).
2
0,22,26
been widely used as surrogates of natural substrates.
Second, since 5-hydroxy triketide thioesters are prone to rapid
For the synthesis of thioether substrate mimics 12 and 13, N-
37
acyl thiazolidinethione 35 was protected and reduced to
aldehyde 37 (Scheme 2a), followed by acetate aldol reaction with
N-methoxy-N-methylacetamide to give Weinreb amides 38 and
21
cyclization to triketide lactones, we sought to replace and
mimic the electrophilic thioester of the native PikKR2
intermediate with an amide or thioether bond. Thus, two series
of substrate mimics with stabilized structures were designed: (1)
incorporation of secondary (10) or tertiary (11) amides as stable
39 (dr = 7:3, separated by chromatography). Weinreb amide 38
was directly converted to chloromethyl ketone 40, followed by
IBX oxidation of the secondary alcohol, S 2 reaction of the
N
27
thioester isosteres and (2) thioether analogs 12 and 13, which
are based upon previous work of us and others.
chloromethyl ketone with N-acetylcysteamine, and deprotection
13,28
40,41
Although
to afford thioether substrate mimic 12.
The synthesis of
thioethers 12 and 13 have one- and two-carbon spacers between
the C1-carbonyl of the triketide and the NAC group, we
anticipated that the long phosphopantetheinyl-binding channel
of PikKR2 could accommodate this variance. Additional support
for this approach was suggested by prior work of others with
malonyl CoA analogs where the thioester was replaced with a
ketone. These malonyl CoA analogs, when added to
fermentation broths, underwent chain elongation, β-processing,
thioether substrate mimic 13 was accomplished in an analogous
fashion from Weinreb amide 38 by nucleophilic addition of vinyl
lithium to produce vinyl ketone 43 (Scheme 2b). IBX oxidation,
Michael addition of N-acetylcysteamine, and deprotection
furnished thioether substrate analog 13. The stereochemistry at
C-3 of aldol products 38 and 39 was determined by conversion to
the products’ acetonides followed by Rychnovsky’s acetonide
3
8,39
analysis (Supporting Information Scheme 3).
29−33
and chain termination to release off-loaded intermediates.
For analysis of the enzymatic reaction of substrate mimics 10−
3, synthesis of both potential diastereomeric products 14−21 of
Synthesis of Product Mimics. All potential products from
PikKR2 reduction of thioether substrate mimics 12 and 13 were
synthesized as authentic standards for analytical comparison to
the enzymatic products (Scheme 3). C3-Hydroxy products of
substrate mimic 12, diols 18 and 20, were synthesized from
Weinreb amides 38 and 39 by protection, conversion of the
1
each substrate mimic was also required. Since thioether substrate
mimics 12 and 13 have two ketones that could potentially be
reduced by PikKR2, we also synthesized diols 22−25 (as
diastereomeric mixtures). Diols 22−25 would result from
reduction of the C1-ketone (polyketide numbering) instead of
expected reduction of the C3-ketone. As C1 reduction is an
unnatural event, and does not occur in the biosynthetic pathway,
Weinreb amides to the chloromethyl ketones, S 2 displacement
N
with N-acetylcysteamine, and deprotection (Scheme 3a).
Synthesis of the potential products resulting from C1-reduction
of substrate mimic 12, C1-alcohols 22 and 24, began with
2
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Scheme 2. Synthesis of Thioether Substrate Analogs (a) 12
and (b) 13
The potential C3-reduction products of thioether substrate
mimic 13, diols 19 and 21, were synthesized from protected
Weinreb amides 46 and 47 in an analogous fashion to 18 and 20
(
Scheme 3c). Synthesis of the potential C1-reduction products
of substrate mimic 13, diols 23 and 25, began with addition of
methylmagesium bromide to Weinreb amide 52, followed by
aldol reaction of the resulting methyl ketone 60 with aldehyde 61
(
synthesized in one step from N-acetylcysteamine and acrolein,
Supporting Information Scheme 5) to give alcohol 62 (dr = 2:1,
inseparable by chromatography), and deprotection to provide
diols 23/25 as a diastereomeric mixture.
