Substrate controlled divergence in polyketide synthase catalysis

Article abstract of DOI:10.1021/ja511743n

Biochemical characterization of polyketide synthases (PKSs) has relied on synthetic substrates functionalized as electrophilic esters to acylate the enzyme and initiate the catalytic cycle. In these efforts, N-acetylcysteamine thioesters have typically been employed for in vitro studies of full PKS modules as well as excised domains. However, substrate engineering approaches to control the catalytic cycle of a full PKS module harboring multiple domains remain underexplored. This study examines a series of alternatively activated native hexaketide substrates on the catalytic outcome of PikAIV, the sixth and final module of the pikromycin (Pik) pathway. We demonstrate the ability to control product formation with greater than 10:1 selectivity for either full module catalysis, leading to a 14-membered macrolactone, or direct cyclization to a 12-membered ring. This outcome was achieved through modifying the type of hexaketide ester employed, demonstrating the utility of substrate engineering in PKS functional studies and biocatalysis.

Full text of DOI:10.1021/ja511743n

This is an open access article published under an ACS AuthorChoice License, which permits
copying and redistribution of the article or any adaptations for non-commercial purposes.
Communication
pubs.acs.org/JACS
Substrate Controlled Divergence in Polyketide Synthase Catalysis
Douglas A. Hansen,‡,† Aaron A. Koch,§,† and David H. Sherman*,‡,†,∥,⊥
‡
§
∥
†Life Sciences Institute, and Department of Medicinal Chemistry, Cancer Biology Graduate Program, Department of Chemistry,
⊥Department of Microbiology & Immunology, University of Michigan, Ann Arbor, Michigan 48109, United States
S
* Supporting Information
Scheme 1. PikAIII and PikAIV, the Final PKS Modules from
the Pikromycin Pathway
ABSTRACT: Biochemical characterization of polyketide
synthases (PKSs) has relied on synthetic substrates
functionalized as electrophilic esters to acylate the enzyme
and initiate the catalytic cycle. In these efforts, N-
acetylcysteamine thioesters have typically been employed
for in vitro studies of full PKS modules as well as excised
domains. However, substrate engineering approaches to
control the catalytic cycle of a full PKS module harboring
multiple domains remain underexplored. This study
examines a series of alternatively activated native
hexaketide substrates on the catalytic outcome of PikAIV,
the sixth and final module of the pikromycin (Pik)
pathway. We demonstrate the ability to control product
formation with greater than 10:1 selectivity for either full
module catalysis, leading to a 14-membered macrolactone,
or direct cyclization to a 12-membered ring. This outcome
was achieved through modifying the type of hexaketide
ester employed, demonstrating the utility of substrate
engineering in PKS functional studies and biocatalysis.
of the phosphopantetheine arm that tethers a growing
polyketide chain.5 In vitro studies of PikAIV with its native
substrate have highlighted a key observation: Specifically, when
incubated directly with N-acetylcysteamine Pik hexaketide 4,
PikAIV afforded a 4:1 ratio of macrolactones 10-dml (2) and
narbonolide (1).6 However, reaction schemes pairing PikAIII/
PikAIV3,7,8 with Pik pentaketide 3 where PikAIII performs an
extension and delivers the hexaketide to PikAIV via an ACP5
thioester favor narbonolide as the major product. Additionally,
optimization of PikAIII (as an unnatural TE fusion9 or when
paired with the final module, PikAIV) demonstrated improved
catalysis with thiophenol thioesters10 over N-acetylcysteamine
thioesters.7 These results suggest that the traditionally
employed N-acetylcysteamine thioester might be a poor choice
for loading of the KS domain and motivated exploratory
substrate engineering approaches with PikAIV. However, the
previously observed instability of the native Pik hexaketide 4
imposes considerable experimental challenges7,11 and required
a practical solution prior to downstream studies (Figure 1).
