Characterization of two novel alcohol short-chain dehydrogenases/reductases from Ralstonia eutropha H16 capable of stereoselective conversion of bulky substrates

• A set of probable short-chain dehydrogenases/reductases and alcohol dehydrogenases of R. eutropha H16 was screened for their functionality.
• Two novel short-chain dehydrogenases/reductases were characterized.
• They are among rare enzymes capable of transforming bulky–bulky substrates and are highly S-enantioselective.
• Computational analysis revealed that SDR A5 and SDR B3 belong to different phylogenetic clusters.
• Homologous protein expression in R. eutropha H16 was performed to provide the optimal functional expression conditions.

Biocatalysis has significant advantages over organic synthesis in the field of chiral molecule production and several types of stereoselective enzymes are already in use in industrial biotechnology. However, there is still a high demand for new enzymes capable of transforming bulky molecules with sufficient operability. In order to reveal novel high-potential biocatalysts, the complete genome of the β-proteobacterium Ralstonia eutropha H16 was screened for potential short-chain dehydrogenases/reductases (SDRs). We were able to identify two (S)-enantioselective SDRs named A5 and B3. These showed clear preference towards long-chain and aromatic secondary alcohols, aldehydes and ketones, with diaryl diketone benzil as one of the best substrates. In addition the phylogenetic analysis of all enzyme types, which are known to facilitate benzil reduction, revealed at least two separate evolutionary clusters. Our results indicate the biotechnological potential of SDRs A5 and B3 for the production of chiral compounds with potential commercial value.