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Terminal Alkene Formation by the Thioesterase of Curacin A Biosynthesis

STRUCTURE OF A DECARBOXYLATING THIOESTERASE*
  • Jennifer J. Gehret
    Affiliations
    From the Departments of Biological Chemistry,

    Life Sciences Institute, University of Michigan, Ann Arbor, Michigan 48109,
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  • Author Footnotes
    1 Present address: Dept. of Genetics, Harvard Medical School, 77 Ave. Louis Pasteur, NRB 232, Boston, MA 02115.
    Liangcai Gu
    Footnotes
    1 Present address: Dept. of Genetics, Harvard Medical School, 77 Ave. Louis Pasteur, NRB 232, Boston, MA 02115.
    Affiliations
    Departments of Medicinal Chemistry,

    Life Sciences Institute, University of Michigan, Ann Arbor, Michigan 48109,
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  • William H. Gerwick
    Affiliations
    the Scripps Institution of Oceanography, University of California at San Diego, La Jolla, California 92093, and
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  • Peter Wipf
    Affiliations
    the Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15260
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  • David H. Sherman
    Affiliations
    Departments of Medicinal Chemistry,

    Departments of Chemistry, and

    Departments of Microbiology and Immunology, and

    Life Sciences Institute, University of Michigan, Ann Arbor, Michigan 48109,
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  • Janet L. Smith
    Correspondence
    To whom correspondence should be addressed. Tel.: 734-615-9564; Fax: 734-763-6492;
    Affiliations
    From the Departments of Biological Chemistry,

    Life Sciences Institute, University of Michigan, Ann Arbor, Michigan 48109,
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  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grants R01 DK42303 (to J. L. S.), R01 CA108874 (to D. H. S., W. H. G., and P. W.), and U01 TW007404, and a Hans W. Vahlteich Professorship (to D. H. S.).
    The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S3 and Tables S1.
    1 Present address: Dept. of Genetics, Harvard Medical School, 77 Ave. Louis Pasteur, NRB 232, Boston, MA 02115.
Open AccessPublished:February 27, 2011DOI:https://doi.org/10.1074/jbc.M110.214635
      Curacin A is a polyketide synthase (PKS)-non-ribosomal peptide synthetase-derived natural product with potent anticancer properties generated by the marine cyanobacterium Lyngbya majuscula. Type I modular PKS assembly lines typically employ a thioesterase (TE) domain to off-load carboxylic acid or macrolactone products from an adjacent acyl carrier protein (ACP) domain. In a striking departure from this scheme the curacin A PKS employs tandem sulfotransferase and TE domains to form a terminal alkene moiety. Sulfotransferase sulfonation of β-hydroxy-acyl-ACP is followed by TE hydrolysis, decarboxylation, and sulfate elimination (Gu, L., Wang, B., Kulkarni, A., Gehret, J. J., Lloyd, K. R., Gerwick, L., Gerwick, W. H., Wipf, P., Håkansson, K., Smith, J. L., and Sherman, D. H. (2009) J. Am. Chem. Soc. 131, 16033–16035). With low sequence identity to other PKS TEs (<15%), the curacin TE represents a new thioesterase subfamily. The 1.7-Å curacin TE crystal structure reveals how the familiar α/β-hydrolase architecture is adapted to specificity for β-sulfated substrates. A Ser-His-Glu catalytic triad is centered in an open active site cleft between the core domain and a lid subdomain. Unlike TEs from other PKSs, the lid is fixed in an open conformation on one side by dimer contacts of a protruding helix and on the other side by an arginine anchor from the lid into the core. Adjacent to the catalytic triad, another arginine residue is positioned to recognize the substrate β-sulfate group. The essential features of the curacin TE are conserved in sequences of five other putative bacterial ACP-ST-TE tridomains. Formation of a sulfate leaving group as a biosynthetic strategy to facilitate acyl chain decarboxylation is of potential value as a route to hydrocarbon biofuels.