While mature natural products typically possess adequate
stability to survive isolation and purification from biological
sources, polyketide intermediates often degrade rapidly through
intramolecular hemiketalization and dehydration pathways
presenting experimental bottlenecks in terms of synthetic
accessibility and limited shelf life.7,11,12 Although the structural
basis remains unclear, polyketide elongation intermediates that
are covalently attached to the ACP domain during biosynthesis
are likely stabilized through sequestration within the PKS
polypeptide scaffold.13
odular type I polyketide synthases (PKSs) are complex
M
bacterial proteins comprised of multiple catalytic
domains. In vivo, PKS modules form multienzyme complexes
leading to the production of numerous therapeutic agents.1
Typically, PKS modules act successively whereby a two-carbon
extension of a growing polyketide chain requires a minimum of
three domains: an acyltransferase (AT) that accepts an acyl-
coenzyme A extender unit and passes it to the acyl carrier
protein (ACP), and a ketosynthase (KS) that accepts a growing
chain from the ACP of the previous module and catalyzes
decarboxylative Claisen condensation to extend the poly-
ketide.1a In addition to KS, AT, and ACP, modules commonly
contain up to three additional domains that tailor the β-keto
functionality prior to the next round of chain extension. Finally,
the terminal module typically contains a thioesterase (TE)
domain located at the C-terminus that is responsible for
polyketide chain release.2 PikAIV is the final module in the
pikromycin (Pik) biosynthetic pathway, containing the core KS,
AT, and ACP domains, as well as a terminal TE domain
responsible for macrolactonization to form either 12-membered
10-deoxymethynolide (10-dml, 2) or the 14-membered macro-
cycle narbonolide (1, Scheme 1).3
In order to study isolated PKS modules in vitro,
investigations have relied upon electrophilic thioesters for
diffusive KS domain loading in lieu of transfer from an
upstream ACP. Historically, N-acetylcysteamine4 (NAC) has
been the thioester of choice, as it mimics the terminal portion
Received: November 14, 2014
© XXXX American Chemical Society
A
DOI: 10.1021/ja511743n
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Communication
appended to 2 by employing 8 with stoichiometric CuBr2.16,17
Attention was then focused on opening the macrolactone ring
known to be particularly recalcitrant toward hydrolysis.6,7 We
reasoned that a two-step global reduction and selective
oxidation would neatly side-step problematic hydrolysis
procedures. Excess LiAlH4 in THF proved sluggish and gave
a mixture of diastereomers, whereas reduction with DIBAL-H
proceeded smoothly to provide single stereoisomers. Selective
oxidation of triols 11 and 12 with TEMPO/PIDA18 adjusted
the oxidation state of the primary hydroxyl group to the
carboxylic acid, and the allylic alcohol to the desired α,β-
unsaturated ketone without oxidizing the homoallylic hydroxyl
group at C-11. With desired seco-acids 13 and 14 in hand, we
synthesized a series of hexaketides with a variety of thio- and
oxoesters. Next, the efficiency of PikAIV loading was evaluated
with each substrate in vitro using methylmalonyl N-acetyl-
cysteamine (MM-NAC)10,19 as the extender unit (Table 1).
Figure 1. Examples of previously studied native chain elongation
intermediates.7,11,12a Hydroxyl groups highlighted in red form
hemiketals, while those highlighted in green are unreactive.
Thus, to protect the pikromycin hexaketide for enhanced
stability to enable downstream biochemical studies, we
considered two distinct stabilization strategies: (i) a sterically
undemanding protecting group that would remain attached
throughout the catalytic cycle, and (ii) a protecting group that
could be removed in a controlled manner to provide the native
hexaketide immediately before use in enzymatic reactions.
Ultimately, a methyl ether protecting group was chosen to
satisfy (i) and a photocleavable 2-nitrobenzyloxymethyl ether
moiety (NBOM)14 was explored to address objective (ii).
