6-DEOXYERYTHRONOLIDE-B SYNTHASE 1 IS SPECIFICALLY ACYLATED BY A DIKETIDE INTERMEDIATE AT THE BETA-KETOACYL-ACYL CARRIER PROTEIN SYNTHASE DOMAIN OF MODULE-2

We have used 6-deoxyerythronolide B synthase (DEBS) as a model system to investigate molecular recognition by a modular polyketide synthase (PKS). DEBS consists of three proteins (DEBS1, -2, and -3) that biosyn thesize the polyketide skeleton of the antibiotic erythromycin from pr opionyl-CoA and methylmalonyl-CoA. Active sites within these multifunc tional proteins are organized into biosynthetic ''modules'', each of w hich catalyzes a discrete round of polyketide chain elongation and adj usts the appropriate level of beta-ketoacylthioester reduction, Using DEBS1, we demonstrate that there is a substantial degree of molecular recognition in the processing of the natural diketide chain elongation intermediate. Exogenously added (2S,3R)-2-methyl-3-hydroxypentanoic a cid N-acetylcysteamine thioester is exclusively recognized by its cogn ate beta-ketoacyl-acyl carrier protein synthase domain in module 2 (KS 2). Labeled diketide specifically acylated DEBS1 in crude protein extr acts and limited proteolysis localized the binding to module 2. The pr ecise site of acylation in DEBS1 was established by the finding that a Cys2200Ala mutant of DEBS1, lacking the KS2 active-site cysteine, did not undergo acylation by the diketide. Pretreatment of the wild-type protein with the beta-ketoacyl-ACP synthase inhibitor cerulenin also b locked acylation. These results indicate that in addition to the purel y organizational consequences resulting from the order of active-site domains, the programming of polyketide biosynthesis by modular PKSs in volves a substantial level of molecular recognition, This conclusion h as important implications for the use of PKSs to rationally design nov el polyketides.