      Introduction

      Natural products display a remarkable chemical diversity, providing advantages for the producing plants and microbes to survive and thrive in particular ecological niches. These secondary metabolites and their derivatives have important applications as pharmaceuticals (
      • Newman D.J.
      • Cragg G.M.
      ), and some have the potential to be developed as biofuels (
      • Fortman J.L.
      • Chhabra S.
      • Mukhopadhyay A.
      • Chou H.
      • Lee T.S.
      • Steen E.
      • Keasling J.D.
      ). Gene clusters encoding assembly line biosynthetic pathways for polyketide and polypeptide natural products are ubiquitous in bacterial and fungal genomes. Polyketide synthase (PKS)
      The abbreviations used are: PKS, polyketide synthase; NRPS, non-ribosomal peptide synthetase; TE, thioesterase; ACP, acyl carrier protein; ST, sulfotransferase; PAPS, adenosine 3′-phosphate 5′-phosphosulfate; PAP, adenosine 3′-phosphate 5′-phosphate; SeMet, selenomethione.
      and non-ribosomal peptide synthetase (NRPS) pathways have a common modular organization in which intermediates tethered to carrier domains by a thioester linkage pass sequentially through modules of the assembly line. The final step in the assembly line is typically a thioesterase-catalyzed off-loading from the final carrier domain to produce a carboxylate, macrolactone, or cyclic peptide.
      In a notable exception to this off-loading paradigm, the curacin A final product contains a terminal alkene moiety. Curacin A, from the marine cyanobacterium Lyngbya majuscula, is a mixed polyketide/non-ribosomal peptide with antimitotic properties (
      • Verdier-Pinard P.
      • Lai J.Y.
      • Yoo H.D.
      • Yu J.
      • Marquez B.
      • Nagle D.G.
      • Nambu M.
      • White J.D.
      • Falck J.R.
      • Gerwick W.H.
      • Day B.W.
      • Hamel E.
      ). The hybrid PKS/NRPS assembly line pathway for curacin A (
      • Chang Z.
      • Sitachitta N.
      • Rossi J.V.
      • Roberts M.A.
      • Flatt P.M.
      • Jia J.
      • Sherman D.H.
      • Gerwick W.H.
      ) generates several unusual chemical groups in addition to the terminal alkene, including a cyclopropyl ring, a thiazoline ring, and a cis double bond. We have investigated the biosynthetic steps leading to several of these segments (
      • Gu L.
      • Geders T.W.
      • Wang B.
      • Gerwick W.H.
      • Håkansson K.
      • Smith J.L.
      • Sherman D.H.
      ,
      • Geders T.W.
      • Gu L.
      • Mowers J.C.
      • Liu H.
      • Gerwick W.H.
      • Håkansson K.
      • Sherman D.H.
      • Smith J.L.
      ,
      • Akey D.L.
      • Razelun J.R.
      • Tehranisa J.
      • Sherman D.H.
      • Gerwick W.H.
      • Smith J.L.
      ,
      • Gu L.
      • Wang B.
      • Kulkarni A.
      • Geders T.W.
      • Grindberg R.V.
      • Gerwick L.
      • Håkansson K.
      • Wipf P.
      • Smith J.L.
      • Gerwick W.H.
      • Sherman D.H.
      ,
      • Gu L.
      • Wang B.
      • Kulkarni A.
      • Gehret J.J.
      • Lloyd K.R.
      • Gerwick L.
      • Gerwick W.H.
      • Wipf P.
      • Håkansson K.
      • Smith J.L.
      • Sherman D.H.
      ). Herein we investigate the structural basis for the unique off-loading strategy leading to the terminal alkene in the curacin A molecule.
      The curacin PKS has an unusual terminal module, the CurM protein, with a C-terminal tridomain comprised of an acyl carrier protein (ACP), a sulfotransferase (ST), and a thioesterase (TE) (Fig. 1A). Annotations of both ST and TE were based on weak sequence similarity to characterized enzymes. The prediction of an ST within a PKS was unprecedented (
      • Chang Z.
