Functional Characterization and Synthetic Application of Is2-SDR, a Novel Thermostable and Promiscuous Ketoreductase from a Hot Spring Metagenome

In a metagenome mining-based search of novel thermostable hydroxysteroid dehydrogenases (HSDHs), enzymes that are able to selectively oxidize/reduce steroidal compounds, a novelshort-chain dehydrogenase/reductase (SDR), named Is2-SDR, was recently discovered. This enzyme,found in an Icelandic hot spring metagenome, shared a high sequence similarity with HSDHs, but,unexpectedly, showed no activity in the oxidation of the tested steroid substrates, e.g., cholic acid.Despite that, Is2-SDR proved to be a very active and versatile ketoreductase, being able to regioand stereoselectively reduce a diversified panel of carbonylic substrates, including bulky ketones,α- and ?-ketoesters, and ?-diketones of pharmaceutical relevance. Further investigations showedthat Is2-SDR was indeed active in the regio- and stereoselective reduction of oxidized steroid derivatives, and this outcome was rationalized by docking analysis in the active site model. Moreover,Is2-SDR showed remarkable thermostability, with an apparent melting temperature (TM) around75 oC, as determined by circular dichroism analysis, and no significant decrease in catalytic activity,even after 5 h at 80 oC. A broad tolerance to both water-miscible and water-immiscible organicsolvents was demonstrated as well, thus, confirming the potential of this new biocatalyst for itssynthetic application.