Mechanism and stereospecificity of a fully saturating polyketide synthase module: Nanchangmycin synthase module 2 and its dehydratase domain

Article abstract of DOI:10.1021/ja1073432

Recombinant nanchangmycin synthase module 2 (NANS module 2), with the thioesterase domain from the 6-deoxyerythronolide B synthase (DEBS TE) appended to the C-terminus, was cloned and expressed in Escherichia coli. Incubation of NANS module 2+TE with (±)-2-methyl-3-keto-butyryl-N-acetylcysteamine thioester (1), the SNAC analog of the natural ACP-bound substrate, with methylmalonyl-CoA (MM-CoA) in the absence of NADPH gave 3,5,6-trimethyl-4- hydroxypyrone (2), identified by direct comparison with synthetic 2 by radio-TLC-phosphorimaging and LC-ESI(+)-MS-MS. The reaction showed k _cat 0.5 ± 0.1 min^-1 and K_m(1) 19 ± 5 mM at 0.5 mM MM-CoA and k_cat(app) 0.26 ± 0.02 min^-1 and K_m(MM-CoA) 0.11 ± 0.02 mM at 8 mM 1. Incubation in the presence of NADPH generated the fully saturated triketide chain elongation product as a 5:3 mixture of (2S,4R)-2,4-dimethyl-5-ketohexanoic acid (3a) and the diastereomeric (2S,4S)-3b. The structure and stereochemistry of each product was established by comparison with synthetic 3a and 3b by a combination of radio-TLC-phosphorimaging and LC-ESI(-)-MS-MS, as well as chiral capillary GC-MS analysis of the corresponding methyl esters 3a-Me and 3b-Me. The recombinant dehydratase domain from NANS module 2, NANS DH2, was shown to catalyze the formation of an (E)-double bond by syn-dehydration of the ACP-bound substrate anti-(2R,3R,4S,5R)-2,4-dimethyl-3,5-dihydroxyheptanoyl-ACP6 (4), generated in situ by incubation of (2S,3R)-2-methyl-3-hydroxypentanoyl-SNAC (5), methylmalonyl-CoA, and NADPH with the recombinant [KS6][AT6] didomain and ACP6 from DEBS module 6 along with the ketoreductase from the tylactone synthase module 1 (TYLS KR1). These results also indirectly establish the stereochemistry of the reactions catalyzed by the KR and enoylreductase (ER) domains of NANS module 2.

Full text of DOI:10.1021/ja1073432

Published on Web 10/06/2010
Mechanism and Stereospecificity of a Fully Saturating Polyketide Synthase
Module: Nanchangmycin Synthase Module 2 and Its Dehydratase Domain
Xun Guo,† Tiangang Liu,†,‡,§ Chiara R. Valenzano,† Zixin Deng,‡ and David E. Cane*,†
Department of Chemistry, Box H, Brown UniVersity, ProVidence, Rhode Island 02912-9108, and Laboratory of
Microbial Metabolism and School of Life Sciences & Biotechnology, Shanghai Jiaotong UniVersity,
Shanghai 200030, China
Received August 15, 2010; E-mail: David_Cane@brown.edu
Abstract: Recombinant nanchangmycin synthase module 2 (NANS
module 2), with the thioesterase domain from the 6-deoxyerythro-
nolide B synthase (DEBS TE) appended to the C-terminus, was
cloned and expressed in Escherichia coli. Incubation of NANS
module 2+TE with (()-2-methyl-3-keto-butyryl-N-acetylcysteam-
ine thioester (1), the SNAC analog of the natural ACP-bound
substrate, with methylmalonyl-CoA (MM-CoA) in the absence of
NADPH gave 3,5,6-trimethyl-4-hydroxypyrone (2), identified by
direct comparison with synthetic 2 by radio-TLC-phosphorimaging
and LC-ESI(+)-MS-MS. The reaction showed kcat 0.5 ( 0.1 min-1
and Km(1) 19 ( 5 mM at 0.5 mM MM-CoA and kcat(app) 0.26 (
0.02 min-1 and Km(MM-CoA) 0.11 ( 0.02 mM at 8 mM 1.
