Journal
CHEMBIOCHEM
Volume 11, Issue 2, Pages 197-207Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/cbic.200900570
Keywords
asymmetric bioreduction; biocatalysis; thermostable enzymes; unsaturated alkenes; UV/Vis spectroscopy; X-ray crystallography
Funding
- UK Biotechnology and Biological Sciences Research Council (BBSRC)
- Biotechnology and Biological Sciences Research Council [BB/E010717/1, BB/D01963X/1] Funding Source: researchfish
- BBSRC [BB/E010717/1, BB/D01963X/1] Funding Source: UKRI
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We report the crystal structure of a thermophilic ene reductase (TOYE) isolated from Thermoanaerobacter pseudethanolicus E39. The crystal structure reveals a tetrameric enzyme and an active site that is relatively large compared to most other structurally determined and related Old Yellow Enzymes. The enzyme adopts higher order oligomeric states (octamers and dodecamers) in solution, as revealed by sedimentation velocity and multiangle laser light scattering. Bead ling indicates that the solution structure is consistent with the basic tetrameric structure observed in crystallographic dies and electron microscopy. TOYE is stable at high temperatures (T-m > 70 degrees C) and shows increased resistance to denaturation in water-miscible organic solvents compared to the mesophilic Old Yellow Enzyme family member, pentaerythritol tetranitrate reductase. TOYE has typical ene-reductase properties of the Old Yellow Enzyme family. There is currently major interest in using Old Yellow Enzyme family members in the preparative biocatalysis of a number of activated alkenes. The increased stability of TOYE in organic solvents is advantageous for bio-transformations in which water-miscible organic solvents and biphasic reaction conditions are required to both deliver novel substrates and minimize product racemisation.
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