An engineered strain of Streptomyces venezuelae ATCC 15439
provided 10-deoxymethynolide (2) from which seco-acids
could be generated (Scheme 2). Methylation of the C3-
Table 1. Evaluation of Stabilized Pik Hexaketides 15 with
a
PikAIV and MM-NAC Extender Unit
Scheme 2. Second-Generation7 Route to Stable Seco-acids
13 and 14 Derived from 10-dml (2)
a
Enzymatic reaction conditions: 1 mM Pik hexaketide, 20 mM MM-
NAC, 8 mM 2-vinylpyridine, 1 mM sodium metabisulfite and 25 mM
ascorbic acid with NBOM photolysis, 0.25 mol % PikAIV (2.5 μM), 4
h, rt. Conversion to 2 (R3 = H) or 9 (R3 = Me) and 1 (R3 = H) or 16
(R3 = Me) was monitored (HPLC) with data represented as the mean
standard deviation where n = 3. ND = not detected.
For NBOM protected substrates, 4-nitrophenol (entry 6)
and N-hydroxysuccinimide (entry 8) substrates decomposed
rapidly upon photolysis and subsequently gave low conversion
to macrolactones. In contrast, the corresponding hexaketide
thiophenol, benzyl mercaptan, and N-acetylcysteamine thio-
esters photolyzed smoothly (entries 2, 4, and 10, respectively)
though benzyl mercaptan thioesters (entry 4) gave lower
overall conversion to either macrolactone. Remarkably, we
observed significant selectivity in product formation depending
on the type of ester employed, where the hexaketide thiophenol
thioester (entry 2) demonstrated greater than 10:1 selectivity
for generation of narbonolide (1), while the hexaketide N-
acetylcysteamine thioester (entry 10) showed greater than 10:1
selectivity for 10-dml (2) production.
hydroxyl group under commonly employed etherification
conditions for sterically demanding alcohols proved challeng-
ing. Only trace etherification of the hindered C3-hydroxyl
group was detected in neat MeI/Ag2O, and decomposition to a
complex mixture of products was observed with excess
Me3OBF4/1,8-bis(dimethylamino)naphthalene in CH2Cl2.15
Ultimately, 1.2 equiv each of MeOTf and 2,6-di-tert-butyl-
pyridine in CH2Cl2/PhMe (2:1, 2 M) at 4 °C furnished the
desired product 9, albeit at an extended reaction time (72 h).
Concurrently, a 2-nitrobenzyloxymethyl (NBOM)14 group was
B
DOI: 10.1021/ja511743n
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Communication
In parallel experiments (Table 1), methylated substrates were
converted to methyl protected 10-dml (9) or methyl protected
narbonolide (16) albeit with selectivity shifted toward methyl
10-dml (9) and reduced overall conversions relative to native
substrates generated through initial NBOM photolysis. To
further establish the basis for macrolactone product distribution
imparted by the thio- or oxoester employed, we altered the
reaction conditions by excluding MM-NAC in PikAIV
reactions,6 and also by examining the excised Pik TE
domain,2,11 eliminating the possibility of generating narbono-
lide (1) or methyl protected narbonolide (19) (Table 2).
methyl protected 10-dml (9) (Table 2, entries 13−14). These
experiments demonstrate that thiophenol thioesters are
inefficient for direct macrolactonization utilizing either PikAIV
or the excised TE domain (Table 2, entries 1−4).
A series of reactions to explore substrate flexibility were
conducted with NBOM protected hexaketides and PikAIV
(excluding MM-NAC) or Pik TE without photolysis, and
yielded surprising conversion to NBOM protected 10-dml (10;
see Supporting Information Table S1). The same general
trends were observed with N-acetylcysteamine giving the
highest levels of conversion, followed by N-hydroxy-
succinimide, with aryl and benzyl thio- and oxoesters giving
uniformly low levels of product formation. Further exploration
with PikAIV (and MM-NAC included) failed to generate
NBOM protected narbonolide, and heat inactivated enzymes
also failed to produce either NBOM protected macrolactone.
Methyl ether mediated stabilization proved to be a viable
strategy for reactions with the excised Pik TE, though
suboptimal for full module catalysis. The NBOM protecting
group proved to effectively stabilize the Pik hexaketide prior to
in vitro reactions and should offer ready stabilization of a range
of advanced polyketide chain elongation substrates for PKS
functional studies. Moreover, its ability to undergo rapid and
efficient photoinduced deprotection demonstrates its role as an
effective tool for in vitro studies of PKS modules.