      • Sitachitta N.
      • Rossi J.V.
      • Roberts M.A.
      • Flatt P.M.
      • Jia J.
      • Sherman D.H.
      • Gerwick W.H.
      ). STs are widely distributed and are known to have detoxification, hormone regulation, or signaling functions (
      • Chapman E.
      • Best M.D.
      • Hanson S.R.
      • Wong C.H.
      ). They catalyze transfer of a sulfonate group from the donor PAPS to a hydroxyl or amine of an acceptor small molecule or protein. CurM TE, although identifiable as a thioesterase, does not resemble any of the previously established PKS or NRPS TE subfamilies (
      • Roongsawang N.
      • Washio K.
      • Morikawa M.
      ).
      Figure thumbnail gr1
      FIGURE 1Chain termination in curacin A biosynthesis. A, off-loading reactions in the final module, CurM. Following extension by the CurM ketosynthase (KS) and acyltransferase (AT) and reduction by the ketoreductase (KR), ST catalyzes transfer of sulfonate to the β-hydroxyl followed by TE hydrolysis of the thioester with concomitant decarboxylation and sulfate elimination. The sulfonate group donor is the PAPS cofactor, which is converted to PAP. B, experimental scheme for the assay of CurM TE. Recombinant CurM ACP loaded with a substrate analog and sulfonated by ST was reacted with excised CurM TE. Activity was monitored by HPLC of the reaction mixture and detection of the holo-ACP product.
      Using a simplified analog of the penultimate pathway intermediate, we recently demonstrated that off-loading and terminal alkene formation require ST-mediated sulfonation of the β-hydroxyl group from the PAPS cofactor (
      • Gu L.
      • Wang B.
      • Kulkarni A.
      • Gehret J.J.
      • Lloyd K.R.
      • Gerwick L.
      • Gerwick W.H.
      • Wipf P.
      • Håkansson K.
      • Smith J.L.
      • Sherman D.H.
      ) (Fig. 1A). This was the first observation of biological substrate activation by formation of a sulfate leaving group. CurM TE acts upon the β-sulfate intermediate to yield a decarboxylated product with a terminal double bond resulting from sulfate elimination (Fig. 1B). CurM TE catalyzes thioester hydrolysis 800-fold more slowly on the corresponding substrate bearing a β-hydroxyl group. The unprecedented requirement of a β-sulfate for thioester hydrolysis as well as the decarboxylation and sulfate elimination suggests a unique catalytic strategy and active site structure for the TE. Moreover, this curacin pathway decarboxylation strategy provides an opportunity to investigate a new biological route to hydrocarbon production from fatty acids. Thus, in addition to interest in curacin biosynthesis as a route to a potent anti-cancer compound (
      • Verdier-Pinard P.
      • Lai J.Y.
      • Yoo H.D.
      • Yu J.
      • Marquez B.
      • Nagle D.G.
      • Nambu M.
      • White J.D.
      • Falck J.R.
      • Gerwick W.H.
      • Day B.W.
      • Hamel E.
      ), the pathway also has relevance to biofuel production.
      Off-loading TEs from many PKSs have been studied including the TEs of the pikromycin (Pik TE) (
      • Giraldes J.W.
      • Akey D.L.
      • Kittendorf J.D.
      • Sherman D.H.
      • Smith J.L.
      • Fecik R.A.
      ,
      • Akey D.L.
      • Kittendorf J.D.
      • Giraldes J.W.
      • Fecik R.A.
      • Sherman D.H.
      • Smith J.L.
      ,
      • Tsai S.C.
      • Miercke L.J.
      • Krucinski J.
      • Gokhale R.
      • Chen J.C.
      • Foster P.G.
      • Cane D.E.
      • Khosla C.
      • Stroud R.M.
      ), erythromycin (
      • Tsai S.C.
      • Miercke L.J.
      • Krucinski J.
      • Gokhale R.
      • Chen J.C.
      • Foster P.G.
      • Cane D.E.
      • Khosla C.
      • Stroud R.M.
      ), and tautomycetin (
