{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,17]],"date-time":"2026-09-17T00:35:37Z","timestamp":1789605337641,"version":"build-2803163510"},"reference-count":48,"publisher":"MDPI AG","issue":"20","license":[{"start":{"date-parts":[[2022,10,12]],"date-time":"2022-10-12T00:00:00Z","timestamp":1665532800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"National Biodiversity Future Center","award":["CN00000033"],"award-info":[{"award-number":["CN00000033"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>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, \u03b1- and \u03b2-ketoesters, and \u03b1-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 \u00b0C, as determined by circular dichroism analysis, and no significant decrease in catalytic activity, even after 5 h at 80 \u00b0C. 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.<\/jats:p>","DOI":"10.3390\/ijms232012153","type":"journal-article","created":{"date-parts":[[2022,10,12]],"date-time":"2022-10-12T20:53:29Z","timestamp":1665608009000},"page":"12153","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":8,"title":["Functional Characterization and Synthetic Application of Is2-SDR, a Novel Thermostable and Promiscuous Ketoreductase from a Hot Spring Metagenome"],"prefix":"10.3390","volume":"23","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-3390-9638","authenticated-orcid":false,"given":"Erica Elisa","family":"Ferrandi","sequence":"first","affiliation":[{"name":"Istituto di Scienze e Tecnologie Chimiche \u201cGiulio Natta\u201d, Consiglio Nazionale delle Ricerche, Via Mario Bianco 9, 20131 Milano, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9589-3689","authenticated-orcid":false,"given":"Ivan","family":"Bassanini","sequence":"additional","affiliation":[{"name":"Istituto di Scienze e Tecnologie Chimiche \u201cGiulio Natta\u201d, Consiglio Nazionale delle Ricerche, Via Mario Bianco 9, 20131 Milano, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Susanna","family":"Bertuletti","sequence":"additional","affiliation":[{"name":"Istituto di Scienze e Tecnologie Chimiche \u201cGiulio Natta\u201d, Consiglio Nazionale delle Ricerche, Via Mario Bianco 9, 20131 Milano, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4753-3571","authenticated-orcid":false,"given":"Sergio","family":"Riva","sequence":"additional","affiliation":[{"name":"Istituto di Scienze e Tecnologie Chimiche \u201cGiulio Natta\u201d, Consiglio Nazionale delle Ricerche, Via Mario Bianco 9, 20131 Milano, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5977-9109","authenticated-orcid":false,"given":"Chiara","family":"Tognoli","sequence":"additional","affiliation":[{"name":"Istituto di Scienze e Tecnologie Chimiche \u201cGiulio Natta\u201d, Consiglio Nazionale delle Ricerche, Via Mario Bianco 9, 20131 Milano, Italy"},{"name":"Pharmaceutical Sciences Department, University of Milan, Via Mangiagalli 25, 20133 Milano, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marta","family":"Vanoni","sequence":"additional","affiliation":[{"name":"Istituto di Scienze e Tecnologie Chimiche \u201cGiulio Natta\u201d, Consiglio Nazionale delle Ricerche, Via Mario Bianco 9, 20131 Milano, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3399-7973","authenticated-orcid":false,"given":"Daniela","family":"Monti","sequence":"additional","affiliation":[{"name":"Istituto di Scienze e Tecnologie Chimiche \u201cGiulio Natta\u201d, Consiglio Nazionale delle Ricerche, Via Mario Bianco 9, 20131 Milano, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2022,10,12]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"5644","DOI":"10.1002\/anie.202001876","article-title":"Biocatalytic Reduction Reactions from a Chemist\u2019s Perspective","volume":"60","author":"Hollmann","year":"2021","journal-title":"Angew. Chem. Int. Ed."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1052","DOI":"10.1021\/acs.chemrev.1c00574","article-title":"Shortening Synthetic Routes to Small Molecule Active Pharmaceutical Ingredients Employing Biocatalytic Methods","volume":"122","author":"Schmermund","year":"2022","journal-title":"Chem. Rev."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"4111","DOI":"10.1021\/cr100334x","article-title":"Redox Reactions Catalyzed by Isolated Enzymes","volume":"111","author":"Monti","year":"2011","journal-title":"Chem. Rev."