Epothilone C macrolactonization and hydrolysis are catalyzed by the isolated thioesterase domain of epothilone polyketide synthase.
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.
Boddy
Christopher N
CN
Departments of Chemical Engineering, Stanford University, Stanford, California 94305-5025, USA.
Schneider
Tanya L
TL
Hotta
Kinya
K
Walsh
Christopher T
CT
Khosla
Chaitan
C
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
D034261:Epothilones; D007783:Lactones; D009097:Multienzyme Complexes; C426624:epothilone C; D004950:Esterases