Biosynthetic characterization and combinatorial biocatalysis of the cysteine protease inhibitor E-64

E-64 is an irreversible and selective cysteine protease inhibitor prominently used in chemical biology and drug discovery. In this work, we uncovered and characterized the NRPS-independent pathway responsible for biosynthesis of E-64, which is widely conserved in fungi. Heterologous reconstitution and biochemical assays show the pathway starts with epoxidation of fumaric acid to the warhead (2 S ,3 S )- trans -epoxysuccinic acid with an α-ketoglutarate (αKG)/Fe(II)-dependent oxygenase, followed by successive condensation with an l -amino acid by an ATP-grasp enzyme, and with an amine by the first characterized amide bond synthetase from fungi. Both amide bond-forming enzymes displayed significant biocatalytic potential, including scalability, stereoselectivity towards the warhead and broader substrate scopes in forming the amide bonds. Combinatorial biocatalysis with the two amide-bond forming enzymes generated a library of cysteine protease inhibitors and led to more potent analogs towards cathepsin B. In addition, preparative synthesis of clinically relevant cysteine protease inhibitors was accomplished from a single reaction mixture. Our work highlights the importance of biosynthetic investigation for enzyme discovery and the potential of amide bond-forming enzymes as biocatalysts for a library synthesis of small molecules.