Substrate scope and catalytic mechanism of α, β-epoxyketone synthase EpnF illuminated by in-situ esterase-mediated deprotection

Abstract

, {beta}-Epoxyketones are an important class of bacterial natural products with wide-ranging potential applications in oncology, immunology, and infectious disease. Their clinical potential derives from the , {beta}-epoxyketone pharmacophore, which covalently modifies the N-terminal catalytic threonine residue of proteasome {beta}-subunits with high selectivity. Although a synthetic , {beta}-epoxyketone (Carfilzomib) is approved for clinical use, stereo-controlled synthesis of the pharmacophore remains challenging, involving either multiple steps and energy-intensive processes, or unsustainable reagents. Unusual flavoenzymes catalyze the assembly of the pharmacophore in , {beta}-epoxyketone biosynthesis. A detailed understanding of the substrate scope and catalytic mechanism of these enzymes has thus far been limited by the intrinsic instability of their - (di)methyl-{beta}-ketoacid substrates. Here, we report the development and application of an esterase-mediated unmasking strategy for in-situ generation of these substrates from the corresponding methyl esters. Using this approach, we demonstrate that EpnF, the epoxyketone synthase involved in eponemycin / TMC-86A biosynthesis, tolerates a broad range of synthetic substrate analogs, including several with N-terminal protecting groups widely used in peptide synthesis. These findings establish that EpnF has the potential to be developed into a useful biocatalyst for the chemoenzymatic synthesis of dipeptidyl epoxyketone precursors of clinically approved drugs and drug candidates. To elucidate the molecular basis for catalysis of , {beta}-epoxyketone formation by EpnF, substrate docking and molecular dynamics simulations were performed on a well-validated AlphaFold model, providing support for a previously proposed decarboxylation-dehydrogenation-monooxygenation mechanism. Site-directed mutagenesis and LC-MS analysis validated the proposed roles of key active-site residues in substrate positioning and catalysis of epoxide formation. Collectively, these results demonstrate that EpnF and related enzymes belong to a new class of internal flavoprotein monooxygenases and provide a foundation for developing epoxyketone synthases into useful biocatalysts for the sustainable synthesis of high-value ,{beta}-epoxyketones.