Iterative chain elongation by a pikromycin monomodular polyketide synthase

Article abstract of DOI:10.1021/ja029974c

The unique ability of the pikromycin polyketide synthase (Pik PKS) to generate 12- and 14-membered ring macrolactones presents an opportunity to explore the fundamental processes of polyketide synthesis, specifically, the mechanistic details of the chain extension process. We have overexpressed and purified PikAIII and PikAIV and demonstrated the ability of these proteins to generate triketide lactone products using ¹⁴C-methylmalonyl-CoA as the sole substrate. Monomodular PikAIII generates TKL (1) when reacted alone, and synthesizes TKL (2) upon reaction in combination with PikAIV. Product formation remains dependent on the enzymatic decarboxylation of methylmalonyl-CoA and transfer of the acyl chain within the enzyme rather than acylation by propionyl-CoA from spontaneous decarboxylation. We propose that synthesis of TKL (1) by PikAIII involves iterative assembly of the triketide chain within a PikAIII homodimer analogous to the nonmodular type I PKS systems. Copyright

Full text of DOI:10.1021/ja029974c

Published on Web 03/28/2003
Iterative Chain Elongation by a Pikromycin Monomodular Polyketide
Synthase
Brian J. Beck,† Courtney C. Aldrich,† Robert A. Fecik,‡ Kevin A. Reynolds,§ and David H. Sherman*,†
Department of Microbiology and Biotechnology Institute, and Department of Medicinal Chemistry,
UniVersity of Minnesota, Minneapolis, Minnesota, and
Department of Medicinal Chemistry and Institute for Structural Biology and Drug DiscoVery,
Virginia Commonwealth UniVersity, Richmond, Virginia 23284
Received December 31, 2002; E-mail: david-s@biosci.umn.edu
Polyketides are a diverse class of natural products that possess
antibiotic, anticancer, and other biological activities.1 Biosynthesis
of polyketides and fatty acids share a common mechanism where
condensation of simple carboxylic acids such as acetate and
propionate are catalyzed by complex multienzyme systems, the
polyketide synthases (PKSs) and fatty acid synthases (FASs),
respectively.2 In bacterial type II FAS and some type II PKS systems
the keto-acid condensation and acyl transfer activities (e.g.,
malonyltransferase) are expressed as separate proteins that function
iteratively until the elongated, processed intermediate is released
from an acyl carrier protein (ACP). Eukaryotic type I FAS and
PKS (e.g., fungal) as well as prokaryotic type I PKS systems
combine up to six catalytic domains (including ketoacyl synthase
(KS), acyltransferase (AT), dehydratase (DH), enoylreductase (ER),
acyl carrier protein (ACP), and thioesterase (TE)) into multifunc-
tional polypeptides. The type I FASs catalyze iterative condensation/
reductive processing reactions to provide palmitic acid. Likewise,
fungal type I PKSs are composed of a single multifunctional protein
that catalyzes iterative chain elongation and processing, leading to
polyketide products such as lovastatin.3 In contrast, the modular
design of bacterial type I PKSs mediates successive elongation
where each distinct set of condensation/reductive processing
activities or “modules” catalyzes a single elongation cycle before
passing the intermediate chain to the next PKS polypeptide. Thus,
it is the number of modules comprising a prokaryotic type I PKS
that determines the length, functionality, and stereochemistry of
the polyketide product.
The mechanistic basis for successive chain elongation in bacterial
type I PKSs has been investigated using purified proteins of the
6-deoxyerythronolide B synthase (DEBS). “Docking” of the
domains at the NH2 and COOH ends of these bimodular proteins
(DEBS1, -2, and -3) have been proposed to promote the sequential
interaction of PKS modules and the channeling of polyketide chain
intermediates.4-7 Although modular PKSs (e.g., DEBS) have
evolved to function successively, several recent reports have
revealed an additional ability to perform polyketide biosynthesis
iteratively. Iterative processing by a type I modular PKS was first
observed in vivo by Leadlay and co-workers who determined that
“stuttering” of the elongating chain intermediate by DEBS2 module
4 leads to the formation of a 16-membered ring macrolactone.8
Interestingly, the recent identification of bacterial type I-like PKSs
for calicheamicin9 and C-102710 suggests that an iterative mech-
anism is responsible for synthesis of these polyketide-derived
enediyne natural products.
The chain elongation/processing functions of modular PKSs have
been studied in vitro using N-acetylcysteamine (NAC) diketide
thioesters as substrates for native bimodular DEBS PKS proteins,
as well as DEBS proteins that have been re-engineered and purified
as nonnative monomodular enzymes.4-7 This work has shown the
ability of unnatural DEBS monomodules to catalyze a single chain
elongation step, resulting in the production of tri- and tetraketide
lactones.
The intriguing observation of in vivo stuttering as well as the in
vitro analysis of an ”unnatural” DEBS monomodular type I PKS
motivated our effort to investigate an iterative function of the native
monomodular type I PKSs from the pikromycin (Pik) PKS11 of
Streptomyces Venezuelae, PikAIII (module 5) and PikAIV (module
6). We propose to define iterative function as a monomodular PKS
that catalyzes a minimum of two condensation reactions. To explore
this possibility in the Pik PKS, we sought to identify iterative
activity using purified PikAIII and PikAIV proteins and exploit
the observation that PKS modules may be able to perform “self-
priming”.12 In a crude extract, DEBS3 was shown to produce a
triketide lactone (TKL) (1) in the absence of an acceptable
elongation intermediate or other DEBS proteins. In this case, TKL
production was dependent on the decarboxylation of methylmalo-
nyl-CoA (or methylmalonyl-ACP) and “priming” of a KS domain
with a propionyl unit that could be extended by modules 5 and 6
of DEBS3.12,13 Thus, we proceeded to investigate the self-priming
ability of PikAIII with the expectation that iterative polyketide
assembly would produce a triketide chain released by spontaneous
lactonization, yielding TKL (1). Moreover, we reasoned that in vitro
pairing of PikAIII and PikAIV enzymes would generate TKL (2)
(Figure 1).
PikAIII and PikAIV were individually coexpressed in Escheri-
chia coli BL21 (DE3) with the Bacillus subtilis sfp gene encoding
a phosphopantetheine transferase14 for posttranslational modification
of the ACP domains. The PikAIII protein was engineered with a
NH2-6×HIS tag, and PikAIV was modified to contain a COOH-
6×HIS tag. Both proteins were purified by Ni-affinity chromatog-
raphy and were assayed with 2-[methyl-14C]-methylmalonyl-CoA
as the substrate. The reaction products, 1 and 2, were visualized
by radio-TLC and rigorously identified using synthetic standards.
We found that PikAIII indeed operates iteratively as a single
multifunctional protein as shown by its ability to synthesize TKL
(1). Reaction of 2-[methyl-14C]-methylmalonyl-CoA with PikAIII
and PikAIV yielded TKL (2), consistent with our hypothesis that
self-priming of PikAIII allows elongation by PikAIII and PikAIV.
The rate of synthesis (kcat) for 1 and 2 was determined to be
100-fold lower than the elongation rates (kcat ) 0.005-0.01 min-1
)
† Department of Microbiology and Biotechnology Institute, University of
Minnesota.
observed for PikAIII and PikAIV primed with a N-acetylcysteamine
(2S,3R)-2-methyl-3-hydroxypentanoic diketide thioester (B. Beck,
‡ Department of Medicinal Chemistry, University of Minnesota.
§ Virginia Commonwealth University.
9
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J. AM. CHEM. SOC. 2003, 125, 4682-4683
10.1021/ja029974c CCC: $25.00 © 2003 American Chemical Society

