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 beta-ketoacyl ACP synthase components known as KSalpha and KSbeta. 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 (kcat = 0.49 min-1, KM = 207 microM). This reaction increases 5-fold in rate in the presence of KSalphaKSbeta (kcat = 2.3 min-1, KM = 215 microM). 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 KSalphaKSbeta can be measured (kcat = 20.6 min-1, KM = 2.4 microM). 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 approximately 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 KSalpha/KSbeta (Kic = 50 microM, Kiu = 137 microM), but apo-ACP does not appear to inhibit MCAT.
Beltran-Alvarez
Pedro
P
School of Chemistry, University of Bristol, Cantock's Close, Bristol, BS8 1TS, UK.
Cox
Russell J
RJ
Crosby
John
J
Simpson
Thomas J
TJ
D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase / Q000378:metabolism*; D000213:Acyl Carrier Protein / Q000378:metabolism*; D000880:Anthraquinones / Q000737:chemistry / Q000378:metabolism; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D003630:Daunorubicin / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D015394:Molecular Structure; D010118:Oxytetracycline / Q000737:chemistry / Q000378:metabolism; D048630:Polyketide Synthases / Q000378:metabolism*; D048372:Streptomyces coelicolor / Q000201:enzymology; D013379:Substrate Specificity; D000095622:Benzoisochromanequinones
D015099:3-Oxoacyl-(Acyl-Carrier-Protein) Synthase; D000213:Acyl Carrier Protein; D000880:Anthraquinones; D003630:Daunorubicin; D010118:Oxytetracycline; D048630:Polyketide Synthases; C087193:polyketide beta-ketoacylsynthase; C013390:actinorhodin; D000095622:Benzoisochromanequinones