Cloning, Expression, and Purification of PikKR2. The
KR2 domain of PikAI was identified by sequence comparison
with other type I PKS KR domains. The PikAI sequence
encoding KR2 (residues 3880−4355) was cloned from the
cosmid pLZ51 into expression vector pMCSG7 and verified by
4
3,44
sequencing.
The N-terminal hexa-His tagged protein was
overexpressed in E. coli BL21(DE3) cells. The protein was
purified by sequential nickel affinity and gel filtration
chromatography to afford approximately 45 mg of purified
enzyme per liter of culture that was >95% pure as judged by SDS-
PAGE (Supporting Information Figure 1). The molecular weight
determined by SDS-PAGE is approximately 55 kDa, while the
monoisotopic mass of PikKR2 determined by electrospray
ionization (ESI) mass spectrometry is 51 430 Da, also consistent
with the calculated value from the amino acid sequence of the
PikKR2 domain and the histidine tag (51 431 Da). A Superdex
2
00 gel filtration, calibrated using a Gel Filtration Calibration Kit
LMW (GE Healthcare), was used to estimate the molecular
weight of the native enzyme as 95 kDa, suggesting the enzyme
exists as a homodimer. The cloning, expression, and purification
of PikKR2-DH2 didomain was conducted in an analogous
fashion.
Grignard addition of allyl magnesium bromide to Weinreb amide
5
2 (synthesized in two steps from thiazolidinethione 35,
Supporting Information Scheme 4) to afford allyl ketone 53
4
2
(
Scheme 3b). Nonselective epoxidation of 53 with m-CPBA,
epoxide opening with N-acetylcysteamine, and deprotection
gave C1 products 22/24 as a distereomeric mixture. The reaction
of epoxide 54 with N-acetylcysteamine was low-yielding due to
competitive formation of the C-2 addition product.
Substrate Specificity of PikKR2. To initially test whether
the synthesized substrate mimics 10−13 were competent
substrates, PikKR2 (5 μM) was incubated overnight (12−16
h) with 10, 11, 12, or 13 (1 mM) and NADPH (2 mM) in
Scheme 3. Synthesis of All Possible Thioether Products (a) 18 and 20, (b) 22/24, (c) 19 and 21, and (d) 23/25
2
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sodium phosphate buffer (100 mM, pH 7.1). After incubation,
each enzymatic reaction was quenched via the addition of
MeCN, and precipitated protein was removed by centrifugation.
The supernatant was analyzed by ESI mass spectrometry to
detect enzymatic reduction products (as seen by the increase of
[
M+2] peak), compared to control reaction (either without
enzyme or without NADPH). The reactions of amides 10 and 11
did not show any appreciable increase of the [M+2] peaks, nor
consumption of the parent ion peaks (data not shown). In
contrast, thioethers 12 and 13 demonstrated substantial turnover
with large increases of the [M+2] peaks (Supporting Information
Figure 2). Exclusion of either NADPH or PikKR2 abolished
product formation with 12 and 13. This assay indicated that
thioethers 12 and 13 are substrates for PikKR2 while amides 10
and 11 are not turned over by the enzyme.
Several factors may contribute to the lack of reactivity of
amides 10 and 11. Unlike the thioester, amides may form either
an unfavorable N−H hydrogen bond (secondary amide 10) or
harbor excess steric bulk (tertiary amide 11), impeding access
into the narrow active site channel. The amide linkage’s restricted
conformations (i.e., s-trans for 10 and s-cis for 11) may also not
enable the substrate mimics to adopt the required catalytically
active conformation. Computational docking by others of a
ketide-bound ACP substrate to a KR domain indicated that the
thioester linkage may not be planar, lending support to the latter
2
4
hypothesis.
Confirmation of Reaction Products by LC-MS/MS. Due
to its intrinsic selectivity and sensitivity, LC-MS/MS was chosen
for assignment of the enzymatic products of PikKR2. We
synthesized all plausible constitutional isomer reduction
products of thioethers 12 and 13 as authentic standards. These
synthetic standards are readily resolved by LC-MS/MS by their
unique HPLC retention times and/or by their parent and
daughter ions in MS/MS (Supporting Information Figure 3 and
Supporting Information Table 1). Standards 19 and 21 are
observed with two retention times by HPLC (Supporting
Information Figure 3), which we hypothesize is caused by the
ring−chain equilibration between the open chain keto tautomer
and the closed ring lactol tautomer. Standards 22/24 and 23/25
are mixtures of two diastereomers, also resulting in two HPLC
retention times.
