Enzymatic synthesis of resorcylic acid lactones by cooperation of fungal iterative polyketide synthases involved in hypothemycin biosynthesis

Article abstract of DOI:10.1021/ja100060k

Hypothemycin is a macrolide protein kinase inhibitor from the fungus Hypomyces subiculosus. During biosynthesis, its carbon framework is assembled by two iterative polyketide synthases (PKSs), Hpm8 (highly reducing) and Hpm3 (nonreducing). These were heterologously expressed in Saccharomyces cerevisiae BJ5464-NpgA, purified to near homogeneity, and reconstituted in vitro to produce (6′S,10′S)-trans-7′,8′-dehydrozearalenol (1) from malonyl-CoA and NADPH. The structure of 1 was determined by X-ray crystallographic analysis. In the absence of functional Hpm3, the reducing PKS Hpm8 produces and offloads truncated pyrone products instead of the expected hexaketide. The nonreducing Hpm3 is able to accept an N-acetylcysteamine thioester of a correctly functionalized hexaketide to form 1, but it is unable to initiate polyketide formation from malonyl-CoA. We show that the starter-unit:ACP transacylase (SAT) of Hpm3 is critical for crosstalk between the two enzymes and that the rate of biosynthesis of 1 is determined by the rate of hexaketide formation by Hpm8.

Full text of DOI:10.1021/ja100060k

Published on Web 03/11/2010
Enzymatic Synthesis of Resorcylic Acid Lactones by Cooperation of Fungal
Iterative Polyketide Synthases Involved in Hypothemycin Biosynthesis
Hui Zhou,† Kangjian Qiao,† Zhizeng Gao,‡ Michael J. Meehan,§ Jesse W.-H. Li,‡ Xiling Zhao,§
Pieter C. Dorrestein,§ John C. Vederas,*,‡ and Yi Tang*,†
Department of Chemical and Biomolecular Engineering, UniVersity of California, Los Angeles, California 90095,
Department of Chemistry, UniVersity of Alberta, Edmonton, AB, Canada T6G 2G2, and Skaggs School of Pharmacy
and Pharmaceutical Sciences and Departments of Pharmacology, Chemistry, and Biochemistry,
UniVersity of California, San Diego, California 92093
Received January 5, 2010; E-mail: john.vederas@ualberta.ca; yitang@ucla.edu
Polyketides of fungal origin represent an important family of
natural products and display a wide range of biological activities.1
Among them are the resorcylic acid lactones (RALs), which are
12- or 14-member macrolides that contain a resorcinol carboxylate
moiety, for example, zearalenone,2 hypothemycin,3 and radicicol.3,4
Hypothemycin (Figure 1) irreversibly inhibits a subset of kinases
at nanomolar concentrations, including mitogen-activated protein
kinase (MEK) and human ERK2.5 Biosynthesis of fungal polyketides
often utilizes iterative type-I polyketide synthases (PKSs) that have
single copies of domains that are used repeatedly in a highly
programmed fashion.6 The representative hypothemycin (Hpm) PKS
from Hypomyces subiculosus contains two iterative PKSs, Hpm8
and Hpm3.3 The highly reducing PKS Hpm8 is proposed to
synthesize the reduced hexaketide (7S,11S,2E,8E)-7,11-dihydroxy-
dodeca-2,8-dienoate (2), which is transferred downstream to the
nonreducing PKS Hpm3. Hpm3 is proposed to extend 2 to a
nonaketide, after which regioselective cyclization and macrolac-
tonization affords 7′,8′-dehydrozearalenol (1). Heterologous expres-
sion of both Hpm PKS genes (but neither gene alone) resulted in
production of a product assigned as the 6′R epimer of 1.3
(Figure 2A). Addition of NADPH to Hpm8 with malonyl-CoA
yielded the tetraketide and pentaketide R-pyrones 4 and 5,
