Stereochemistry of catalysis by the ketoreductase activity in the first extension module of the erythromycin polyketide synthase.

Multiple ketoreductase activities play a crucial role in establishing the stereochemistry of the products of modular polyketide synthases (PKSs), but there has been little systematic scrutiny of catalysis by individual ketoreductases. To allow this, a diketide synthase, consisting of the loading module, first extension module, and the chain-terminating thioesterase of the erythromycin-producing PKS of Saccharopolyspora erythraea, has been expressed and purified. The DNA encoding the ketoreductase-1 domain in this construct is flanked by unique restriction sites so that another ketoreductase domain can be readily substituted. The purified recombinant diketide synthase catalyzes, at a very low rate (k(cat) equals 2.5 x 10(-3) s(-1)), the specific production of the diketide (2S,3R)-2-methyl-3-hydroxypentanoic acid. The activity of the ketoreductase domain in this model synthase was analyzed using as a model substrate (+/-)-2-methyl-3-oxopentanoic acid N-acetylcysteaminyl (NAC) ester for which k(cat)/K(m) was 21.7 M(-1) s(-1). The NAC thioester of (2S,3R)-2-methyl-3-hydroxypentanoic acid was the major product and was strongly preferred over other stereoisomers as a substrate in the reverse reaction. The bicyclic ketone (9RS)-trans-1-decalone, a known substrate for ketoreductase in fatty acid synthase, was found also to be an effective substrate for the ketoreductase of the diketide synthase. Only the (9R)-trans-1-decalone was reduced, selectively and reversibly, to the (1S,9R)-trans-decalol. The stereochemical course of reduction and oxidation is exactly as found previously for the ketoreductase of animal fatty acid synthase, an additional indication of the close similarity of these enzymes.