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-A CP synthase (ACPS), is described. Holo-ACPs were enzymatically synthes ized from coenzyme A and apo-ACPs using Escherichia coil ACPS. Frenoli cin and granaticin holo-ACPs formed in this manner were shown to be fu lly functional together with the other components of the minimal actin orhodin polyketide synthase (act PKS), resulting in synthesis of the s ame 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-coenzym e A is as efficient an ACPS substrate as coenzyme A, with k(cat) and K -m values of 20 min(-1) and 25 mu M, respectively, In contrast to acet yl-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 me chanisms and molecular recognition features of polyketide biosynthesis .