respectively. The structures of 4 and 5 were confirmed by
comparison to authentic standards made by chemical synthesis. Both
4 and 5 are shunt products that arise from derailment of tailoring
steps during later stages of chain extension, and they are offloaded
by Hpm8 via lactonization, in similar fashion as LovB.6b Hexaketide
2 was not detected in the reaction mixture, indicating that offloading
of this product requires the partner protein Hpm3. In contrast,
incubation of Hpm3 with acetyl- and malonyl-CoA did not produce
any detectable polyketide products, suggesting the inability of this
protein to initiate polyketide biosynthesis as a stand-alone PKS.
The two Hpm PKSs are therefore proposed to be chemically “modular”,
with each iterative PKS specializing in generating polyketide backbones
having different structural features. Crosstalk between the two PKSs
transfers the correct intermediate 2 from the upstream Hpm8 to the
downstream Hpm3 and is a crucial step in the biosynthesis. The
protein-protein interaction between two megasynthases is similarly
required in the biosynthesis of other RALs, the aflatoxin precursor
norsolorinic acid7 and the recently discovered asperfuranone.8 In this study,
we have accomplished the complete reconstitution of Hpm8-Hmp3
activities in vitro to synthesize 1 and shown that the N-terminal starter-
unit:ACP transacylase (SAT) domain plays a key role in facilitating acyl
transfer of 2 bound to Hpm8.
Both Hpm8 (254 kDa) and Hpm3 (223 kDa) were solubly
expressed with C-terminal hexahistidine tags in Saccharomyces
cereVisiae BJ5464-NpgA6b and purified to near homogeneity with
yields of 1.5 mg/L and 2 mg/L, respectively (Figure S1 in the
Supporting Information). The Hpm8 acyl-carrier protein (ACP)
domain was mapped with Fourier transform mass spectrometry
(FTMS), and phosphopantetheinylation was confirmed by Ppant
ejection assays (Figures S2-S4).9 The activities of the minimal
PKS domains of Hpm8 were first probed in the presence of 2 mM
malonyl-CoA without NADPH. LC-MS analysis of the organic
extract showed accumulation of triketide R-pyrone 3, which is
formed through spontaneous cyclization of the unreduced triketide
Figure 1. Biosynthesis of (6′S,10′S)-7′,8′-dehydrozearalenol (1) by Hpm8
and Hpm3. Hpm8 consists of ketosynthase (KS), malonyl-CoA:ACP
acyltransferase (MAT), dehydratase (DH), core, enoylreductase (ER),
ketoreductase (KR), and acyl-carrier protein (ACP); Hpm3 consists of
starter-unit:ACP transacylase (SAT), KS, MAT, product template (PT),
ACP, and thioesterase (TE). Hpm3 can accept a hexaketide starter unit either
from 2-Hpm8 via the SAT domain or from 2-SNAC directly.
Upon addition of 2 mM NADPH and malonyl-CoA to a reaction
mixture containing both Hpm8 and Hpm3, a new peak (m/z 317, [M
- H]-) with a UV absorbance pattern characteristic of resorcylic acid
emerged in the organic extract [retention time (RT) ) 23.3 min] (Figure
2B). Performing the Hpm8-Hpm3 reaction in the presence of
[2-13C]malonate and the MatB system10 yielded a peak at the same
RT and m/z 326 ([M - H]-). The increase of 9 mass units is consistent
with the expected nonaketide backbone, which is shown below to be
1. In order to obtain sufficient 1 for structural characterization, the
† University of California, Los Angeles.
‡ University of Alberta.
§ University of California, San Diego.
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10.1021/ja100060k  2010 American Chemical Society

