Utilization of enzymatically phosphopantetheinylated acyl carrier proteins and acetyl-acyl carrier proteins by the actinorhodin polyketide synthase.
The functional reconstitution of two purified proteins of an aromatic polyketide synthase pathway, the acyl carrier protein (ACP) and holo-ACP synthase (ACPS), is described. Holo-ACPs were enzymatically synthesized from coenzyme A and apo-ACPs using Escherichia coli ACPS. Frenolicin and granaticin holo-ACPs formed in this manner were shown to be fully functional together with the other components of the minimal actinorhodin polyketide synthase (act PKS), resulting in synthesis of the same aromatic polyketides as those formed by the act PKS in vivo. ACPS also catalyzed the transfer of acetyl-, propionyl-, butyryl-, benzoyl-, phenylacetyl-, and malonylphosphopantetheines to apo-ACPs from their corresponding coenzyme As, as detected by electrophoresis and/or mass spectrometry. A steady state kinetic study showed that acetyl-coenzyme A is as efficient an ACPS substrate as coenzyme A, with kcat and Km values of 20 min-1 and 25 microM, respectively. In contrast to acetyl-coenzyme A, enzymatically synthesized acetyl-ACPs were shown to be efficient substrates for the act PKS, indicating that acetyl-ACP is a chemically competent intermediate of aromatic polyketide biosynthesis. Together, these methods provide a valuable tool for dissecting the mechanisms and molecular recognition features of polyketide biosynthesis.
Carreras
C W
CW
Department of Chemical Engineering, Stanford University, Stanford, California 94305-5025, USA.
Gehring
A M
AM
Walsh
C T
CT
Khosla
C
C
D000213:Acyl Carrier Protein / Q000378:metabolism*; D000217:Acyltransferases / Q000378:metabolism*; D001426:Bacterial Proteins; D002384:Catalysis; D007700:Kinetics; D008956:Models, Chemical; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D048630:Polyketide Synthases; D013302:Streptomyces; D017855:Transferases (Other Substituted Phosphate Groups) / Q000378:metabolism*
D000213:Acyl Carrier Protein; D001426:Bacterial Proteins; D048630:Polyketide Synthases; D000217:Acyltransferases; C087193:polyketide beta-ketoacylsynthase; D017855:Transferases (Other Substituted Phosphate Groups); C021364:holo-(acyl-carrier-protein) synthase; D010727:Phosphoric Diester Hydrolases; C018754:(acyl-carrier-protein) phosphodiesterase