Excision of the Epothilone Synthetase B Cyclization Domain and Demonstration of in Trans Condensation/Cyclodehydration Activity^†

The epothilones are potent anticancer natural products produced by a polyketide synthase (PKS)−nonribosomal peptide synthetase (NRPS) hybrid involving proteins EpoA−F. The single NRPS module of the epothilone assembly line, EpoB, is a distinct subunit of approximately 160 kDa and consists of four successive domains:  cyclization, adenylation, oxidation, and peptidyl carrier protein (Cy−A−Ox−PCP). The cyclization domain is responsible for introduction of the thiazoline heterocycle into the growing polyketide/nonribosomal peptide chain from the precursors malonyl-CoA and cysteine through the multiple steps of condensation, cyclization, and dehydration. This enzyme-bound thiazoline intermediate is subsequently oxidized to a thiazole by the EpoB Ox domain. The EpoB module was dissected to provide 57 kDa EpoB(Cy) and 102 kDa EpoB(A−Ox−PCP) as subunit fragments to evaluate Cy as a free-standing domain. EpoB was reconstituted by these fragments in trans to generate the methylthiazole product. Using this system, apparent kinetic constants for the upstream acyl donor EpoA(ACP) and EpoB(Cy) were determined, providing a measure of affinity for the naturally occurring interface of the amino terminus of EpoB and the EpoA carboxy terminus. Site-directed mutants in excised EpoB(Cy) were prepared and used to examine residues involved in condensation and heterocycle formation. This work demonstrates the ability to define a functional Cy domain by excision from its native NRPS module, and examine both its protein−protein interactions and mechanism of activity.