Dissecting the role of acyltransferase domains of modular polyketide synthases in the choice and stereochemical fate of extender units

Modular polyketide synthases (PKSs), such as the 6-deoxyerythronolide B syn thase (DEBS), are large multifunctional enzyme complexes that are organized into modules, where each module carries the domains needed to catalyze the condensation of an extender unit onto a growing polyketide chain. Each mod ule also dictates the stereochemistry of the chiral centers introduced into the backbone during the chain elongation process. Here we used domain muta genesis to investigate the role of the acyl transferase (AT) domains of ind ividual modules in the choice and stereochemical fate of extender units. Ou r results indicate that the AT domains of DEBS do not influence epimerizati on of the (2S)-methylmalonyl-CoA extender units. Hence, stereochemical cont rol of the methyl-branched centers generated by DEBS most likely resides in the ketosynthase (KS) domains of the individual modules. In contrast, seve ral recent studies have demonstrated that extender unit specificity can be altered by AT domain substitution. In some of these examples, the resulting polyketide was produced at considerably lower titers than the correspondin g natural product. We analyzed one such attenuated mutant of DEBS, in which the methylmalonyl transferase domain of module 2 was replaced with a malon yl transferase domain. As reported earlier, the resulting PKS produced only small quantities of the expected desmethyl analogue of 6-deoxyerythronolid e B. However, when the same hybrid module was placed as the terminal module in a truncated 2-module PKS, it produced nearly normal quantities of the e xpected desmethyl triketide lactone. These results illustrate the limits to modularity of these multifunctional enzymes. To dissect the role of specif ic amino acids in controlling AT substrate specificity, we exchanged severa l segments of amino acids between selected malonyl and methylmalonyl transf erases, and found that a short (23-35 amino acid) C-terminal segment presen t in all AT domains is the principal determinant of their substrate specifi city. Interestingly, its length and amino acid sequence vary considerably a mong the known AT domains. We therefore suggest that the choice of extender units by the PKS modules is influenced by a "hypervariable region", which could be manipulated via combinatorial mutagenesis to generate novel AT dom ains possessing relaxed or altered substrate specificity.