Modifying a loop region in ene-reductase to boost catalytic activity

Flavin mononucleotide (FMN)-dependent ene-reductases (ERs) belonging to the Old Yellow Enzyme (OYE) superfamily, are versatile biocatalysts with great potential for the production of fine and specialty chemicals, such as (-)-menthol. However, limited catalytic activity of OYEs hampers their practical application. To address this issue, loop engineering strategy was employed to modify the loop region (Loop 6) in close proximity to the substrate tunnel in ERs by introducing insertion and deletion (indel) mutation. The resulting indel mutants of OYE2p from Saccharomyces cerevisiae YJM1341, exhibited improved catalytic activity toward the model substrate citral compared to the wild-type (WT) OYE2p. The representative insertion mutant F298insGGG and the deletion mutant (F298-L299-T300) del exhibited enhanced specific activity toward the diverse tested alkenes. Molecular dynamics simulations revealed that the indel mutants result the reduction in key catalytic distances, the reshaped substrate-binding pocket and increased flexibility of Loop 6. Moreover, a generalizability test of Loop 6 engineering was conducted in other OYEs, eliciting comparable catalytic activity enhancement for OYE1, OYE3 from Saccharomyces cerevisiae and NCR from Zymomonas mobilis. This study not only revealed the vital roles of Loop 6 in ERs but also provided a general strategy for improving catalytic activity through rational redesign of the loop architecture.