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 (KR0) domains that are implicated in the generation of C2-epimerized (2S)-2-methyl-3-ketoacyl-ACP intermediates. Evidence that the natural substrate for the polyether KR0domains is, as predicted, a (2R)-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 (2R)-2-methyl-3-ketopentanoyl-ACP (7a) product. In tandem equilibrium isotope exchange experiments, incubation of [2-2H]-(2R,3S)-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 NanKR10or NanKR50, from modules 1 and 5 of the nanchangmycin synthase, or recombinant SalKR70from 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 KR0-catalyzed isotope exchange of the reversibly generated, transiently formed oxidation product [2-2H]-(2R)-2-methyl-3-ketopentanoyl-ACP (7a), consistent with the proposed epimerase activity of each of the KR0domains. Although they belong to the superfamily of short chain dehydrogenase-reductases, the epimerase-active KR0domains from polyether synthases lack one or both residues of the conserved Tyr-Ser dyad that has previously been implicated in KR-catalyzed epimerizations.
Department of Chemistry, Brown University , Box H, Providence, Rhode Island 02912-9108, United States.
Xinqiang
X
Xie
Department of Chemistry, Brown University , Box H, Providence, Rhode Island 02912-9108, United States.
Ashish
A
Garg
Departments of Chemical Engineering, Chemistry, and Biochemistry, Stanford University , Stanford, California 94305, United States.
Chaitan
0000-0001-6529-495X
C
Khosla
Department of Chemistry, Brown University , Box H, Providence, Rhode Island 02912-9108, United States.
David E
0000-0002-1492-9529
DE
Cane
D004987:Ethers / Q000737:chemistry / Q000378:metabolism*; D008968:Molecular Conformation; D048630:Polyketide Synthases / Q000378:metabolism*; D061065:Polyketides / Q000737:chemistry* / Q000378:metabolism; D011108:Polymers / Q000737:chemistry / Q000378:metabolism*; D013237:Stereoisomerism
D004987:Ethers; D061065:Polyketides; D011108:Polymers; D048630:Polyketide Synthases