C O M M U N I C A T I O N S
Figure 1. Proposed self-priming mechanism for triketide lactone formation by PikAIII and PikAIV.
unpublished results). As expected, PikAIV alone did not produce
a TKL product because the enzyme lacks a KR domain required
to generate the hydroxyl group necessary for nucleophilic attack
leading to lactonization. Reaction of these proteins with radiolabeled
[1-14C]-propionyl-CoA and unlabeled methylmalonyl-CoA sub-
strates failed to produce radiolabeled TKLs, indicating that the
priming reaction requires enzymatic decarboxylation of methyl-
malonyl-CoA.
The finding that a type I monomodular PKS can perform iterative
polyketide synthesis provides another evolutionary link between
the diverse classes of polyketide biosynthetic systems. This
characteristic may reflect the functional remnant of a progenitor
FAS or PKS enzyme that has subsequently evolved into a bacterial
type I modular system. Investigations focused on the evolution of
these unique mechanisms of small-molecule assembly may provide
new strategies to generate greater diversity of polyketide compounds
through combinatorial biosynthesis.
The processing of a polyketide chain intermediate by a PKS
module has been shown to proceed through a homodimeric active
site.13,15,16 Accordingly, once primed with a propionyl unit, the first
extension reaction leading to diketide formation by PikAIII would
be predicted to follow the established pathway. However, the final
extension reaction producing a triketide intermediate could occur
through two possible mechanisms. Iterative cycling of the diketide
from ACP5 to the KS5 domain on the same homodimer would
precede condensation with a new methylmalonyl-CoA extender unit
producing the triketide chain elongation intermediate. Alternatively,
an inter-modular transfer could occur, whereby a PikAIII ho-
modimer with a diketide attached to the ACP5 domain reacts with
a separate PikAIII homodimer loaded with methylmalonyl-CoA.
To distinguish between these two mechanisms we reasoned that
polyketide chain transfer between PikAIII and PikAIV would be
the favored path for the diketide intermediate rather than inter-
enzyme transfer between PikAIII homodimers. Supporting this
assumption is the demonstration that docking domains between
interacting modules ensure sequential interaction of PKS modules
and channeling of substrates.4-6 Additionally, the purified PikAIII
protein contains an NH2-6×HIS tag sequence (13 amino acids) that
may interfere with the interaction between PikAIII homodimers,
whereas the COOH-terminal location of the PikAIV HIS tag
presumably does not interfere with PikAIII-PikAIV interaction.
When 2-[methyl-14C]-methylmalonyl-CoA was reacted with
PikAIII and PikAIV, only TKL (2) was produced. The exclusive
synthesis of TKL (2) provides experimental evidence that a diketide
intermediate formed by self-priming of PikAIII is preferentially
elongated by PikAIV rather than being iteratively processed by
PikAIII. However, when PikAIII was incubated with a mutant form
of PikAIV with KS and ACP catalytic domains inactivated by site-
directed mutagenesis, no inhibition of TKL (1) synthesis was
observed. Since PikAIII would be expected to interact preferentially
with PikAIV rather than another PikAIII homodimer, the synthesis
of comparable amounts of TKL (1) by PikAIII alone and in the
presence of inactive PikAIV is consistent with the proposed iterative
processing of the diketide chain intermediate by PikAIII. Thus, the
inability to transfer to a catalytically active module results in the
iterative processing of the polyketide chain by PikAIII.
Acknowledgment. This research was supported by NIH Grant
GM48562 and NSF (NSF/BES-0118926) to D.H.S.
Supporting Information Available: Construction of overexpression
vectors, protein purification methods, and assay conditions; radio-TLC
data of TKL product formation as well as the synthetic schemes used
to make reference compounds (PDF). This material is available free of
charge via the Internet at http://pubs.acs.org.
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