Dissecting the Component Reactions Catalyzed by the Actinorhodin Minimal Polyketide Synthase

The actinorhodin (act) minimal polyketide synthase (PKS) from Streptomyces coelicolor consists of three proteins: an acyl carrier protein (ACP) and two β-ketoacyl ACP synthase components known as KSα and KSβ. The act minimal PKS catalyzes at least 18 separate reactions which can be divided into loading, initiation, extension, and cyclization and release phases. Two quantitative kinetic assays were developed and used to measure individual rate and Michaelis constants for loading, initiation and extension steps. In the minimal PKS, the reaction between malonyl CoA and ACP to form malonyl ACP (loading) is the rate-limiting step (k cat = 0.49 min-1, K M = 207 μM). This reaction increases 5-fold in rate in the presence of KSαKSβ (k cat = 2.3 min-1, K M = 215 μM). In the presence of S. coelicolor malonyl CoA:ACP transacylase (MCAT), the rate of loading increases and the kinetic parameters of malonyl-ACP as a substrate of KSαKSβ can be measured (k cat = 20.6 min-1, K M = 2.4 μM). Under these conditions, it appears that decarboxylation of malonyl-ACP to form acetyl-ACP (initiation) is the rate-limiting step. When an excess of acetyl ACP is supplied, either chain extension, cyclization, or release steps become rate limiting (k ≈ 60 min-1). No ACP-bound intermediates could be observed, suggesting that partially or fully extended chains do not accumulate because chain extension is rate limiting under these conditions and that cyclization and release are fast. apo-ACP acts as a mixed inhibitor of malonyl ACP binding to KSα/KSβ (K ic = 50 μM, K iu = 137 μM), but apo-ACP does not appear to inhibit MCAT.