Incubation of thioether 12 with PikKR2 using conditions
described above resulted in two reaction products with retention
times of 4.00 min (red trace, transition m/z 292 → 216) and 4.70
min (blue trace, transition m/z 314 → 198) corresponding to the
C1-reduction product (22 or 24) and C3-reduction product 20,
respectively (Figure 3a). The normalized product ratio of 22/20
or 24/20 is 1:4 based on a standard curve generated from the
authentic standards. The larger peak for 22 or 24 at 4.00 min
actually represents the minor product, a consequence of its high
ionization efficiency. Similarly, incubation of thioether 13 with
PikKR2 resulted in two reaction products. The peaks at 5.88 and
Figure 3. LC-MS/MS analysis of PiKKR2 activity. (a) LC-MS/MS
enzymatic product analysis of substrate 12; red trace represents MRM
(
m/z 292 → 216); blue trace represents MRM (m/z 314 → 198). (b)
LC-MS/MS analysis for enzymatic product of substrate 13; red trace
represents MRM (m/z 306 → 230); blue trace represents MRM (m/z
3
28 → 212).
the minor reduction (C1 reduction) is conducted in a complete
diastereoselective fashion.
To investigate the stereoselectivity of KR in a more native
context, substrate 13 was incubated with the PikKR2-DH2
didomain and analyzed in an analogous way. The LC-MS/MS
trace of didomain products showed exactly the same retention
times as observed by the single domain, demonstrating the same
enzymatic products, D-alcohol at C3-reduction and one single
diastereomer at C1-reduction (Supporting Information Figure
4
(
1
). The ratio of C3 reduction (natural event) to C1 reduction
unnatural event) by PikKR2-DH2 didomain was increased to
8:1, suggesting that larger portions of the PKS module
preferably conduct the natural reaction (C3 reduction) over
the competitive side reactions (C1 reduction).
Steady-State Kinetic Analysis of PikKR2. To study the
substrate specificity of PikKR2, the steady-state kinetic
parameters of the triketide substrate mimics 12 and 13, as well
as widely used substrates diketide 63 and trans-decalone 64, were
6.93 min (blue trace, transition m/z 328 → 212) correspond to
standard 21, while the peak at 5.68 min (red trace, transition m/z
306 → 230) corresponds to one of the diastereomers in standard
23/25 (Figure 3b). The normalized product ratio of (23 or
25):21 is 1:6. These results indicate that PikKR2 preferentially
6,45
determined (Table 1 and Supporting Information Figure 5).
Incubations were carried out with PikKR2 (5 μM), saturating
NADPH (0.5 mM), and varying substrate concentrations under
initial velocity conditions. Apparent kinetic parameters for
NADPH (Table 1) were determined with 40 mM 63 (this is
nonsaturating as a result of the limited solubility of this
reduces the C3-ketone in substrates 12 and 13 with exquisite
stereoselectivity to afford the D-configured alcohol products 20
and 21, respectively. Intriguingly, only a single peak was observed
by LC-MS/MS for C1-reduction products, which corresponds to
22 or 24 (Figure 3a) and 23 or 25 (Figure 3b), indicating even
substrate). The initial rates, v , at a given [S] were determined
0
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Table 1. Steady-State Kinetic Parameters for PikKR2 Substrates
a
6
5 was isolated as a 4:1 mixture of D- and L-alcohols.
by single-time point stopped-time incubations at 12 min for 12
and 13 (the reaction velocity remained linear up to 30 min). The
reaction products 20 and 21 were quantified by LC-MS/MS
employing a standard curve generated from synthetic standards.
For substrates 63 and 64, whose turnover was substantially faster
than 12 and 13, we were able to employ a continuous assay that
monitors consumption of NADPH by a decrease in absorbance
at 340 nm. The enzymatic products of diketide 63 were identified
as a mixture of D- and L-alcohols, in a ratio of 4 to 1, by chiral
HPLC (Supporting Information Figure 6).