Table 2. Evaluation of Stabilized Pik Hexaketides 15 with
PikAIV and Pik TE without MM-NAC Extender Unit
a
Present
The work described herein provides further insight into
substrate loading parameters in modular PKS catalysis using
thiophenol thioesters,10 indicating that product formation can
be influenced by the type of substrate ester employed. While
reactions with the native, upstream acyl-ACP likely offer the
highest level of biosynthetic fidelity through docking domain
mediated chain transfer,20 thiophenol thioesters appear to be
highly effective for achieving full module catalysis. We propose
that substrate control can be explained, at least in part, through
the observation that thiophenol thioesters suffer diminished
conversion when incubated with the excised Pik TE domain
(Table 2, entries 2, 4) relative to the corresponding NAC
thioesters (Table 2, entries 18, 20). Moreover, we11 and
others21 have shown previously that PKS TE domains function
as flexible hydrolases when substrate macrolactonization cannot
be achieved. PikAIV is unique among terminal PKS modules as
the TE domain is able to form both 12- and 14-membered
rings. However, the likely result of errant TE acylation in typical
PKS modules would be substrate hydrolysis and decreased
conversion to the desired macrocyclic product. As such,
optimizing PKS substrates through substrate engineering may
minimize hydrolysis and other undesired side reactions.
a
Enzymatic reaction conditions: 1 mM Pik hexaketide, 8 mM 2-
Further exploration of these strategies in other type I PKS
pathways are underway with the expectation that substrate
engineering will serve to better emulate natural PKS function
when studying these complex enzymes in vitro, and for
chemoenzymatic synthesis of secondary metabolites.
vinylpyridine, 1 mM sodium metabisulfite and 25 mM ascorbic acid
with NBOM photolysis, 0.25 mol % PikAIV (2.5 μM) (∗) or 1 mol %
PikAIV (10 μM) (∗∗) or 1 % TE (10 μM), 4 h, rt. Conversion to 2
(R3 = H) or 9 (R3 = Me) was monitored (HPLC) with data
represented as the mean standard deviation where n = 3.
ASSOCIATED CONTENT
■
Incubation of hexaketides with PikAIV in the absence of
MM-NAC or with the excised TE domain demonstrated
variation in macrolactonization efficiency to 10-dml (2) or
methyl 10-dml (9) dictated by the ester employed (Table 2).
Consistent with PikAIV reactions where MM-NAC was
present, the N-acetylcysteamine thioester (Table 2, entries
17−20) gave the highest conversion to 10-dml (2) or methyl
protected 10-dml (9) under all conditions tested, with N-
hydoxysuccinimide esters providing moderate conversion to
S
* Supporting Information
Full experimental details and spectroscopic data. This material
is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*davidhs@umich.edu
C
DOI: 10.1021/ja511743n
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Communication
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This research was supported by Rackham Merit Predoctoral
Fellowships (D.A.H. and A.A.K.), American Foundation for
Pharmaceutical Education Predoctoral Fellowship (D.A.H.),
NIH T-32-CA009676 (A.A.K.), NIH Grant GM076477, and
the Hans W. Vahlteich Professorship (D.H.S.). We are grateful
to Dr. J. W. Kampf for small molecule X-ray crystallography.
REFERENCES
■
(1) (a) Fischbach, M. A.; Walsh, C. T. Chem. Rev. 2006, 106, 3468.
(b) Newman, D. J.; Cragg, G. M. J. Nat. Prod. 2012, 75, 311.
(2) Tsai, S.-C.; Lu, H.; Cane, D. E.; Khosla, C.; Stroud, R. M.
Biochemistry 2002, 41, 12598.
(3) Kittendorf, J. D.; Beck, B. J.; Buchholz, T. J.; Seufert, W.;
Sherman, D. H. Chem. Biol. 2007, 14, 944.
(4) (a) Cane, D. E.; Yang, C. C. J. Am. Chem. Soc. 1987, 109, 1255.
