Discovery and Biosynthesis of Pseudoamides Reveal Enzymatic Cyclization of the Polyene Precursor to 5-5 Bicyclic Tetramate Macrolactams

Polycyclic tetramate macrolactams (PoTeMs) comprise a family of pharmacologically promising macrolactams that possess a tetramic acid moiety and various carbocyclic ring systems. Despite the progress in elucidating the biosynthetic pathways of representative PoTeMs, the mechanism by which the bicyclic system is formed remains unclear. Here, we report the targeted discovery of 5-5 bicyclic pseudoamides A-C (3-5) through heterologous expression of the recombinant pel cluster, which is a novel PoTeM cluster for products bearing a C13-C20 cyclization pattern. We also demonstrated that two FMN-dependent oxidoreductases, Pel1 and Pel3, are required for the generation of the 5-5 bicyclic system. Pel1 catalyzes a reductive cyclization reaction to form the outer 5-membered ring, which involves the incorporation of the hydride of FMNH2 and one proton from water. Pel3 represents the unique cyclase catalyzing a C13-C20 cyclization coupled with the C-12 hydroxylation and is proposed to transfer oxygen from FMN-C4a-hydroxyperoxide to the substrate. Thus, this work establishes key biochemical insights into the formation of the 5-5 bicyclic system in PoTeM biosynthesis.