Incubation in the presence of NADPH generated the fully
saturated triketide chain elongation product as a 5:3 mixture of
(2S,4R)-2,4-dimethyl-5-ketohexanoic acid (3a) and the diastereo-
meric (2S,4S)-3b. The structure and stereochemistry of each
Figure 1. (A) Nanchangmycin. (B) Chain elongation, ketoreduction,
dehydration, and enoyl reduction catalyzed by NANS module 2.
product was established by comparison with synthetic 3a and 3b
by a combination of radio-TLC-phosphorimaging and LC-ESI(-)-
MS-MS, as well as chiral capillary GC-MS analysis of the
corresponding methyl esters 3a-Me and 3b-Me. The recombinant
dehydratase domain from NANS module 2, NANS DH2, was
shown to catalyze the formation of an (E)-double bond by syn-
dehydration of the ACP-bound substrate anti-(2R,3R,4S,5R)-2,4-
dimethyl-3,5-dihydroxyheptanoyl-ACP6 (4), generated in situ by
incubation of (2S,3R)-2-methyl-3-hydroxypentanoyl-SNAC (5),
methylmalonyl-CoA, and NADPH with the recombinant [KS6][AT6]
didomain and ACP6 from DEBS module 6 along with the
ketoreductase from the tylactone synthase module 1 (TYLS KR1).
These results also indirectly establish the stereochemistry of the
reactions catalyzed by the KR and enoylreductase (ER) domains
of NANS module 2.
biochemical reactions remain covalently tethered to acyl carrier
protein domains throughout the entire cycle, with the end product
of each module being passed to the appropriate downstream module
for the next round of chain elongation and modification. Since none
of the intermediates are released from the modular PKS prior to
the final thioesterase (TE)-catalyzed hydrolysis or lactonization
reaction, the structure and stereochemistry of the product generated
by each module are normally deduced from the structure of the
corresponding segment of the ultimate, full-length polyketide. By
contrast, the structure and stereochemistry of the specific intermedi-
ates of a single round of chain elongation and modification, which
may not be evident in the structure of the eventually formed full-
length polyketide, are usually inferred from the specific domain
composition of the responsible module, by analogy to the well-
established mode of action of the individual chain modifying
proteins of a Type II fatty acid synthase, according to which a
ketosynthase (KS) domain is expected to generate a ꢀ-ketoacyl-
ACP intermediate, a ketoreductase (KR) domain, a ꢀ-hydroxyacyl-
ACP intermediate, a dehydratase (DH) domain, a 2,3-enoyl-ACP
intermediate, and an enoylreductase (ER) domain, the fully saturated
acyl-ACP chain elongation product.1b These models have been
validated by targeted deletion of one or more catalytic domains of
the PKS and the determination of the structure of the resulting
modified polyketide product.2
Modular polyketide synthases (PKSs) are exceptionally large,
multifunctional proteins responsible for the elaboration of an
enormous variety of complex, branched-chain, polyoxygenated
polyketides of microbial origin, many of which play important roles
in human and veterinary medicine as antibiotics, immunosuppres-
sants, or antitumor, antifungal, or antiparasitic agents.1 Each PKS
protein is composed of one or more modules, assemblages of three
or more catalytic centers, termed domains, that together are
responsible for a single round of polyketide chain elongation and
functional group modification. In the assembly line biosynthesis
catalyzed by a modular PKS, the intermediates of the individual
An exceptionally powerful tool for directly probing the mech-
anism of polyketide chain elongation and for determination of the
structures of polyketide chain elongation intermediates has been
to utilize recombinant forms of individual PKS modules as well as
their constituent KS, AT, KR, DH, and ACP domains.3,4 Unfor-
tunately, no PKS module containing the full complement of active
† Brown University.
‡ Shanghai Jiaotong University.
§ Current address: School of Pharmacy, Wuhan University, 185 Donghu Rd.,
Wuhan 430071, China.