(b) Yue, S.; Duncan, J. S.; Yamamoto, Y.; Hutchinson, C. R. J. Am.
Chem. Soc. 1987, 109, 1253.
(5) Nonhydrolyzable NAC and CoA analogs have been employed
previously; see: (a) Tosin, M.; Betancor, L.; Stephens, E.; Li, W. M.;
Spencer, J. B.; Leadlay, P. F. ChemBioChem 2010, 11, 539. (b) Spiteller,
D.; Waterman, C. L.; Spencer, J. B. Angew. Chem., Int. Ed. 2005, 44,
7079.
(6) Wu, J.; He, W.; Khosla, C.; Cane, D. E. Angew. Chem., Int. Ed.
2005, 44, 7557.
(7) Aldrich, C. C.; Beck, B. J.; Fecik, R. A.; Sherman, D. H. J. Am.
Chem. Soc. 2005, 127, 8441.
(8) Aldrich et al. (ref 7) monitored 10-dml (2) formation using Pik
pentaketide (3) with PikAIII-TE and PikAIII/PikAIV constructs, but
not with Pik hexaketide (4) and PikAIV due to limitations of the assay
employed.
(9) Yin, Y.; Lu, H.; Khosla, C.; Cane, D. E. J. Am. Chem. Soc. 2003,
125, 5671.
(10) Hansen, D. A.; Rath, C. M.; Eisman, E. B.; Narayan, A. R.;
Kittendorf, J. D.; Mortison, J. D.; Yoon, Y. J.; Sherman, D. H. J. Am.
Chem. Soc. 2013, 135, 11232.
(11) Aldrich, C. C.; Venkatraman, L.; Sherman, D. H.; Fecik, R. A. J.
Am. Chem. Soc. 2005, 127, 8910.
(12) (a) Mortison, J. D.; Kittendorf, J. D.; Sherman, D. H. J. Am.
Chem. Soc. 2009, 131, 15784. (b) Harris, T. M.; Harris, C. M.
Tetrahedron 1977, 33, 2159.
(13) (a) Crosby, J.; Crump, M. P. Nat. Prod. Rep. 2012, 29, 1111.
(b) Whicher, J. R.; Dutta, S.; Hansen, D. A.; Hale, W. A.; Chemler, J.
A.; Dosey, A. M.; Narayan, A. R. H.; Hakansson, K.; Sherman, D. H.;
̊
Smith, J. L.; Skiniotis, G. Nature 2014, 510, 560.
(14) Schwartz, M. E.; Breaker, R. R.; Asteriadis, G. T.; deBear, J. S.;
Gough, G. R. Bioorg. Med. Chem. Lett. 1992, 2, 1019.
(15) Evans, D. A.; Ratz, A. M.; Huff, B. E.; Sheppard, G. S.
Tetrahedron Lett. 1994, 35, 7171.
(16) Sawada, D.; Ito, Y. Tetrahedron Lett. 2001, 42, 2501.
(17) Early attempts to install a 2-nitrobenzyl moiety onto the C3-
hydroxyl group of 10-dml (2) with 2-nitrobenzyl bromide, 2-
nitrobenzyl trifluoromethylsulfonate, or 2-nitrobenzyl trichloro-
acetimidate were met with failure.
(18) Epp, J. B.; Widlanski, T. S. J. Org. Chem. 1998, 64, 29.
(19) Pohl, N. L.; Gokhale, R. S.; Cane, D. E.; Khosla, C. J. Am. Chem.
Soc. 1998, 120, 11206.
(20) Broadhurst, R. W.; Nietlispach, D.; Wheatcroft, M. P.; Leadlay,
P. F.; Weissman, K. J. Chem. Biol. 2003, 10, 723.
(21) (a) Gokhale, R. S.; Hunziker, D.; Cane, D. E.; Khosla, C. Chem.
Biol. 1999, 6, 117. (b) Aggarwal, R.; Caffrey, P.; Leadlay, P. F.; Smith,
C. J.; Staunton, J. J. Chem. Soc., Chem. Commun. 1995, 15, 1519.
D
DOI: 10.1021/ja511743n
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX