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Structural basis for biosynthetic programming of fungal aromatic polyketide cyclization


Polyketides are a class of natural products with diverse structures and biological activities. The structural variability of aromatic products of fungal nonreducing, multidomain iterative polyketide synthases (NR-PKS group of IPKSs) results from regiospecific cyclizations of reactive poly-β-keto intermediates1,2,3. How poly-β-keto species are synthesized and stabilized, how their chain lengths are determined, and, in particular, how specific cyclization patterns are controlled have been largely inaccessible and functionally unknown until recently4. A product template (PT) domain is responsible for controlling specific aldol cyclization and aromatization of these mature polyketide precursors, but the mechanistic basis is unknown. Here we present the 1.8 Å crystal structure and mutational studies of a dissected PT monodomain from PksA, the NR-PKS that initiates the biosynthesis of the potent hepatocarcinogen aflatoxin B1 in Aspergillus parasiticus. Despite having minimal sequence similarity to known enzymes, the structure displays a distinct ‘double hot dog’ (DHD) fold. Co-crystal structures with palmitate or a bicyclic substrate mimic illustrate that PT can bind both linear and bicyclic polyketides. Docking and mutagenesis studies reveal residues important for substrate binding and catalysis, and identify a phosphopantetheine localization channel and a deep two-part interior binding pocket and reaction chamber. Sequence similarity and extensive conservation of active site residues in PT domains suggest that the mechanistic insights gleaned from these studies will prove general for this class of IPKSs, and lay a foundation for defining the molecular rules controlling NR-PKS cyclization specificity.

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Figure 1: Biosynthesis of norsolorinic acid anthrone (1) by PksA.
Figure 2: The crystal structure of the PT domain from PksA.
Figure 3: PT structures with bound palmitate or substrate analogue HC8.
Figure 4: Proposed mechanism of cyclizations catalysed by the PT domain.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

The atomic coordinates of palmitate-bound and HC8-bound PT have been deposited in the Protein Data Bank under accession code 3HRQ and 3HRR.


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We thank T. M. Harris for his gift of HC8. The work at Johns Hopkins was supported by the US National Institutes of Health grant ES001670 awarded to C.A.T. S.-C.T. is supported by the Pew Foundation. Portions of this research were carried out at the Stanford Synchrotron Radiation Laboratory (SSRL), a national user facility operated by Stanford University on behalf of the US Department of Energy, Office of Basic Energy Sciences. The Advanced Light Source is supported by the Director, Office of Science, Office of Basic Energy Sciences, of the US Department of Energy under Contract No. DE-AC02-05CH11231. J.M.C. is currently a fellow supported by the Damon Runyon Cancer Research Foundation (DRG-2002-09, Harvard Medical School).

Author Contributions J.M.C. carried out biochemical experiments and provided recombinant proteins. A.L.V. carried out all mutational studies. T.P.K. assisted by O.K.-B. determined the PT X-ray crystal structures. E.A.H. prepared substrate and intermediate analogues for co-crystallization experiments. J.W.L. and T.P.K. conducted in silico docking studies. J.M.C., T.P.K. and J.W.L. analysed data and contributed to the writing of the paper, and J.W.L. refined the proposed PT mechanism. S.-C.T. and C.A.T. directed the research, provided funding and edited the final manuscript.

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Correspondence to Shiou-Chuan Tsai or Craig A. Townsend.

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Crawford, J., Korman, T., Labonte, J. et al. Structural basis for biosynthetic programming of fungal aromatic polyketide cyclization. Nature 461, 1139–1143 (2009).

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