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C O M M U N I C A T I O N S
Scheme 2. (A) Stereochemistry of NANS DH2-Catalyzed
KS, KR, DH, and ER domains has yet been expressed in Vitro as
an active single modular protein. To address this problem, we chose
to examine the nanchangmycin synthase (NANS) which has been
shown to be responsible for the multistep biosynthesis of the
polyketide backbone of the anticoccidial polyether nanchangmycin
(Figure 1A).5 Within this PKS, the monomodular protein NANS
module 2, which harbors the full complement of ꢀ-carbon-
processing KR, DH, and ER domains, is predicted to catalyze the
second round of polyketide chain elongation, resulting in the
formation of a saturated (2R,4S)-2,4-dimethyl-5-ketohexanoyl-ACP
triketide intermediate (Figure 1B).5,6 We therefore cloned and
expressed recombinant NANS module 2, appending the TE domain
of the 6-deoxyerythronolide B synthase (DEBS) to the C-terminus
so as to promote release of the anticipated NANS ACP2-bound
triketide products and to thus allow multiple catalytic turnovers.
Incubation of recombinant NANS module 2+TE with the
N-acetylcysteamine thioester of (()-2-methyl-3-ketobutyric acid (1),
the SNAC analog of the natural (2S)-2-methyl-3-ketobutyryl-ACP1
diketide produced by NANS module 1, with methylmalonyl-CoA
(MM-CoA) in the absence of NADPH, afforded 3,5,6-trimethyl-
4-hydroxypyrone (2), derived by TE- or buffer-catalyzed cyclization
of the presumed unreduced (2R,4S)-2,4-dimethyl-3,5-diketohex-
anoyl-ACP2 intermediate, as established by direct comparison of
the enzymatically generated product with authentic synthetic 27a
by both radio-TLC-phosphorimaging and LC-ESI(+)-MS-MS
(Scheme 1, reaction A). The reaction exhibited kcat 0.5 ( 0.1 min-1
and Km(1) 19 ( 5 mM at 0.5 mM MM-CoA and kcat(app) 0.26 (
0.02 min-1 and Km(MM-CoA) 0.11 ( 0.02 mM at 8 mM 1.8 When
the incubation was carried out in the presence of NADPH, the
resulting product consisted instead of a 5:3 mixture of the natural
reduced (2S,4R)-2,4-dimethyl-5-ketohexanoic acid (3a) and the
diastereomeric (2S,4S)-3b accompanied by trace amounts of pyrone
2 (Scheme 1, reaction B), as established by comparison with
authentic synthetic standards (Supporting Information)7 by a
combination of TLC-phosphorimaging and LC-ESI(-)-MS-MS.
The diastereomeric composition of the ketotriketide acid product
3a and 3b was unambiguously established by chiral capillary GC-
EI(+)-MS analysis of the derived methyl esters 3a-Me and 3b-
Me and direct comparison with a synthetic mixture of established
absolute configuration. The formation of both the (4R)- and (4S)-
methyl diastereomers of 3 indicates that recombinant NANS module
2 does not cleanly discriminate between the two enantiomers of
the SNAC analogue of its natural substrate. This apparent lack of
substrate stereoselectivity would not be critical to the natural
assembly line biosynthesis in the intact nanchangmycin PKS since
only the correct stereoisomer of the diketide intermediate, (2S)-1-
ACP1, will be delivered by NANS module 1 to downstream NANS
KS2.
