Determinants of substrate specificity in mint pulegone reductase, a double bond reductase of the medium-chain dehydrogenase/reductase superfamily.

Pulegone reductase from peppermint (Mentha × piperita; MpPulR) is an NADPH-dependent medium-chain dehydrogenase/reductase that catalyzes the asymmetric reduction of the C4-C7 double bond of (+)-pulegone to (-)-menthone and (+)-isomenthone. To define the structural determinants of substrate recognition and catalytic turnover, we integrated structure-based modeling (docking and molecular dynamics (MD) simulations) with experimental mutagenesis and comprehensive substrate profiling. Residues positioned closely to (+)-pulegone in homology-based structural models were subjected to L-Ala-scanning mutagenesis. Four substitutions (Y53A, F66A, Y78A, Y257A) caused severe activity losses, consistent with a binding pocket in which Y78 supports carbonyl anchoring via hydrogen bonding and aromatic residues (Y53, F66, and Y257) creating a hydrophobic cavity that enforces a productive substrate orientation. Other residues, including L56, I63, M135, F281, and V282, line the perimeter of the active-site cavity, thus providing architectural constraints for substrate binding. Screening of 40 candidate monoterpenoids revealed measurable activity with only six substrates, and kinetic analysis demonstrated exceptionally high catalytic efficiency toward piperitenone and piperitenone oxide, moderate efficiency with (+)-pulegone, and lower efficiency with citral, (-)-pulegone, and isoegomaketone. MD simulations of productive versus non-productive ligands indicated binding requirements that include stable carbonyl-Y78 hydrogen bonding, enzyme-ligand complex stability, and favorable cofactor-double bond alignment. Finally, an essential oil survey across diverse accessions of the genus Mentha and functional characterization of 2 Mentha longifolia orthologs indicate that MpPulR-like enzymes are promiscuous reductases likely contributing to the interspecific diversification of essential oil profiles.