Mechanism and Stereochemistry of Polyketide Chain Elongation and Methyl Group Epimerization in Polyether Biosynthesis

The polyketide synthases responsible for the biosynthesis of the polyether antibiotics nanchangmycin ( 1 ) and salinomycin ( 4 ) harbor a number of redox-inactive ketoreductase (KR 0 ) domains that are implicated in the generation of C2-epimerized (2 S )-2-methyl-3-ketoacyl-ACP intermediates. Evidence that the natural substrate for the polyether KR 0 domains is, as predicted, a (2 R )-2-methyl-3-ketoacyl-ACP intermediate, came from a newly developed coupled ketosynthase (KS)-ketoreductase (KR) assay that established that the decarboxylative condensation of methylmalonyl-CoA with S -propionyl- N -acetylcysteamine catalyzed by the Nan[KS1][AT1] didomain from module 1 of the nanchangmycin synthase generates exclusively the corresponding (2 R )-2-methyl-3-ketopentanoyl-ACP ( 7a ) product. In tandem equilibrium isotope exchange experiments, incubation of [2- 2 H]-(2 R,3 S )-2-methyl-3-hydroxypentanoyl-ACP ( 6a ) with redox-active, epimerase-inactive EryKR6 from module 6 of the 6-deoxyerythronolide B synthase and catalytic quantities of NADP + in the presence of redox-inactive, recombinant NanKR1 0 or NanKR5 0, from modules 1 and 5 of the nanchangmycin synthase, or recombinant SalKR7 0 from module 7 of the salinomycin synthase, resulted in first-order, time-dependent washout of deuterium from 6a . Control experiments confirmed that this washout was due to KR 0 -catalyzed isotope exchange of the reversibly generated, transiently formed oxidation product [2- 2 H]-(2 R )-2-methyl-3-ketopentanoyl-ACP ( 7a ), consistent with the proposed epimerase activity of each of the KR 0 domains. Although they belong to the superfamily of short chain dehydrogenase-reductases, the epimerase-active KR 0 domains from polyether synthases lack one or both residues of the conserved Tyr-Ser dyad that has previously been implicated in KR-catalyzed epimerizations.