Molecular insights into the substrate recognition and quasi-unidirectional catalysis of OpaE, an atypical fungal enzyme from the Asp/Glu racemase family

Enzymes of the Asp/Glu racemase family, mostly found in bacteria, typically catalyze bidirectional reactions with similar rate because of their pseudosymmetrical structure and minimal differences between substrate isomers. We recently identified OpaE, an atypical fungal enzyme that catalyzes the d-to-l conversion of nonribosomal peptide derivatives in oxepinamide E/F biosynthesis from Aspergillus ustus. However, the structure and catalytic mechanism remained poorly understood. In this study, we present the crystal structure of OpaE, which reveals a pseudosymmetrical architecture characteristic of bidirectional racemases, yet it catalyzes the "quasi-unidirectional" d-to-l conversion, yielding approximately 90% of the l-form. This "quasi-unidirectional" activity arises from structural divergence between substrate isomers, which leads to differences in binding modes, energy barriers, and intermediate stabilities, as well as distinct thermodynamic properties, as revealed by crystal structure, docking, molecular dynamics, and quantum mechanics/molecular mechanics-based metadynamics simulations. Phylogenetic analysis revealed that OpaE has evolved specific structural adaptations, including hydrophobic pockets and loop shifts, to optimize substrate binding and catalysis, which ultimately favor "quasi-unidirectional" activity and reflect a distinct evolutionary path. In summary, our study deepens our understanding of the structural and mechanistic diversity within the Asp/Glu family and highlights OpaE's potential for biotechnological applications, particularly in chiral substrate transformations.