The specificity constants (k /K ) of the triketide substrate
cat
M
−1
−1
mimics 12 and 13 are 12.1 and 14.5 min M , respectively
while the k /K values of diketide 63 is 66 min
decalone 64 is 718 min M . Thus, the native substrate mimics
−
1
−1
M
and trans-
cat
M
−1
−1
1
2 and 13 are processed between 4−60 fold less efficiently than
the unnatural substrates 63 and 64. Examination of the kinetic
parameters reveals that 12 and 13 possess improved K values
M
that are 2−10 fold lower than 63 and 64, likely due to their
similarity to the natural acyl chain. Consequently, the lower
specificity constants for 12 and 13 are exclusively caused by the
greatly reduced turnover of these substrates. Indeed the kcat
values for 12 and 13 are 15−336 fold lower than 63 and 64. To
explain the slow turnover of substrates 12 and 13, we initially
tested for product inhibition (see Supporting Information).
However, product inhibition was not observed, thus we
considered an alternative rationale for attenuation of kcat values.
We believe the low activity of the thioether substrates 12 and 13
may be caused by their β-diketone moieties, which exist to a large
1
extent in the nonreducable enol forms (35−85% observed in H
NMR (CDCl ), see Supporting Information) as a result of the
3
greater acidity of this functional group relative to a β-
ketothioester.
Structure and Mechanism of A- and B-type KRs. KRs can
be classified into two types based on the stereochemistry of the
reduction product. A-type KRs generate L-hydroxyacyl products
and contain a highly conserved tryptophan (W motif) while B-
type KRs yield D-hydroxyacyl products and harbor a LDD motif
Figure 4. Signature motif comparison of A- and B-type KRs. (a)
Sequence alignment of PikKR5, EryKR2, AmpKR1, TylKR2, PikKR1,
EryKR1, and PikKR2; conserved catalytic tyrosine shown in yellow; W
motif in cyan; LDD motif in pink. (b) Active site of A-type PlmKR1 with
+
NADP bound; W motif shown in cyan; M366 shown in purple. (c)
+
Active site of B-type EryKR1 with NADP bound; LDD motif shown in
(or often LXD motif). After aligning the protein sequence of
25
pink. Parts b and c were created in Pymol using PDB codes 4HXY and
PikKR2 with other characterized KRs, an LSD motif (a variation
of the LDD motif) is found (Figure 4a), indicative of a B-type KR
with a D-hydroxyl product. The results here substantiate the
prediction for PikKR2, which also agrees with the experimental
41
2
FR0.
motifs, there have been many efforts to obtain KR domain
structural information. Recent crystallographic studies have
shown that all KRs bind the cofactor NADPH in the same
orientation, which transfers the 4-pro-S-hydride to the β-carbonyl
16
approach using an engineered Pik module 2.
To rationalize the relationship between observed stereo-
chemical outcome and the empirically deduced amino acid
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2
1,22,24,46,47
carbon in the polyketide intermediate.
As a result, the
eduction step and resulted in an unexpected product due to the
competition between the slower, unnatural KR domain and more
difference in stereochemical outcome must arise from a reversed
presentation of the β-ketoacyl substrate in the active site,
56
rapid downstream enzymes. Clearly, prerequisite knowledge of
the mechanism, kinetic parameters, stereoselectivity, and
substrate specificity of each domain is crucial for applications
in combinatorial biosynthesis and metabolic engineering.
Conclusion. This study demonstrates that chemical-based
approaches to the study of PKS KR domains provide access to
valuable information crucial for understanding the limitations of
current approaches in combinatorial biosynthesis. Utilizing a
small number of probes designed as substrate analogs, we were
able to expose a limitation in accepted substrate linkages
(secondary and tertiary amides) and a flexible catalytic reduction
site (C1 vs C3 reduction in diketone thioether substrates),
indicating a stringent preference in linkage region with a flexible
reduction locale in the active site, respectively. The extremely
high stereoselectivity of PikKR2 when presented with a variety of
substrates exhibits its potential use in combinatorial biosynthesis
and chemoenzymatic synthesis. This technique is expected to be
broadly applicable to other PKS KR domains, hopefully unveiling
fundamental knowledge required for the rational engineering of
PKS systems for drug discovery.
