Programming of erythromycin biosynthesis by a modular polyketide synthase.

Since the discovery and sequencing of 6-deoxyerythronolide B synthase 20 years ago, this exceptionally large, multifunctional protein remains the paradigm for the understanding of the structure and biochemical function of modular polyketide synthases. The broad-spectrummacrolide antibiotic erythromycin is one of several hundred closely related, branched chain, polyoxygenated polyketides, many of which are widely used in human and veterinarymedicine as antibiotic, immunosuppressant, antitumor, antifungal, and antiparasitic agents. The multistep assembly of the parent macrocyclic aglycon, 6-deoxyerythronolide B (6-dEB), is controlled by a large (2 MDa) modular protein known as 6-dEB synthase (DEBS) (1–4). The biosynthetic intermediates never leave thepolyketide synthase (PKS) but are passed along theDEBS assembly line from one acyl carrier protein (ACP) domain to the next. In fact, DEBS has served as the prototype of modular PKS gene clusters, dozens of which of both known and unknown function have now been sequenced from bacterial sources (5–7). Each homodimeric DEBS subunit contains two 160–200-kDa protein modules, each responsible for a single round of polyketide chain extension and functional group modification. Within each module are several catalytic domains of 100–400 amino acids each that are analogous in structure, function, and organization to the corresponding fatty acid synthase (FAS) components (8–10). All six DEBS modules contain three core domains consisting of 1) a -ketoacyl-ACP synthase (the ketosynthase (KS) domain) that catalyzes the key polyketide chainbuilding reaction, a decarboxylative condensation of a methylmalonyl-ACP building block with the polyketide chain provided by the upstream PKSmodule (see Figs. 1 and 2); 2) an acyltransferase (AT) domain that specifically loads the methylmalonyl-CoA extender unit onto the flexible 18-Å phosphopantetheine arm of the ACP domain; and 3) the ACP domain itself, which carries the polyketide biosynthetic intermediates from domain to domain and then delivers the resulting product to the KS domain of the downstreammodule. Additional FASlike domains are responsible for modification of the oxidation state and stereochemistry of the growing ACP-bound intermediates: a -ketoacyl-ACP reductase (KR) domain, a dehydratase (DH) domain, and an enoyl reductase (ER) domain. At the N terminus of the most upstream module is a loading didomain that primes the KS domain of module 1 with the propionyl starter unit. Finally, cyclization of the full-length macrocyclic polyketide and release of 6-dEB are controlled by a dedicated thioesterase domain located at the C terminus of the most downstream module.