C O M M U N I C A T I O N S
synthesis of 1 (Figure S7). These results illustrate that the SAT-mediated
protein-protein interactions are highly specific.
To probe whether an alternative priming pathway exists for
Hpm3 in the presence of small-molecule precursors, we chemically
synthesized the N-acetylcysteamine thioester hexaketide, 2-SNAC
(Scheme S3). LC-MS analysis revealed that 1 was produced by
Hpm3 in the presence of 2-SNAC in high yield (Figure 2D). The
Hpm3-SAT0 mutant was similarly primed with 2-SNAC and
produced 1 in comparable yield to the wild type (Figure 2E). This
result shows that although the SAT domain is necessary for protein
interaction, small-molecule precursors can be directly captured by
Hpm3 to initiate biosynthesis of 1. This SAT-independent pathway
is probably facilitated by the direct priming of the KS domain
(Figure 1). This is consistent with the SAT domains being
dispensable in the precursor-directed biosynthesis of polyketide
analogues by PKS1312 and Gibberella fujikuroi PKS4.13
Synthesis and structural confirmation of 1 have demonstrated that
all ∼30 catalytic steps in the synthesis of 1 were reconstituted, including
(i) formation of the reduced ketide acyl intermediate 2 by Hpm8, (ii)
communication between the two proteins and successful transfer of
the acyl intermediate, and (iii) correct processing of the reduced
intermediate by Hpm3. The presence of the single RAL 1 also
illustrates that only the completed hexaketide can be transferred to
the downstream enzyme and elongated into an RAL. Despite this, free
2-SNAC can be transformed to 1 by Hpm3.
Figure 2. Reconstitution of the Hpm iterative PKSs. HPLC analysis (300
nm) of polyketides synthesized by (A) Hpm8, (B) Hpm8 and Hpm3, (C) Hpm8
and Hpm3-SAT0, (D) Hpm3 with 2 mM 2-SNAC, and (E) Hpm3-SAT0
with 2 mM 2-SNAC. Megasynthases were added to 10 µM. In reactions
(A-C), 2 mM NADPH and malonyl-CoA were added to PBS buffer (pH 7.4).
BJ5464-NpgA host was transformed with expression plasmids for both
Hpm8 and Hpm3 and cultured as described by Reeves et al.3 The extract
of the culture afforded a compound with the same RT on a chiral HPLC
column and the UV absorbance and mass fragmentation pattern as the in
vitro-synthesized compound shown in Figure 2B. Purification and NMR
analysis supported the structure of 1. It was then crystallized, and
subsequent X-ray analysis showed 1 to have 6′S,10′S stereochemistry
(Figure S5). Although the 10′S stereochemistry was expected, as the
absolute stereochemistry of Hpm is known,5b the 6′S configuration was
surprising. If the KR domain were to catalyze all of the the keto reduction
steps with the same facial stereochemistry, one would expect the final
compound to be the 6′R,10′S diastereomer, as initially proposed.3
To assess the kinetics of the synthesis of 1, we quantified the product
level by using [2-14C]malonyl-CoA and radioactive thin-layer chroma-
tography. When Hpm3 was fixed at 5 µM, the initial velocity of formation
of 1 varied linearly with increasing concentrations of Hpm8, yielding an
apparent rate constant of 0.11 min-1 (Figure S6A). In contrast, when the
Hpm8 concentration was fixed at 10, 20, or 30 µM, the initial velocities
of formation of 1 at different Hpm3 concentrations were essentially
constant at 1.1, 2.04, and 3.88 µM min-1, respectively (Figure S6B). The
overall reaction velocity was independent of Hpm3 concentration within
this concentration range. Thus, synthesis of the hexaketide intermediate
from malonyl-CoA catalyzed by Hpm8 is the rate-limiting step. Transfer
of this acyl intermediate to Hpm3 and the subsequent formation of 1 are
faster, resulting in the rapid scavenging and offloading of 2-Hpm8 by
Hpm3.
The SAT domain at the N-terminus of Hpm3 is the most likely
candidate to facilitate crosstalk between the two enzymes.11 To probe
the role of SAT, the Hpm3 point mutant Hpm3-SAT0 was constructed
by mutating the putative active site Ser121 within the GXSXG motif
to alanine. Synthesis of 1 was completely abolished in the in vitro
reaction containing Hpm3-SAT0 and Hpm8 (Figure 2C), confirming
that the catalytic activity of the SAT domain is essential for acyl transfer
between the proteins. To examine the specificity of the SAT domain
toward the partner PKS, we paired Hpm8 with the functionally
equivalent PKS13 from the Gibberella zeae zearalenone pathway.12
Surprisingly, they failed to function in tandem, and no RAL products
were detected. We then constructed a hybrid downstream PKS, PKSH1,
in which the Hpm3 SAT domain was replaced with the noncognate PKS13
SAT domain. While PKSH1 was solubly expressed and all of the the
domains were active in the presence of 2-SNAC (see below), the hybrid
enzyme was not able to communicate with Hpm8 with respect to the
Acknowledgment. We thank Dr. Chris Reeves for the generous
gifts of pKOS518-120A and pKOS518-118A. Crystallographic analysis
of 1 was done by Dr. Michael Ferguson (University of Alberta). This
work was supported by NIH Grant 1R01GM085128, a David and
Lucile Packard Fellowship to Y.T., the Natural Sciences and Engineer-
ing Research Council of Canada (NSERC), and the Canada Research
Chair in Bioorganic and Medicinal Chemistry.
Note Added after ASAP Publication. The structure of hypothe-
mycin in Figure 1 was corrected on March 16, 2010.
Supporting Information Available: Experimental details and
spectroscopic information. This material is available free of charge via
the Internet at http://pubs.acs.org.
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