Comparative Analysis of the Substrate Specificity of trans- versus cis-Acyltransferases of Assembly Line Polyketide Synthases

Polyketides are a large class of medicinally relevant natural products, many of which are produced in an assembly line fashion by multimodular polyketide synthases (PKSs). Each PKS module is composed of several enzymatic domains that together are responsible for one round of polyketide chain elongation and processing. The acyltransferase (AT) domains within each module are responsible for selection of coenzyme A (CoA)-linked extender units for incorporation into the growing polyketide chain.1 Because this specificity influences the structural diversity and biological activity of the natural product, AT domains are often targeted in the engineering of PKSs for the production of novel polyketides.2 The reaction catalyzed by AT domains is shown in Scheme 1. Each AT transfers an acyl group from an α-carboxyacyl-CoA (carboxyacyl-CoA) extender unit to the phosphopantetheine arm of an acyl carrier protein (ACP) domain. Assembly line PKSs are known to harbor two types of AT domains. In systems such as the 6-deoxyerythronolide B synthase (DEBS), each AT domain is paired with its own ACP domain; these ATs are typically referred to as cis-acting AT domains (Scheme 1A). In contrast, trans-acting ATs exist as stand-alone enzymes; they catalyze extender unit transfer onto one or more ACPs of a multimodular PKS assembly line (Scheme 1B,C).3 Examples of PKSs harboring trans-acting AT domains include assembly lines that synthesize disorazole,4 leinamycin,5 and bryostatin.6 Scheme 1 Reactions Catalyzed by Three Acyltransferase (AT) Domains trans-Acting AT domains have the potential to be useful engineering tools: a cis-AT could in principle be inactivated by site-directed mutagenesis, and the resulting PKS could be complemented with a trans-AT that transfers a different extender unit onto the ACP domain of the module. However, rational implementation of such a strategy requires a firm understanding of the carboxyacyl-CoA and ACP specificities of these AT domains. Most trans-AT domains are specific for malonyl-CoA. A trans-AT involved in the biosynthesis of kirromycin, however, was shown to preferentially utilize ethylmalonyl-CoA extender units.7 Indeed, this enzyme was able to load its cognate ACP with a range of non-natural extender units.8trans-Acting ATs may also have good tolerance for unnatural ACP substrates.9,10 For example, the AT from the disorazole synthase (DSZS) had higher specificity constants (kcat/KM) for ACP domains from DEBS than did an AT from the DEBS assembly line.10 In this study, we therefore sought to exploit a recently developed fluorometric assay11 to compare the specificity of the DSZS AT, the ethylmalonyl-specific kirromycin AT (KirCII), and a representative AT domain from DEBS for alternative carboxyacyl-CoA and ACP substrates. In turn, these quantitative insights led us to predict the efficacy with which the two trans-AT domains would complement a mutant of the DEBS assembly line in which a single AT domain had been catalytically inactivated. Our predictions were verified in vitro using a recently established LC-MS assay.12