(582bk) Characterization and Engineering of Acyltransferase Domains in the Pursuit of Novel Polyketide Therapeutics

Natural products have historically been an invaluable resource in the discovery and development of therapeutically active compounds.  The polyketide class of natural products has seen huge success in the commercial drug arena, acting as antibiotics, anticancer agents, immunosuppressants, and other widely-used therapeutics. This biological diversity stems largely from the structural diversity of these small molecules, many of which are produced in an assembly-line fashion by large enzymatic complexes called modular polyketide synthases (PKSs). Each module of a PKS is comprised of several domains, and each domain plays a distinct role in extending and chemically modifying the growing polyketide chain. The acyltransferase domain (AT) is responsible for selection and incorporation of simple Coenzyme A- (CoA-) linked building blocks, and therefore acts as a gatekeeper to polyketide structural diversity. Engineering of the AT domain for incorporation of novel building blocks is an attractive approach in the production of new therapeutic polyketides.

This work presents the characterization of an AT domain from the 6-deoxyerythronolide B synthase (DEBS), the PKS responsible for producing the aglycone precursor to the antibiotic erythromycin. DEBS is arguably the most highly studied PKS and often acts as a model system for understanding modular polyketide biosynthesis. We have utilized a continuous, coupled enzymatic assay to examine the steady state kinetics of AT-catalyzed transacylation. The transacylation reaction includes the formation of an acyl-AT intermediate and subsequent attack by a nucleophilic phosphopantetheine prosthetic group on the adjacent acyl carrier protein (ACP). We discuss the substrate (α-carboxyacyl-CoA) and ACP specificity of the transacylation reaction, focusing on the mechanistic determinants of this specificity. This knowledge is necessary for successful engineering of the specificity of AT domains.

Previous attempts to engineer AT domains for novel extender unit incorporation have focused primarily on the swapping of an entire domain for one with alternate substrate specificity. These attempts often come at the expense of the appropriate protein-protein interactions necessary for successful polyketide chain extension and therefore lead to drastically decreased product titers. A more conservative and increasingly popular approach for engineering AT domain specificity involves the targeted mutagenesis of the active site or adjacent residues. We present kinetic data on the “best” AT site-directed mutants characterized in the literature to date, and reveal that these mutants have drastically attenuated activity in vitro. This decreased activity (rather than enhanced specificity) is likely what allows these mutants to incorporate a nonnative substrate in vivo. This reveals extensive room for improvement in the engineering of AT specificity by site-directed mutagenesis. We present briefly our future plans for tackling this engineering challenge, with the concomitant goal of generating novel polyketides by incorporation of diverse building blocks.