Reconstituting modular activity from separated domains of 6-deoxyerythronolide B synthase

The hallmark of a type I polyketide synthase (PKS), such as the 6-deoxyerythronolide B synthase (DEBS), is the presence of catalytic modules comprised of covalently fused domains acting together to catalyze one round of chain elongation. In addition to an obligate ketosynthase (KS), acyl transferase (AT), and acyl carrier protein (ACP), a module may also include a ketoreductase (KR), dehydratase (DH), and/or enoyl reductase (ER) domain. The size, flexibility, and fixed domain-domain stoichiometry of these PKS modules present challenges for structural, mechanistic, and protein-engineering studies. Here, we have harnessed the power of limited proteolysis and heterologous protein expression to isolate and characterize individual domains of module 3 of DEBS, a 150-kD protein consisting of a KS, an AT, an ACP, and an inactive KR domain. Two interdomain boundaries were identified via limited proteolysis, which led to the production of a 90-kD KS-AT, a 142-kD KS-AT-KR(0), and a 10-kD ACP as structurally stable stand-alone proteins. Each protein was shown to possess the requisite catalytic properties. In the presence of the ACP, both the KS-AT and the KS-AT-KR(0) proteins were able to catalyze chain elongation as well as the intact parent module. Separation of the KS from the ACP enabled direct interrogation of the KS specificity for both the nucleophilic substrate and the partner ACP. Malonyl and methylmalonyl extender units were found to be equivalent substrates for chain elongation. Whereas ACP2 and ACP4 of DEBS could be exchanged for ACP3, ACP6 was a substantially poorer partner for the KS. Remarkably, the newly identified proteolytic sites were conserved in many PKS modules, raising the prospect of developing improved methods for the construction of hybrid PKS modules by engineering domain fusions at these interdomain junctions.