Kinetic analysis of the actinorhodin aromatic polyketide synthase.
Type II polyketide synthases (PKSs) are bacterial multienzyme systems that catalyze the biosynthesis of a broad range of natural products. A core set of subunits, consisting of a ketosynthase, a chain length factor, an acyl carrier protein (ACP) and possibly a malonyl CoA:ACP transacylase (MAT) forms a "minimal" PKS. They generate a poly-beta-ketone backbone of a specified length from malonyl-CoA derived building blocks. Here we (a) report on the kinetic properties of the actinorhodin minimal PKS, and (b) present further data in support of the requirement of the MAT. Kinetic analysis showed that the apoACP is a competitive inhibitor of minimal PKS activity, demonstrating the importance of protein-protein interactions between the polypeptide moiety of the ACP and the remainder of the minimal PKS. In further support of the requirement of MAT for PKS activity, two new findings are presented. First, we observe hyperbolic dependence of PKS activity on MAT concentration, saturating at very low amounts (half-maximal rate at 19.7 +/- 5.1 nM). Since MAT can support PKS activity at less than 1/100 the typical concentration of the ACP and ketosynthase/chain length factor components, it is difficult to rule out the presence of trace quantities of MAT in a PKS reaction mixture. Second, an S97A mutant was constructed at the nucleophilic active site of the MAT. Not only can this mutant protein support PKS activity, it is also covalently labeled by [(14)C]malonyl-CoA, demonstrating that the serine nucleophile (which has been the target of PMSF inhibition in earlier studies) is dispensible for MAT activity in a Type II PKS system.
Dreier
J
J
Department of Chemical Engineering, Stanford University, Stanford, California 94305-5025, USA.
Shah
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AN
Khosla
C
C
D000213:Acyl Carrier Protein / Q000378:metabolism / Q000494:pharmacology; D051080:Acyl-Carrier Protein S-Malonyltransferase; D000217:Acyltransferases / Q000235:genetics / Q000378:metabolism; D000880:Anthraquinones / Q000378:metabolism; D001059:Apoproteins / Q000494:pharmacology; D029968:Escherichia coli Proteins; D054889:Fatty Acid Synthase, Type II; D007700:Kinetics; D008316:Malonyl Coenzyme A / Q000378:metabolism; D015394:Molecular Structure; D009097:Multienzyme Complexes / Q000737:chemistry / Q000378:metabolism*; D009154:Mutation; D013302:Streptomyces; D000095622:Benzoisochromanequinones
D000213:Acyl Carrier Protein; D051080:Acyl-Carrier Protein S-Malonyltransferase; D000217:Acyltransferases; D000880:Anthraquinones; D001059:Apoproteins; D029968:Escherichia coli Proteins; D054889:Fatty Acid Synthase, Type II; D008316:Malonyl Coenzyme A; D009097:Multienzyme Complexes; C040157:acpP protein, E coli; C013390:actinorhodin; D000095622:Benzoisochromanequinones