| Summary: | Aiming at expanding the biocatalytic toolbox of ene-reductase enzymes, we decided to explore photosynthetic extremophile microorganisms as unique reservoir of (new) biocatalytic activities. We selected a new thermophilic ene-reductase homologue in <i>Chloroflexus aggregans</i>, a peculiar filamentous bacterium. We report here on the functional and structural characterization of this new enzyme, which we called <i>Ca</i>OYE. Produced in high yields in recombinant form, it proved to be a robust biocatalyst showing high thermostability, good solvent tolerance and a wide range of pH optimum. In a preliminary screening, <i>Ca</i>OYE displayed a restricted substrate spectrum (with generally lower activities compared to other ene-reductases); however, given the amazing metabolic ductility and versatility of <i>Chloroflexus aggregans</i>, further investigations could pinpoint peculiar chemical activities. X-ray crystal structure has been determined, revealing conserved features of Class III (or thermophilic-like group) of the family of Old Yellow Enzymes: in the crystal packing, the enzyme was found to assemble as dimer even if it behaves as a monomer in solution. The description of <i>Ca</i>OYE catalytic properties and crystal structure provides new details useful for enlarging knowledge, development and application of this class of enzymes.
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