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Structure‐Based Mechanism of Oleate Hydratase from Elizabethkingia meningoseptica |
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| Authors: | Matthias Engleder Dr Tea Pavkov‐Keller Dr Anita Emmerstorfer Altijana Hromic Sabine Schrempf Dr Georg Steinkellner Dr Tamara Wriessnegger Prof Erich Leitner Dr Gernot A Strohmeier Dr Iwona Kaluzna Dr Daniel Mink Dr Martin Schürmann Dr Silvia Wallner Prof Peter Macheroux Prof Karl Gruber Dr Harald Pichler |
| Affiliation: | 1. Institute of Molecular Biotechnology, NAWI Graz, Graz University of Technology, Petersgasse 14/2, 8010 Graz (Austria);2. ACIB‐Austrian Centre of Industrial Biotechnology, Petersgasse 14/2, 8010 Graz (Austria);3. Institute of Molecular Biosciences, NAWI Graz, University of Graz, Humboldtstrasse 50/3, 8010 Graz (Austria);4. Institute of Analytical Chemistry and Food Chemistry, NAWI Graz, Graz University of Technology, Stremayrgasse 9, 8010 Graz (Austria);5. Institute of Organic Chemistry, NAWI Graz, Graz University of Technology, Stremayrgasse 9, 8010 Graz (Austria);6. DSM Chemical Technology R&D B.V., Innovative Synthesis, Urmonderbaan 22, 6167 RD Geleen (The Netherlands);7. Institute of Biochemistry, NAWI Graz, Graz University of Technology, Petersgasse 12, 8010 Graz (Austria) |
| Abstract: | Hydratases provide access to secondary and tertiary alcohols by regio‐ and/or stereospecifically adding water to carbon‐carbon double bonds. Thereby, hydroxy groups are introduced without the need for costly cofactor recycling, and that makes this approach highly interesting on an industrial scale. Here we present the first crystal structure of a recombinant oleate hydratase originating from Elizabethkingia meningoseptica in the presence of flavin adenine dinucleotide (FAD). A structure‐based mutagenesis study targeting active site residues identified E122 and Y241 as crucial for the activation of a water molecule and for protonation of the double bond, respectively. Moreover, we also observed that two‐electron reduction of FAD results in a sevenfold increase in the substrate hydration rate. We propose the first reaction mechanism for this enzyme class that explains the requirement for the flavin cofactor and the involvement of conserved amino acid residues in this regio‐ and stereoselective hydration. |
| Keywords: | catalytic site flavin cofactors oleate hydratases protein structures redox chemistry |
