title: Structural basis for olivetolic acid formation by a polyketide cyclase from Cannabis sativa.
abstract: In polyketide biosynthesis, ring formation is one of the key diversification steps. Olivetolic acid cyclase (OAC) from Cannabis sativa, involved in cannabinoid biosynthesis, is the only known plant polyketide cyclase. In addition, it is the only functionally characterized plant α+β barrel (DABB) protein that catalyzes the C2-C7 aldol cyclization of the linear pentyl tetra-β-ketide CoA as the substrate, to generate olivetolic acid (OA). Herein, we solved the OAC apo and OAC-OA complex binary crystal structures at 1.32 and 1.70 Å resolutions, respectively. The crystal structures revealed that the enzyme indeed belongs to the DABB superfamily, as previously proposed, and possesses a unique active-site cavity containing the pentyl-binding hydrophobic pocket and the polyketide binding site, which have never been observed among the functionally and structurally characterized bacterial polyketide cyclases. Furthermore, site-directed mutagenesis studies indicated that Tyr72 and His78 function as acid/base catalysts at the catalytic center. Structural and/or functional studies of OAC suggested that the enzyme lacks thioesterase and aromatase activities. These observations demonstrated that OAC employs unique catalytic machinery utilizing acid/base catalytic chemistry for the formation of the precursor of OA. The structural and functional insights obtained in this work thus provide the foundation for analyses of the plant polyketide cyclases that will be discovered in the future.
  
  
  METHODS
  Structural data reported in this paper are available in the Protein Data Bank under the accession numbers 5B08 for the OAC apo, 5B09 for the OAC-OA binary complex and 5B0A, 5B0B, 5B0C, 5B0D, 5B0E, 5B0F and 5B0G for the OAC His5Q, Ile7F, Tyr27F, Tyr27W, Val59M, Tyr72F and His78S mutant enzymes, respectively.
authors.affiliation: Institute of Natural Medicine, University of Toyama, Japan.
authors.forename: Xinmei
authors.initials: X
authors.lastname: Yang
authors.affiliation: Institute of Natural Medicine, University of Toyama, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Matsui
authors.affiliation: Institute of Natural Medicine, University of Toyama, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Kodama
authors.affiliation: Graduate School of Pharmaceutical Sciences, University of Tokyo, Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Mori
authors.affiliation: Institute of Natural Medicine, University of Toyama, Japan.
authors.forename: Xiaoxi
authors.initials: X
authors.lastname: Zhou
authors.affiliation: Graduate School of Medicine and Pharmaceutical Sciences, Faculty of Pharmacy and Pharmaceutical Sciences, University of Toyama, Japan.
authors.forename: Futoshi
authors.initials: F
authors.lastname: Taura
authors.affiliation: School of Pharmaceutical Sciences, University of Shizuoka, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Noguchi
authors.affiliation: Graduate School of Pharmaceutical Sciences, University of Tokyo, Japan.
authors.forename: Ikuro
authors.initials: I
authors.lastname: Abe
authors.affiliation: Institute of Natural Medicine, University of Toyama, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Morita
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002188:Cannabis / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D007535:Isomerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D061065:Polyketides / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012459:Salicylates / Q000737:chemistry / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
chemical_list: D010940:Plant Proteins; D061065:Polyketides; D011994:Recombinant Proteins; D012459:Salicylates; C112458:olivetolic acid; D007535:Isomerases
keywords: Cannabis sativa; DABB protein; crystal structure; enzyme mechanism; olivetolic acid cyclase