The ene-reductase (ERED) enzyme family is widely used in biocatalysis for the asymmetric reduction of carbon-carbon double bonds and has recently gained importance in sustainable synthesis. Some extremophilic enoate reductases (EnoRs) uniquely combine both FAD and FMN flavin cofactors with [4Fe–4S] clusters, suggesting unexplored catalytic potential beyond the classical hydride transfer mechanism characteristic of most EREDs.

This thesis investigates five thermophilic EnoRs: SsOYE, StOYE, SiOYE, AmOYE, and NbOYE. Successful heterologous expression in E. coli and subsequent purification were achieved for SiOYE, SsOYE, and AmOYE, with SiOYE exhibiting the highest purification yield. Biochemical characterization of SiOYE demonstrated a temperature-dependent increase in specific activity, with optimal performance observed in phosphate buffer. Biocatalytic conversions revealed that SiOYE and SsOYE are highly efficient in reducing small enones, achieving up to 95% conversion of 3-buten-2-one at mild temperatures (30 °C) under both aerobic and anaerobic conditions. The consistency between aerobic and anaerobic yields highlights a remarkable oxygen tolerance. However, limitations in the substrate scope were observed for sterically demanding or poorly activated substrates; conversions were unsuccessful for β-methyl substituted cyclic enones, methyl crotonate, and mono- and dicarboxylic acids such as methacrylic and itaconic acid. Alongside these findings, an high performance liquid chromatography (HPLC) method was developed and optimized for the quantification of FAD and FMN in purified enzyme samples, and electron paramagnetic resonance (EPR) spectroscopy was employed to probe the iron-sulfur cluster composition of the enzymes. Together, these results expand the functional understanding of thermophilic EnoRs, highlighting their robust stability and synthetic utility while laying the groundwork for further exploration of their multi-cofactor reactivity in asymmetric biocatalysis.
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