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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 KS_α and KS_β. 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 KS_αKS_β (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 …