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"900554","DOI":"10.3389\/fctls.2022.900554","article-title":"Alcohol Dehydrogenases as Catalysts in Organic Synthesis","volume":"2","author":"Milagre","year":"2022","journal-title":"Front. Catal."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"3895","DOI":"10.1007\/s00018-008-8588-y","article-title":"Medium- and Short-Chain Dehydrogenase\/Reductase Gene and Protein Families","volume":"65","author":"Kavanagh","year":"2008","journal-title":"Cell. Mol. Life Sci."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"236","DOI":"10.1016\/j.cbi.2014.09.024","article-title":"The Aldo-Keto Reductases (AKRs): Overview","volume":"234","author":"Penning","year":"2015","journal-title":"Chem. Biol. Interact."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"593","DOI":"10.1016\/S0168-6445(03)00068-8","article-title":"Alcohol Dehydrogenases from Thermophilic and Hyperthermophilic Archaea and Bacteria","volume":"27","author":"Radianingtyas","year":"2003","journal-title":"FEMS Microbiol. Rev."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"711","DOI":"10.1007\/s10295-017-1920-5","article-title":"Improving the \u2018Tool Box\u2019 for Robust Industrial Enzymes","volume":"44","author":"Littlechild","year":"2017","journal-title":"J. Ind. Microbiol. Biotechnol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"fnx211","DOI":"10.1093\/femsle\/fnx211","article-title":"Metagenomics: Novel Enzymes from Non-Culturable Microbes","volume":"364","author":"Berini","year":"2017","journal-title":"FEMS Microbiol. Lett."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"630013","DOI":"10.3389\/fmicb.2021.630013","article-title":"Bioprospecting of Novel Extremozymes From Prokaryotes\u2014The Advent of Culture-Independent Methods","volume":"12","author":"Sysoev","year":"2021","journal-title":"Front. Microbiol."},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Sousa, J., Silv\u00e9rio, S.C., Costa, A.M.A., and Rodrigues, L.R. (2022). Metagenomic Approaches as a Tool to Unravel Promising Biocatalysts from Natural Resources: Soil and Water. Catalysts, 12.","DOI":"10.3390\/catal12040385"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"2879","DOI":"10.1111\/febs.13328","article-title":"Discovery and Characterization of Thermophilic Limonene-1,2-Epoxide Hydrolases from Hot Spring Metagenomic Libraries","volume":"282","author":"Ferrandi","year":"2015","journal-title":"FEBS J."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"2077","DOI":"10.1016\/j.biotechadv.2018.09.004","article-title":"Discovering Novel Hydrolases from Hot Environments","volume":"36","author":"Wohlgemuth","year":"2018","journal-title":"Biotechnol. Adv."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"144","DOI":"10.3389\/fbioe.2018.00144","article-title":"New Thermophilic \u03b1\/\u03b2 Class Epoxide Hydrolases Found in Metagenomes from Hot Environments","volume":"6","author":"Ferrandi","year":"2018","journal-title":"Front. Bioeng. Biotechnol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1023","DOI":"10.1007\/s00253-010-2507-5","article-title":"Molecular Cloning and Characterization of a Novel Metagenome-Derived Multicopper Oxidase with Alkaline Laccase Activity and Highly Soluble Expression","volume":"87","author":"Ye","year":"2010","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"1103","DOI":"10.1007\/s00253-010-2934-3","article-title":"A Bacterial Laccase from Marine Microbial Metagenome Exhibiting Chloride Tolerance and Dye Decolorization Ability","volume":"89","author":"Fang","year":"2011","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"4963","DOI":"10.1007\/s00253-017-8228-2","article-title":"Novel Thermostable Amine Transferases from Hot Spring Metagenomes","volume":"101","author":"Ferrandi","year":"2017","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"2000125","DOI":"10.1002\/biot.202000125","article-title":"Discovery and Characterization of a Novel Thermostable \u03b2-Amino Acid Transaminase from a Meiothermus Strain Isolated in an Icelandic Hot Spring","volume":"15","author":"Ferrandi","year":"2020","journal-title":"Biotechnol. J."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"4463","DOI":"10.1002\/ejoc.202000192","article-title":"Hydroxysteroid Dehydrogenases: An Ongoing Story","volume":"2020","author":"Ferrandi","year":"2020","journal-title":"Eur. J. Org. Chem."