Stereospecificity of ketoreductase domains 1 and 2 of the tylactone modular polyketide synthase.
Tylactone synthase (TYLS) is a modular polyketide synthase that catalyzes the formation of tylactone (1), the parent aglycone precursor of the macrolide antibiotic tylosin. TYLS modules 1 and 2 are responsible for the generation of antidiketide and triketide intermediates, respectively, each bound to an acyl carrier protein (ACP) domain. Each module harbors a ketoreductase (KR) domain. The stereospecificity of TYLS KR1 and TYLS KR2 has been determined by incubating each of the recombinant ketoreductase domains with reconstituted ketosynthase-acyltransferase [KS][AT] and ACP domains from the 6-deoxyerythronolide B synthase (DEBS) in the presence of the N-acetylcysteamine thioester of syn-(2S,3R)-2-methyl-3-hydroxypentanoate (6), methylmalonyl-CoA, and NADPH resulting in the exclusive formation of the ACP-bound (2R,3R,4S,5R)-2,4-methyl-3,5-dihydroxyhepanoyl triketide, as established by GC-MS analysis of the TMS ether of the derived triketide lactone 7. Both TYLS KR1 and KR2 therefore catalyze the stereospecific reduction of the 2-methyl-3-ketoacyl-ACP substrate from the re-face, with specificity for the reduction of the (2R)-methyl (D) diastereomer. The dehydration that is catalyzed by the dehydratase (DH) domains of TYLS module 2 to give the unsaturated (2E,4S,5R)-2,4-dimethyl-5-hydroxyhept-2-enoyl-ACP2 is therefore a syn elimination of water.
Castonguay
Roselyne
R
Department of Chemistry, Box H, Brown University, Providence, Rhode Island 02912-9108, USA.
Valenzano
Chiara R
CR
Chen
Alice Y
AY
Keatinge-Clay
Adrian
A
Khosla
Chaitan
C
Cane
David E
DE
D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D048630:Polyketide Synthases / Q000737:chemistry* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D013237:Stereoisomerism; D013379:Substrate Specificity; D015645:Tylosin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism
D001426:Bacterial Proteins; C027411:protylonolide; D048630:Polyketide Synthases; D000429:Alcohol Oxidoreductases; C087192:polyketide synthase ketoreductase; D015645:Tylosin