Dehydration and (B) NANS DH2 Syn-Dehydration Mechanism
The requisite anti-(2R,3R)-2-methyl-3-hydroxyacyl-ACP substrate
4 was generated, as previously described, by incubation of (2S,3R)-
2-methyl-3-hydroxypentanoyl-SNAC (5), methylmalonyl-CoA, and
NADPH with the recombinant [KS6][AT6] didomain and ACP6
from DEBS module 6 along with the KR1 reductase from the
tylactone synthase (TYLS) module 1 (Scheme 2A).4c,e,9 Coincu-
bation with NANS DH2 resulted in formation, after basic hydrolysis
and acidification, of (4R,5R)-(E)-2,4-dimethyl-5-hydroxy-2-hep-
tenoic acid (6), as established by direct comparison with synthetic
6 by TLC-phosphorimaging and chiral GC-EI(+)-MS analysis of
the derived methyl ester (6-Me). A control incubation (Supporting
Information) in which DEBS KR6 was substituted for TYLS KR1
so as to generate the corresponding syn-(2R,3S)-2-methyl-3-
hydroxyacyl-ACP6 stereoisomer9a did not yield any dehydration
product, generating only the derived triketide lactone, as expected.9a
Taken together these results firmly establish that the NANS DH2
domain (a) utilizes an ACP-bound anti-(2R,3R)-2-methyl-3-hy-
droxyacyl substrate and (b) produces an (E)-2-methylenoyl-ACP
product by a (c) net syn dehydration.10 NANS DH2 contains the
universally conserved active site motifs HXXXDXXXXP and
DXXXQ, in which H49 (H977 of full-length NANS module 2) is
thought to serve as the active site base, with D219, acting as the
general acid to donate a proton to the 3-hydroxyl leaving group,
resulting in a net syn elimination of water (Scheme 2B).4d,11 We
have also established the same specificity for anti-(2R,3R)-2-methyl-
3-hydroxyacyl-ACP substrates and syn dehydration stereochemistry
for both the DEBS DH4 and TYLS DH2 domains.9b,c It can
therefore be predicted with confidence that NANS KR2, the natural
partner of NANS DH2, will generate the anti-(2R,3R)-2-methyl-
3-hydroxy-acyl-ACP2 triketide intermediate from the corresponding
KS2-generated (2R)-2-methyl-3-ketoacyl-ACP2 substrate, while the
downstream NANS ER2 domain will reduce the (E)-2-methylenoyl-
ACP2 unsaturated triketide, with capture of a proton on the 2re-
face of the double bond, to give a reduced product with the observed
(2S)-methyl configuration. We have thus established the stereo-
chemistry of every step of the NANS module 2-catalyzed chain
elongation and saturation process, with the exception of the still
cryptic stereochemistry of ER2-catalyzed hydride addition to C-3
of the unsaturated triketide. Experiments are in progress to
characterize directly NANS KR2, whose structural and catalytic
subdomains flank the ER2 domain.
Scheme 1. Incubation of NANS Module 2+TE with 1 and
Methylmalonyl-CoA (A) without NADPH and (B) with NADPH
To establish the structure and stereochemistry of the individual
triketide intermediates generated by NANS module 2, we used a
synthetic gene with codons optimized for expression in E. coli to
express NANS DH2 domain as a recombinant standalone protein
corresponding to the region from G929 to G1241 of NANS module
2 which is flanked by the corresponding AT2 and KR2 domains.4d,5
Acknowledgment. This work was supported by the NIH
(GM22172, D.E.C.), the Ministry of Science and Technology of
China (973 and 863, Z.D.), and the National Science Foundation
of China (Z.D.). We thank Tun-Li Shen for assistance with mass
spectrometry and Russell Hopson for assistance with NMR.
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C O M M U N I C A T I O N S
Methods Enzymol. 2009, 459, 187–214. (c) Genbank Accession No.
AF521085.
Supporting Information Available: Experimental procedures,
phosphorimaging, GC-MS, and LC-MS data. This material is available
free of charge via the Internet at http://pubs.acs.org.
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also generate the same 2,4-dimethyl-5-ketoacyl-ACP triketide intermediate,
both display similar architecture and a high degree of amino acid sequence
similarity to NANS module 2. Cf. (a) Oliynyk, M.; Stark, C. B.; Bhatt, A.;
Jones, M. A.; Hughes-Thomas, Z. A.; Wilkinson, C.; Oliynyk, Z.;
Demydchuk, Y.; Staunton, J.; Leadlay, P. F. Mol. Microbiol. 2003, 49,
1179–1190. (b) Harvey, B. M.; Mironenko, T.; Sun, Y.; Hong, H.; Deng,
Z.; Leadlay, P. F.; Weissman, K. J.; Haydock, S. F. Chem. Biol. 2007, 14,
703–714.
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