Actinorhodin (ACT) produced by
Streptomyces coelicolor A3(2) is an aromatic polyketideantibiotic, whose basic carbon skeleton is derived from type II polyketide synthase (PKS). Although anacyl carrier protein (ACP) serves as an anchor of nascent intermediates during chain elongation in thetype II PKS complex, it generally remains unknown when an ACP-free intermediate is released from thecomplex to post-PKS modification ("tailoring") steps. In ACT biosynthesis, a stereospecific ketoreductase(RED1) encoded by
actVI-ORF1 reduces the 3
-keto group of a proposed bicyclic intermediate to an (
S)secondary alcohol. The bicyclic intermediate is formed from the steps of PKS and its closely associatedenzymes and lies at the interface toward ACT-tailoring steps. To clarify whether RED1 recognizes theACP-bound bicyclic intermediate or the ACP-free bicyclic intermediate, recombinant RED1 was purifiedfor enzymatic characterization. RED1 was heterologously expressed in
Escherichia coli and purified usingNi-chelate and gel filtration column chromatographies to homogeneity in soluble form. Enzymatic studies
in vitro on RED1 with synthetic analogues, in place of an unstable bicyclic intermediate, showed thatRED1 recognizes 3-oxo-4-naphthylbutyric acid (ONBA) as a preferred substrate and not its
N-acetylcysteamine thioester. This strongly suggests that RED1 recognizes ACP-free bicyclic
-keto acidas the first committed intermediate of tailoring steps. Kinetic studies of RED1 showed high affinity withONBA, consistent with the requirement for an efficient reduction of a labile
-keto carboxylic acid.Interestingly, the methyl ester of ONBA acted as a competitive inhibitor of RED1, indicating the presenceof strict substrate recognition toward the terminal acid functionality.