BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS Two new ene-reductases from photosynthetic extremophiles enlarge the panel of old yellow enzymes: CtOYE and GsOYE Marina Simona Robescu 1 & Mattia Niero 1 & Mélanie Hall 2 & Laura Cendron 1 & Elisabetta Bergantino 1 Received: 19 June 2019 /Revised: 21 November 2019 /Accepted: 28 November 2019 # Springer-Verlag GmbH Germany, part of Springer Nature 2020 Abstract Looking for new ene-reductases with uncovered features beneficial for biotechnological applications, by mining genomes of photosynthetic extremophile organisms, we identified two new Old Yellow Enzyme homologues: CtOYE, deriving from the cyanobacterium Chroococcidiopsis thermalis, and GsOYE, from the alga Galdieria sulphuraria. Both enzymes were produced and purified with very good yields and displayed catalytic activity on a broad substrate spectrum by reducing α,β-unsaturated ketones, aldehydes, maleimides and nitroalkenes with good to excellent stereoselectivity. Both enzymes prefer NADPH but demonstrate a good acceptance of NADH as cofactor. CtOYE and GsOYE represent robust biocatalysts showing high thermo- stability, a wide range of pH optimum and good co-solvent tolerance. High resolution X-ray crystal structures of both enzymes have been determined, revealing conserved features of the classical OYE subfamily as well as unique properties, such as a very long loop entering the active site or an additional C-terminal alpha helix in GsOYE. Not surprisingly, the active site of CtOYE and GsOYE structures revealed high affinity toward anions caught from the mother liquor and trapped in the anion hole where electron-withdrawing groups such as carbonyl group are engaged. Ligands (para-hydroxybenzaldehyde and 2-methyl- cyclopenten-1-one) added on purpose to study complexes of GsOYE were detected in the enzyme catalytic cavity, stacking on top of the FMN cofactor, and support the key role of conserved residues and FMN cofactor in the catalysis. Keywords Biocatalysis . Ene-reductases . Extremophiles . OYE Introduction Ene-reductases (ERs) from the flavin mononucleotide (FMN)-containing old yellow enzyme (OYE) family of oxi- doreductases (EC 1.6.99.1) constitute the main class of en- zymes involved in C=C-double bond reduction reactions, per- formed at the expense of nicotinamide cofactors. These biocatalysts act on C=C-double bonds activated by at least one electron-withdrawing group (EWG); ideal substrates in- clude α,β-unsaturated carbonyl and nitro compounds, and maleimides (Stuermer et al. 2007; Toogood et al. 2010). Based on sequence alignment, thus on typical fingerprint mo- tifs (Oberdorfer et al. 2011; Litthauer et al. 2014), and quater- nary structure, ERs have been historically clustered into two different classes: classical and thermophilic-like enzymes, with the latter generally considered more attractive for indus- trial applications due to their higher thermostability and wider pH and co-solvent tolerance. Few years ago, the increasing number of ERs being characterized inspired a phylogenetic analysis that distinguished three, instead of the two previously established, comprehensive groups: class I, containing many classical OYEs from plants, bacteria and cyanobacteria; class II, regarding exclusively classical enzymes from fungi; class III, grouping the traditional thermophilic-like OYEs. Such classification also seemed to better account for the differences in substrate preference among the diverse enzymes; a number of sequences, however, remained unassigned to any class (Scholtissek et al. 2017a). Indeed, the latter ‘lost proteins’ have been included in a very new and extended classification Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00253-019-10287-2) contains supplementary material, which is available to authorized users. * Laura Cendron laura.cendron@unipd.it * Elisabetta Bergantino elisabetta.bergantino@unipd.it 1 Department of Biology, University of Padova, Viale G. Colombo 3, 35131 Padova, Italy 2 Department of Chemistry, University of Graz, Heinrichstrasse 28, 8010 Graz, Austria Applied Microbiology and Biotechnology https://doi.org/10.1007/s00253-019-10287-2