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"2474","DOI":"10.1002\/adsc.202000120","article-title":"Insights into the Substrate Promiscuity of Novel Hydroxysteroid Dehydrogenases","volume":"362","author":"Bertuletti","year":"2020","journal-title":"Adv. Synth. Catal."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"411","DOI":"10.1007\/s00248-015-0576-9","article-title":"Comparative Metagenomics of Eight Geographically Remote Terrestrial Hot Springs","volume":"70","author":"Menzel","year":"2015","journal-title":"Microb. Ecol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"3992","DOI":"10.1002\/ejoc.202100174","article-title":"Biocatalytic Approaches to the Enantiomers of Wieland\u2013Miescher Ketone and Its Derivatives","volume":"2021","author":"Bertuletti","year":"2021","journal-title":"Eur. J. Org. Chem."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"e202200105","DOI":"10.1002\/cbic.202200105","article-title":"Stereoselective Biocatalyzed Reductions of Ginger Active Components Recovered from Industrial Wastes","volume":"23","author":"Nasti","year":"2022","journal-title":"ChemBioChem"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"4948","DOI":"10.1002\/cctc.202101307","article-title":"Synthesis of \u03c9-Muricholic Acid by One-Pot Enzymatic Mitsunobu Inversion Using Hydroxysteroid Dehydrogenases","volume":"13","author":"Bertuletti","year":"2021","journal-title":"ChemCatChem"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"14659","DOI":"10.1021\/bi0203684","article-title":"Structure of Bacterial 3\u03b2\/17\u03b2-Hydroxysteroid Dehydrogenase at 1.2 \u00c5 Resolution: A Model for Multiple Steroid Recognition","volume":"41","author":"Benach","year":"2002","journal-title":"Biochemistry"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"W252","DOI":"10.1093\/nar\/gku340","article-title":"SWISS-MODEL: Modelling Protein Tertiary and Quaternary Structure Using Evolutionary Information","volume":"42","author":"Biasini","year":"2014","journal-title":"Nucleic Acids Res."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"675","DOI":"10.1007\/s10529-019-02678-2","article-title":"FabG: From a Core to Circumstantial Catalyst","volume":"41","author":"Shanbhag","year":"2019","journal-title":"Biotechnol. Lett."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"859","DOI":"10.1002\/prot.25036","article-title":"Structural and Biochemical Insights into 7\u03b2-Hydroxysteroid Dehydrogenase Stereoselectivity","volume":"84","author":"Savino","year":"2016","journal-title":"Proteins Struct. Funct. Bioinf."},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Gao, M., Nie, K., Qin, M., Xu, H., Wang, F., and Liu, L. (2021). Molecular Mechanism Study on Stereo-Selectivity of \u03b1 or \u03b2 Hydroxysteroid Dehydrogenases. Crystals, 11.","DOI":"10.3390\/cryst11030224"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"2375","DOI":"10.1111\/j.1742-4658.2010.07656.x","article-title":"Classification of the Short-Chain Dehydrogenase \u2044 Reductase Superfamily Using Hidden Markov Models","volume":"277","author":"Kallberg","year":"2010","journal-title":"FEBS J."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"10610","DOI":"10.1021\/acs.est.0c02071","article-title":"Anaerobic Degradation of Paraffins by Thermophilic Actinobacteria under Methanogenic Conditions","volume":"54","author":"Liu","year":"2020","journal-title":"Environ. Sci. Technol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"147671","DOI":"10.1155\/2015\/147671","article-title":"Archaeal Enzymes and Applications in Industrial Biocatalysts","volume":"2015","author":"Littlechild","year":"2015","journal-title":"Archaea"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"2209","DOI":"10.1128\/AEM.03873-12","article-title":"Thermostable Alcohol Dehydrogenase from Thermococcus kodakarensis KOD1 for Enantioselective Bioconversion of Aromatic Secondary Alcohols","volume":"79","author":"Wu","year":"2013","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"3062","DOI":"10.1046\/j.1432-1327.2001.02201.x","article-title":"Genetic and Biochemical Characterization of a Short-Chain Alcohol Dehydrogenase from the Hyperthermophilic Archaeon Pyrococcus furiosus","volume":"268","author":"Voorhorst","year":"2001","journal-title":"Eur. J. Biochem."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"6003","DOI":"10.1021\/jo800849d","article-title":"Stereoselective Bioreduction of Bulky-Bulky Ketones by a Novel ADH from Ralstonia sp","volume":"73","author":"Lavandera","year":"2008","journal-title":"J. Org. Chem."