Enzymatic assembly of the salinosporamide γ-lactam-β-lactone anticancer warhead

The marine microbial natural product salinosporamide A (Marizomib) is a potent proteasome inhibitor currently in clinical trials for the treatment of brain cancer. Salinosporamide A is characterized by a complex and densely functionalized [Formula: see text]-lactam- [Formula: see text]-lactone bicyclic warhead, the assembly of which has long remained a biosynthetic mystery. Here, we report an enzymatic route to the salinosporamide core catalyzed by a standalone ketosynthase, SalC. Chemoenzymatic synthesis of carrier protein-tethered substrates, as well as intact proteomics, allowed us to probe the reactivity of SalC and understand its role as an intramolecular aldolase/β-lactone synthase with roles in both transacylation and bond forming reactions. Additionally, we present the 2.85 Å SalC crystal structure that, combined with site-directed mutagenesis, allowed us to propose a bicyclization reaction mechanism. This work challenges our current understanding of the role of ketosynthase enzymes and establishes a basis for future efforts towards streamlined production of a clinically relevant chemotherapeutic.