Modular Oxime Formation by a trans‐AT Polyketide Synthase

Modular trans‐acyltransferase polyketide synthases (trans‐AT PKSs) are enzymatic assembly lines that biosynthesize complex polyketide natural products. Relative to their better studied cis‐AT counterparts, the trans‐AT PKSs introduce remarkable chemical diversity into their polyketide products. A notable example is the lobatamide A PKS, which incorporates a methylated oxime. Here we demonstrate biochemically that this functionality is installed on‐line by an unusual oxygenase‐containing bimodule. Furthermore, analysis of the oxygenase crystal structure coupled with site‐directed mutagenesis allows us to propose a model for catalysis, as well as identifying key protein‐protein interactions that support this chemistry. Overall, our work adds oxime‐forming machinery to the biomolecular toolbox available for trans‐AT PKS engineering, opening the way to introducing such masked aldehyde functionalities into diverse polyketides. Benzolactone enamides, a number of which incorporate a methylated oxime moiety, are produced by a range of organisms, and constitute a family of cytotoxic natural products. Here, we determine how this capped oxime group is installed during assembly of the model polyketide lobatamide by a modular trans‐AT polyketide synthase and provide molecular insight into the responsible mono‐oxygenase domain by X‐ray crystallography.