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"333","DOI":"10.1016\/0141-0229(88)90011-7","article-title":"Enzymatic Oxidoreduction of Steroids in Two-Phase Systems: Effects of Organic Solvents on Enzyme Kinetics and Evaluation of the Performance of Different Reactors","volume":"10","author":"Carrea","year":"1988","journal-title":"Enzyme Microb. Technol."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"102","DOI":"10.1016\/S0167-7799(84)80006-2","article-title":"Biocatalysis in Water-Organic Solvent Two-Phase Systems","volume":"2","author":"Carrea","year":"1984","journal-title":"Trends Biotechnol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"89","DOI":"10.3389\/fbioe.2019.00089","article-title":"Stereoselectivity Switch in the Reduction of \u03b1-Alkyl-\u03b2-Arylenones by Structure-Guided Designed Variants of the Ene Reductase OYE1","volume":"7","author":"Crotti","year":"2019","journal-title":"Front. Bioeng. Biotechnol."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"539","DOI":"10.1038\/msb.2011.75","article-title":"Fast, Scalable Generation of High-quality Protein Multiple Sequence Alignments Using Clustal Omega","volume":"7","author":"Sievers","year":"2011","journal-title":"Mol. Syst. Biol."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"W475","DOI":"10.1093\/nar\/gkr201","article-title":"Interactive Tree of Life v2: Online Annotation and Display of Phylogenetic Trees Made Easy","volume":"39","author":"Letunic","year":"2011","journal-title":"Nucleic Acids Res."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"283","DOI":"10.1107\/S0021889892009944","article-title":"PROCHECK: A Program to Check the Stereochemical Quality of Protein Structures","volume":"26","author":"MacArthur","year":"1993","journal-title":"J. Appl. Crystallogr."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"164","DOI":"10.1126\/science.1853201","article-title":"A Method to Identify Protein Sequences That Fold into a Known Three-Dimensional Structure","volume":"253","author":"Bowie","year":"1991","journal-title":"Science"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"727","DOI":"10.1006\/jmbi.1996.0897","article-title":"Development and Validation of a Genetic Algorithm for Flexible Docking","volume":"267","author":"Jones","year":"1997","journal-title":"J. Mol. Biol."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"252","DOI":"10.1080\/10242422.2021.1887150","article-title":"Biocatalysed Reductions of \u03b1-Ketoesters Employing Cyrene TM as Cosolvent","volume":"40","year":"2022","journal-title":"Biocatal. Biotransf."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"2545","DOI":"10.1002\/adsc.201200362","article-title":"Application of Tethered Ruthenium Catalysts to Asymmetric Hydrogenation of Ketones, and the Selective Hydrogenation of Aldehydes","volume":"354","author":"Jolley","year":"2012","journal-title":"Adv. Synth. Catal."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"1887","DOI":"10.1002\/ejoc.201500146","article-title":"Chiral (Cyclopentadienone)Iron Complexes for the Catalytic Asymmetric Hydrogenation of Ketones","volume":"2015","author":"Gajewski","year":"2015","journal-title":"Eur. J. Org. Chem."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"515","DOI":"10.1002\/adsc.201500933","article-title":"Asymmetric Transfer Hydrogenation of Ketones with Modified Grubbs Metathesis Catalysts: On the Way to a Tandem Process","volume":"358","author":"Gajewski","year":"2016","journal-title":"Adv. Synth. Catal."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"352","DOI":"10.1016\/j.jsbmb.2010.08.010","article-title":"Transformation of a Series of Saturated Isomeric Steroidal Diols by Aspergillus tamarii KITA Reveals a Precise Stereochemical Requirement for Entrance into the Lactonization Pathway","volume":"122","author":"Hunter","year":"2010","journal-title":"J. Steroid Biochem. Mol. Biol."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/23\/20\/12153\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T00:52:37Z","timestamp":1760143957000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/23\/20\/12153"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,10,12]]},"references-count":48,"journal-issue":{"issue":"20","published-online":{"date-parts":[[2022,10]]}},"alternative-id":["ijms232012153"],"URL":"https:\/\/doi.org\/10.3390\/ijms232012153","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,10,12]]}}}