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

Lars H Østergaard, Laurenz Kellenberger, Jesús Cortés, Marc P Roddis, Matthew Deacon, James Staunton, Peter F Leadlay

Cambridge Centre for Molecular Recognition and Departments of Biochemistry and Chemistry, University of Cambridge, Cambridge, U.K.

Biochemistry 2002 Feb 26

filter terms:

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.

Citation

Lars H Østergaard, Laurenz Kellenberger, Jesús Cortés, Marc P Roddis, Matthew Deacon, James Staunton, Peter F Leadlay. Stereochemistry of catalysis by the ketoreductase activity in the first extension module of the erythromycin polyketide synthase. Biochemistry. 2002 Feb 26;41(8):2719-26