24
exposing re or si faces of the β-keto group to NADPH. One
hypothesis is that the W motif promotes entry of the polyketide
intermediate through the southeast opening of the active site
groove in A-type KR (Figure 4b), yielding a product with an L
orientation. In an analogous way, in B-type KR, the LDD motif
directs the substrate to access the active site groove from the
northwest side to generate D-hydroxyl product (Figure 4c).
However, the W and LDD motifs are not the only factors
contributing to stereocontrol based on the distance from the
active site (W) and inability to occlude active site openings
Analysis of crystal structures of both A- and B-
type KRs led to a corollary to the direction-of-entry hypothesis
by noting that cofactor binding (NADPH or NADP ) in A-type
KRs may generate a tight and well-ordered conformation at the
active site through a hydrogen bonding network. In this
catalysis-ready conformation, Met366 is pushed into the active
site to block the entry from the northwest. Thus, the substrate
could penetrate the active site groove only from the southeast
2
1,22,48
(
LDD).
+
22
(
Figure 4b). In B-type KRs, cofactor binding is loose, allowing for
polyketide entry through both channel openings. It is proposed
that only binding of substrate from the northwest side could set a
tight, catalysis-ready conformation at the active site (Figure
ASSOCIATED CONTENT
■
*
S
Supporting Information
2
2
4c). Further biochemical and structural experiments may be
Methods are reported in the Supporting Information, which
contains Supporting Information Schemes 1−5, Supporting
Information Figures 1−6, Supporting Information Table 1,
general biology procedures, cloning, protein expression and
purification, NADPH consumption kinetic assay, kinetic assay
and analysis of PikKR2 reaction products by LC-MS/MS,
general chemistry procedures and experimental procedures,
spectral data, and copies of H and C NMR spectra for all new
compounds. This material is available is available free of charge
via the Internet at http:/pubs.acs.org.
needed to better understand the mechanism of the stereo-
selectivity of KRs.
Application in Chemoenzymatic Synthesis. Excised
enzymes from natural product biosynthetic pathways have
shown great promise in chemoenzymatic synthesis providing
2
0,49−51
diverse natural product analogs.
Due to their promiscuity
for a variety of substrates, these enzymes are able to accept
unnatural substrates and conduct highly stereoselective reac-
tions, which are usually difficult to accomplish by purely
synthetic methods. Enzymes provide an economically efficient
and environmentally friendly strategy in organic synthesis by
eliminating the need for organic solvents, chiral auxiliaries, or
expensive chiral ligands. More importantly, enzymes can catalyze
asymmetric chemical reactions in excellent yield and enantio-
meric (or diastereomeric) purity under mild conditions and can
1
13
AUTHOR INFORMATION
■
Corresponding Authors
E-mail: aldri0015@umn.edu.
E-mail: fecik001@umn.edu.
*
*
52
reduce the number of reactions in a synthetic sequence.
Notes
Asymmetric ketoreduction is a promising area for biocatalyst
development as many pharmaceuticals contain stereogenic
hydroxyl groups. PikKR2, in this study, has shown relaxed
substrate flexibility toward unnatural substrates by reducing
diketones to alcohols while affording an extremely high level of
ketoreduction stereoselectivity at both C1 and C3 keto groups.
Combinatorial Biosynthesis. A deeper mechanistic under-
standing of polyketide biosynthesis can aid the efficient
production of “unnatural” natural product libraries for drug
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors thank B. Witthuhn (Center for Mass Spectrometry
and Proteomics, University of Minnesota) for assistance with
LC-MS/MS and N. Bleeker (Institute for Therapeutics
Discovery and Development, University of Minnesota) for the
help with HPLC. Financial support from NIH GM081544-01 (to
J.L.S. and R.A.F.), the Department of Medicinal Chemistry
53,54
discovery by combinatorial biosynthesis.
This topic has
(
University of Minnesota), and support from a Chemistry
drawn much attention and continuous efforts over the past three
decades. Although tremendous breakthroughs have been made
to create libraries of natural product analogs, full exploitation of
Biology Interface training grant (to S.M.B.) is gratefully
acknowledged.
55
combinatorial biosynthesis remains a largely unattained goal.
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