title: Purification and in vitro reconstitution of the essential protein components of an aromatic polyketide synthase.
abstract: A minimal set of proteins which catalyze the synthesis of aromatic polketides from malonyl CoA has been purified and partially characterized. Plasmid-encoded actinorhodin (act) ketosynthase/chain-length factor (KS/CLF) complex was purified from Streptomyces coelicolor CH999/pSEK38, and assayed with purified aromatic PKS holo-ACPs which were overproduced and purified from Escherichia coli and phosphopantetheinylated in vitro using purified E. coli holo-ACP synthase. When highly purified preparations of KS/CLF, and holo-ACP failed to catalyze polyketide biosynthesis, a fourth protein was sought and purified from the S. coelicolor CH999 host on the basis of its ability to complement KS, CLF, and holo-ACP in polyketide synthesis. N-terminal sequencing identified this protein as the fatty acid synthase (fabD) malonyl CoA:ACP transacylase (MAT), recruited from primary metabolism. A alpha2/beta2 structure was shown for the act KS/CLF complex, and three malonyl-enzyme biosynthetic intermediates were identified, defining an escorted path followed by malonyl groups en route from CoA to polyketide.
authors.affiliation: Department of Chemical Engineering, Stanford University, Stanford, California 94305-5025, USA. Carreras@Kosan.com
authors.forename: C W
authors.initials: CW
authors.lastname: Carreras
authors.forename: C
authors.initials: C
authors.lastname: Khosla
mesh_terms: D000217:Acyltransferases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D001426:Bacterial Proteins; D004926:Escherichia coli / Q000235:genetics; D008316:Malonyl Coenzyme A / Q000378:metabolism; D008956:Models, Chemical; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D048630:Polyketide Synthases; D013302:Streptomyces / Q000201:enzymology / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D009097:Multienzyme Complexes; D008316:Malonyl Coenzyme A; D048630:Polyketide Synthases; D000217:Acyltransferases; C087193:polyketide beta-ketoacylsynthase