      • Scaglione J.B.
      • Akey D.L.
      • Sullivan R.
      • Kittendorf J.D.
      • Rath C.M.
      • Kim E.S.
      • Smith J.L.
      • Sherman D.H.
      ) PKS. PKS off-loading TEs typically catalyze either hydrolysis to produce a linear carboxylic acid or the attack of an intramolecular hydroxyl to produce a large-ring macrolactone. PKS TEs are members of the α/β-hydrolase superfamily with a catalytic triad active site located at the top of an α/β core and covered by an α-helical lid subdomain. All PKS off-loading TEs of known structure are dimers in which two N-terminal α-helices in the lid form a lid-to-lid dimer interface (
      • Giraldes J.W.
      • Akey D.L.
      • Kittendorf J.D.
      • Sherman D.H.
      • Smith J.L.
      • Fecik R.A.
      ,
      • Akey D.L.
      • Kittendorf J.D.
      • Giraldes J.W.
      • Fecik R.A.
      • Sherman D.H.
      • Smith J.L.
      ,
      • Tsai S.C.
      • Miercke L.J.
      • Krucinski J.
      • Gokhale R.
      • Chen J.C.
      • Foster P.G.
      • Cane D.E.
      • Khosla C.
      • Stroud R.M.
      ,
      • Scaglione J.B.
      • Akey D.L.
      • Sullivan R.
      • Kittendorf J.D.
      • Rath C.M.
      • Kim E.S.
      • Smith J.L.
      • Sherman D.H.
      ). A classic Ser-His-Asp catalytic triad is positioned at the center of a narrow tunnel formed by the lid. The tunnel architecture with open ends is fixed by the dimer interface. In contrast to these dimeric off-loading TEs, many PKS and NRPS pathways also have a second monomeric thioesterase called a TE II, which performs an editing function within the pathway. TE IIs, as well as NRPS off-loading TEs, are monomers with a flexible lid domain that appears to control access to the active site (
      • Bruner S.D.
      • Weber T.
      • Kohli R.M.
      • Schwarzer D.
      • Marahiel M.A.
      • Walsh C.T.
      • Stubbs M.T.
      ,
      • Samel S.A.
      • Wagner B.
      • Marahiel M.A.
      • Essen L.O.
      ,
      • Koglin A.
      • Löhr F.
      • Bernhard F.
      • Rogov V.V.
      • Frueh D.P.
      • Strieter E.R.
      • Mofid M.R.
      • Güntert P.
      • Wagner G.
      • Walsh C.T.
      • Marahiel M.A.
      • Dötsch V.
      ,
      • Frueh D.P.
      • Arthanari H.
      • Koglin A.
      • Vosburg D.A.
      • Bennett A.E.
      • Walsh C.T.
      • Wagner G.
      ,
      • Claxton H.B.
      • Akey D.L.
      • Silver M.K.
      • Admiraal S.J.
      • Smith J.L.
      ). The curacin TE sequence has low similarity to sequences in all parts of the TE phylogenic tree (
      • Roongsawang N.
      • Washio K.
      • Morikawa M.
      ), lacks an N-terminal extension for dimerization, and has a longer internal lid than other PKS off-loading TEs.
      To gain further insights and enable mechanistic studies of the novel decarboxylation and sulfate elimination, we report here the crystal structure of CurM TE. The structure of the TE lid and an unusual dimer interface appear to fix the active site in a perpetually open state. A model for β-sulfate recognition was tested by site-directed mutagenesis. The similarity of CurM to other conserved ACP-ST-TE tridomain sequences strongly suggests that CurM TE is part of a new subfamily of thioesterases.

      Acknowledgments

      We thank Drs. Amol Kulkarni and Sarang Kulkarni for the synthesis of substrate analogs. Beamline 23ID-D is supported by the National Institutes of Health, National Institute of General Medical Sciences Grant Y1-GM-1104 and National Cancer Institute Grant Y1-CO-1020 through the GM/CA Collaborative Access Team at the APS, which is supported by the United States Department of Energy.

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