Functional characterization and stability improvement of a ‘thermophilic-like’ ene-reductase from Rhodococcus opacus 1CP

Ene-reductases (ERs) are widely applied for the asymmetric synthesis of relevant industrial chemicals. A novel ER OYE Ro 2 was found within a set of 14 putative old yellow enzymes (OYEs) obtained by genome mining of the actinobacterium Rhodococcus opacus 1CP. Multiple sequence alignment suggested that the enzyme belongs to the group of ‘thermophilic-like’ OYEs. OYE Ro 2 was produced in Escherichia coli and biochemically characterized. The enzyme is strongly NADPH dependent and uses non-covalently bound FMNH 2 for the reduction of activated α,β-unsaturated alkenes. In the active form OYE Ro 2 is a dimer. Optimal catalysis occurs at pH 7.3 and 37°C. OYE Ro 2 showed highest specific activities (45-50 U mg -1 ) on maleimides, which are efficiently converted to the corresponding succinimides. The OYE Ro 2-mediated reduction of prochiral alkenes afforded the ( R )-products with excellent optical purity ( ee > 99%). OYE Ro 2 is not as thermo-resistant as related OYEs. Introduction of a characteristic intermolecular salt bridge by site-specific mutagenesis raised the half-life of enzyme inactivation at 32°C from 28 to 87 min and improved the tolerance toward organic co-solvents. The suitability of OYE Ro 2 for application in industrial biocatalysis is discussed.