Defining the boundaries of substrate recognition within PKS domains is crucial to the rationally engineered biosynthesis of novel polyketide products, many of which could be prepared only with great difficulty, if at all, by direct chemical synthesis or semi-synthesis. Our results suggest that the KS2 domain of DEBS1 has a relatively relaxed specificity that can be exploited for the design and synthesis of medicinally important polyketide products.
A fourth fatty acid condensing enzyme was isolated from Eschenchia coli by its ability to restore elongating activity to a protein extract which had been treated with cerulenin, a condensing enzyme-specific inhibitor. The purified 3-ketoacyl-[acyl carrier protein] (ACP) synthase IV [3-oxoacyl-ACP synthase; acyl-ACP:malonyl-ACP C-acyltransferase (decarboxylating), EC 2.3
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 biosynthesize the polyketide skeleton of the antibiotic erythromycin from propionyl-CoA and methylmalonyl-CoA. Active sites within these multifunctional proteins are organized into biosynthetic "modules", each of which catalyzes a discrete round of polyketide chain elongation and adjusts 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 acid N-acetylcysteamine thioester is exclusively recognized by its cognate beta-ketoacyl-acyl carrier protein synthase domain in module 2 (KS2). Labeled diketide specifically acylated DEBS1 in crude protein extracts and limited proteolysis localized the binding to module 2. The precise site of acylation in DEBS1 was established by the finding that a Cys2200 Ala 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 blocked acylation. These results indicate that in addition to the purely organizational consequences resulting from the order of active-site domains, the programming of polyketide biosynthesis by modular PKSs involves a substantial level of molecular recognition. This conclusion has important implications for the use of PKSs to rationally design novel polyketides.
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