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

Abstract

In a metagenome mining-based search of novel thermostable hydroxysteroid dehydrogenases (HSDHs), enzymes that are able to selectively oxidize/reduce steroidal compounds, a novel short-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 regio- and stereoselectively reduce a diversified panel of carbonylic substrates, including bulky ketones, α- and β-ketoesters, and α-diketones of pharmaceutical relevance. Further investigations showed that 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) around 75 °C, as determined by circular dichroism analysis, and no significant decrease in catalytic activity, even after 5 h at 80 °C. A broad tolerance to both water-miscible and water-immiscible organic solvents was demonstrated as well, thus, confirming the potential of this new biocatalyst for its synthetic application.

Promiscuity and Selectivity of Hydroxysteroid Dehydrogenases in the Biocatalyzed Reduction of 1,2‐Diketones

“…It comprised 13 HSDHs from various sources and showing different stereo‐ and regioselectivity, i. e., 7α‐, 7β‐ and 12α‐HSDHs, along with the broad scope reductase Is2‐SDR recently discovered by us in a hot spring metagenome. This enzyme was included in the study because it shows some phylogenetic relationship with known HSDHs and is active as a 3β‐reductase with different bile acids and steroidal substrates [15] . All these enzymes were obtained by heterologous expression in Escherichia coli and purified to homogeneity by affinity chromatography.…”

“…This enzyme was included in the study because it shows some phylogenetic relationship with known HSDHs and is active as a 3β-reductase with different bile acids and steroidal substrates. [15] All these enzymes were obtained by heterologous expression in Escherichia coli and purified to homogeneity by affinity chromatography. It is worth noting that none of them show any relevant sequence similarity to those previously mentioned in the Introduction, such as 2,3-butanediol dehydrogenases [17,18] and benzil reductases.…”

“…The 3D structural model of Is2‐SDR was previously generated by using the SWISS‐MODEL automated server and the structure of the homo tetrameric 3‐oxoacyl‐[acyl‐carrier protein] reductase from Listeria monocytogenes (Lm‐FabG, PDB ID: 4JRO) as template as described in [15] .…”

“…Interesting results were obtained with our HSDHs library when it was applied to the reduction of Wieland‐Miescher ketone (Figure 1D) [12] and of other complex bioactive natural products, like 6‐gingerol (Figure 1E), [13] as well as towards “bulky‐bulky” ketones such as aryl decorated γ‐keto esters, synthetic precursors of valuable bioactive metabolites (Figure 1F). [14,15] …”

“…[11] Interesting results were obtained with our HSDHs library when it was applied to the reduction of Wieland-Miescher ketone (Figure 1D) [12] and of other complex bioactive natural products, like 6-gingerol (Figure 1E), [13] as well as towards "bulky-bulky" ketones such as aryl decorated γ-keto esters, synthetic precursors of valuable bioactive metabolites (Figure 1F). [14,15] In this work, to get further insights into HSDHs promiscuity, we considered the biocatalyzed reduction of 1,2-diketones to optically active α-hydroxy ketones, a topic previously investigated by various groups who used generic alcohol dehydro-genases (ADHs) to achieve the enzyme-mediated preparation of these useful chiral building blocks. [16] Concerning aliphatic vicinal diketones, the selective asymmetric reduction of these substrates has been mainly studied using 2,3-butanediol dehydrogenases (BDHs).…”