Structural and Mechanistic Basis for Orthoester Formation by αKG-Free Endoperoxide Isomerase in Novofumigatonin Biosynthesis

Orthoester-containing natural products possess unique oxygen-rich architectures; however, the enzymatic mechanism responsible for constructing these functional groups has remained unclear. In this study, we report the structure-function analysis of NvfE, a nonheme iron enzyme from <i>Aspergillus novofumigatus</i>, which catalyzes the Fe(II)-dependent isomerization of a reactive endoperoxide to generate the orthoester fumigatonoid C. Structural analysis of NvfE revealed that, although structurally similar to Fe(II)/αKG-dependent oxygenases, NvfE lacks the canonical αKG-binding pocket and instead features a nonconserved Glu149 residue that governs substrate recognition. Site-directed mutagenesis and QM/MM calculations confirmed the critical role of Glu149 in catalysis. Notably, Glu149 variants produced an alternative orthoester isomer, indicating its importance for product selectivity. Based on these results, we propose a mechanism for the unique αKG-free NvfE-catalyzed orthoester formation reaction. These results unveil a remarkable catalytic strategy for orthoester biogenesis and demonstrate the functional diversification of nonheme iron enzymes beyond oxidative chemistry.