Precursor-Directed Biosynthesis: Stereospecificity for Branched-Chain Diketides of the β-Ketoacyl-ACP Synthase Domain 2 of 6-Deoxyerythronolide B Synthase
✍ Scribed by Kenji Kinoshita; Chaitan Khosla; David E. Cane
- Book ID
- 102256224
- Publisher
- John Wiley and Sons
- Year
- 2003
- Tongue
- German
- Weight
- 330 KB
- Volume
- 86
- Category
- Article
- ISSN
- 0018-019X
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✦ Synopsis
Abstract
Modular polyketide synthases such as 6‐deoxyerythronolide B synthase (DEBS) catalyze the biosynthesis of structurally complex natural products. Streptomyces coelicolor CH999/pJRJ2 harbors a plasmid encoding DEBS(KS1^0^), a mutant form of 6‐deoxyerythronolide B synthase that is blocked in the formation of 6‐deoxyerythronolide B (1, 6‐dEB) due to a mutation in the active site of the ketosynthase (KS1) domain that normally catalyzes the first polyketide chain‐elongation step of 6‐dEB biosynthesis. Administration of (2__S__,3__R__,4__S__)‐ and (2__S__,3__R__,4__R)‐3‐hydroxy‐2,4‐dimethylhexanoic acid N‐acetylcysteamine (SNAC) thioesters (=S‐[2‐(acetylamino)ethyl] (2__S,3__R__,4__S__)‐ and (2__S__,3__R__,4__R__)‐3‐hydroxy‐2,4‐dimethylhexanethioates) 3 and 4 in separate experiments to cultures of Streptomyces coelicolor CH999/pJRJ2 led to production of the corresponding (14__S__)‐ and (14__R__)‐14‐methyl analogues of 6‐dEB, 10 and 11, respectively. Unexpectedly, when a 3 : 2 mixture of 4 and 3 was fed under the same conditions, exclusively branched‐chain macrolactone 11 was isolated. In similar experiments, feeding of 3 and 4 to S. coelicolor CH999/pCK16, an engineered strain harboring DEBS1+TE(KS1^0^), resulted in formation of the branched‐chain triketide lactones 13 and 14, while feeding of the 3 : 2 mixture of 4 and 3 gave exclusively 14. The biochemical basis for this stereochemical discrimination was established by using purified DEBS module 2+TE to determine the steady‐state kinetic parameters for 3 and 4, with the k~cat~/K~M~ for 4 shown to be sevenfold greater than that of 3.