An Unusual Fatty Acyl:Adenylate Ligase (FAAL)–Acyl Carrier Protein (ACP) Didomain in Ambruticin Biosynthesis

The divinylcyclopropane (DVC) fragment of the ambruticins is proposed to be formed via a unique polyene cyclization mechanism in which the unusual didomain AmbG plays a key role. It is proposed to activate the branched thioester carboxylic acid resulting from the polyene cyclisation and to transfer it to its associated ACP. After oxidative decarboxylation, the intermediate is channeled back into polyketide synthase (PKS) processing. AmbG was previously annotated as an adenylation-thiolation didomain with a very unusual substrate selectivity code, but has not yet been biochemically studied. Based on sequence and homology model analysis, we reannotate AmbG as a fatty acyl:adenylate ligase-acyl carrier protein didomain with unusual substrate specificity. The expected adenylate-forming activity on fatty acids was confirmed by in vitro studies. AmbG also adenylates a number of structurally diverse carboxylic acids, including functionalized fatty acids, unsaturated and aromatic carboxylic acids. HPLC-MS analysis and competition experiments showed that AmbG preferentially acylates its ACP with long-chain hydrophobic acids and tolerates a p-system and a branch near the carboxylic acid. AmbG is the first characterized example of an FAAL-ACP didomain that is centrally located in a PKS and apparently activates a polyketidic intermediate. This is an important step towards deeper biosynthetic studies such as a partial reconstitution of the ambruticin pathway to elucidate DVC formation.