Polyketide Double Bond Biosynthesis.Mechanistic Analysis of the Dehydratase-Containing Module 2 of the Picromycin/ Methymycin Polyketide Synthase

摘要

Picromycin/methymycin synthase(PICS)is a modular polyketide synthase(PKS)that is responsible for the biosynthesis of both 10-deoxymethynolide(1)and narbonolide(2),the parent 12-and 14-membered aglycone precursors of the macrolide antibiotics methymycin and picromycin,respectively.PICS module 2 is a dehydratase(DH)-containing module that catalyzes the formation of the unsaturated triketide intermediate using malonyl-CoA as the chain extension substrate.Recombinant PICS module 2+TE,with the PICS thioesterase domain appended to the C-terminus to allow release of polyketide products,was expressed in Escherichia coli.Purified PICS module 2+TE converted malonyl-CoA and 4,the N-acetylcysteamine thioester of(2S,3R)-2-methyl-3-hydroxypentanoic acid,to a 1:2 mixture of the triketide acid(4S,5R)-4-methyl-5-hydroxy-2-heptenoic acid(5)and(3S,4S,5R)-3,5-dihydroxy-4-methyl-n-heptanoic acid-delta-lactone(10)with a combined k_(cat)of 0.6 min~(-1).The triketide lactone 10 is formed by thioesterase-catalyzed cyclization of the corresponding D-3-hydroxyacyl-SACP intermediate,a reaction which competes with dehydration catalyzed by the dehydratase domain.PICS module 2+TE showed a strong preference for the syn-diketide-SNAC 4,with a 20-fold greater k_(cat)/K_m than the anti-(2S,3S)-diketide-SNAC 14,and a 40-fold advantage over the syn-(2R,3S)-diketide-SNAC 13.PICS module 2(DH~0)+TE,with an inactivated DH domain,produced exclusively 10,while three PICS module 2(KR~0)+TE mutants,with inactivated KR domains,produced exclusively or predominantly the unreduced triketide ketolactone,(4S,5R)-3-oxo-4-methyl-5-hydroxy-n-heptanoic acid-delta-lactone(7).These studies establish for the first time the structure and stereochemistry of the intermediates of a polyketide chain elongation cycle catalyzed by a DH-containing module,while confirming the importance of key active site residues in both KR and DH domains.