title: Epothilone C macrolactonization and hydrolysis are catalyzed by the isolated thioesterase domain of epothilone polyketide synthase.
abstract: Epothilone C is produced by the combined action of one nonribosomal peptide synthetase (NRPS) and nine polyketide synthase (PKS) modules in a multienzyme system. The final step in the biosynthesis is the thioesterase (TE)-catalyzed cyclorelease of epothilone from the EpoF protein. It has been unclear whether isolated PKS TE domains could exhibit macrolactonization activity. Here we demonstrate that the excised epothilone TE domain can catalyze the efficient cyclization of the N-acetylcysteamine thioester of seco-epothilone C to generate epothilone C (kcat/KM = 0.41 +/- 0.03 min-1 mM-1). The TE domain also catalyzes the hydrolysis of both the N-acetylcysteamine thioester of seco-epothilone C (kcat = 0.087 +/- 0.005 min-1, KM = 291 +/- 53 muM) and that of the epothilone C (kcat = 0.67 +/- 0.01 min-1, KM = 117 +/- 5 muM) to form seco-epothilone C.
authors.affiliation: Departments of Chemical Engineering, Stanford University, Stanford, California 94305-5025, USA.
authors.forename: Christopher N
authors.initials: CN
authors.lastname: Boddy
authors.forename: Tanya L
authors.initials: TL
authors.lastname: Schneider
authors.forename: Kinya
authors.initials: K
authors.lastname: Hotta
authors.forename: Christopher T
authors.initials: CT
authors.lastname: Walsh
authors.forename: Chaitan
authors.initials: C
authors.lastname: Khosla
mesh_terms: D002384:Catalysis; D034261:Epothilones / Q000737:chemistry* / Q000378:metabolism; D004950:Esterases / Q000737:chemistry / Q000378:metabolism; D006868:Hydrolysis; D007783:Lactones / Q000737:chemistry / Q000378:metabolism; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000378:metabolism; D017434:Protein Structure, Tertiary
chemical_list: D034261:Epothilones; D007783:Lactones; D009097:Multienzyme Complexes; C426624:epothilone C; D004950:Esterases