title: Structure-Based Mechanism of Oleate Hydratase from Elizabethkingia meningoseptica.
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.
authors.affiliation: Institute of Molecular Biotechnology, NAWI Graz, Graz University of Technology, Petersgasse 14/2, 8010 Graz (Austria).|ACIB-Austrian Centre of Industrial Biotechnology, Petersgasse 14/2, 8010 Graz (Austria).
authors.forename: Matthias
authors.initials: M
authors.lastname: Engleder
authors.affiliation: ACIB-Austrian Centre of Industrial Biotechnology, Petersgasse 14/2, 8010 Graz (Austria).|Institute of Molecular Biosciences, NAWI Graz, University of Graz, Humboldtstrasse 50/3, 8010 Graz (Austria).
authors.forename: Tea
authors.initials: T
authors.lastname: Pavkov-Keller
authors.affiliation: ACIB-Austrian Centre of Industrial Biotechnology, Petersgasse 14/2, 8010 Graz (Austria).
authors.forename: Anita
authors.initials: A
authors.lastname: Emmerstorfer
authors.affiliation: ACIB-Austrian Centre of Industrial Biotechnology, Petersgasse 14/2, 8010 Graz (Austria).|Institute of Molecular Biosciences, NAWI Graz, University of Graz, Humboldtstrasse 50/3, 8010 Graz (Austria).
authors.forename: Altijana
authors.initials: A
authors.lastname: Hromic
authors.affiliation: Institute of Molecular Biotechnology, NAWI Graz, Graz University of Technology, Petersgasse 14/2, 8010 Graz (Austria).
authors.forename: Sabine
authors.initials: S
authors.lastname: Schrempf
authors.affiliation: ACIB-Austrian Centre of Industrial Biotechnology, Petersgasse 14/2, 8010 Graz (Austria).
authors.forename: Georg
authors.initials: G
authors.lastname: Steinkellner
authors.affiliation: ACIB-Austrian Centre of Industrial Biotechnology, Petersgasse 14/2, 8010 Graz (Austria).
authors.forename: Tamara
authors.initials: T
authors.lastname: Wriessnegger
authors.affiliation: Institute of Analytical Chemistry and Food Chemistry, NAWI Graz, Graz University of Technology, Stremayrgasse 9, 8010 Graz (Austria).
authors.forename: Erich
authors.initials: E
authors.lastname: Leitner
authors.affiliation: ACIB-Austrian Centre of Industrial Biotechnology, Petersgasse 14/2, 8010 Graz (Austria).|Institute of Organic Chemistry, NAWI Graz, Graz University of Technology, Stremayrgasse 9, 8010 Graz (Austria).
authors.forename: Gernot A
authors.initials: GA
authors.lastname: Strohmeier
authors.affiliation: DSM Chemical Technology R&D B.V., Innovative Synthesis, Urmonderbaan 22, 6167 RD Geleen (The Netherlands).
authors.forename: Iwona
authors.initials: I
authors.lastname: Kaluzna
authors.affiliation: DSM Chemical Technology R&D B.V., Innovative Synthesis, Urmonderbaan 22, 6167 RD Geleen (The Netherlands).
authors.forename: Daniel
authors.initials: D
authors.lastname: Mink
authors.affiliation: DSM Chemical Technology R&D B.V., Innovative Synthesis, Urmonderbaan 22, 6167 RD Geleen (The Netherlands).
authors.forename: Martin
authors.initials: M
authors.lastname: Schürmann
authors.affiliation: Institute of Biochemistry, NAWI Graz, Graz University of Technology, Petersgasse 12, 8010 Graz (Austria).
authors.forename: Silvia
authors.initials: S
authors.lastname: Wallner
authors.affiliation: Institute of Biochemistry, NAWI Graz, Graz University of Technology, Petersgasse 12, 8010 Graz (Austria).
authors.forename: Peter
authors.initials: P
authors.lastname: Macheroux
authors.affiliation: ACIB-Austrian Centre of Industrial Biotechnology, Petersgasse 14/2, 8010 Graz (Austria). karl.gruber@uni-graz.at.|Institute of Molecular Biosciences, NAWI Graz, University of Graz, Humboldtstrasse 50/3, 8010 Graz (Austria). karl.gruber@uni-graz.at.
authors.forename: Karl
authors.initials: K
authors.lastname: Gruber
authors.affiliation: Institute of Molecular Biotechnology, NAWI Graz, Graz University of Technology, Petersgasse 14/2, 8010 Graz (Austria). harald.pichler@tugraz.at.|ACIB-Austrian Centre of Industrial Biotechnology, Petersgasse 14/2, 8010 Graz (Austria). harald.pichler@tugraz.at.
authors.forename: Harald
authors.initials: H
authors.lastname: Pichler
mesh_terms: D020134:Catalytic Domain; D041965:Flavobacteriaceae / Q000201:enzymology*; D006836:Hydro-Lyases / Q000737:chemistry* / Q000378:metabolism*; D015394:Molecular Structure; D019301:Oleic Acid / Q000737:chemistry*
chemical_list: D019301:Oleic Acid; D006836:Hydro-Lyases
keywords: catalytic site; flavin cofactors; oleate hydratases; protein structures; redox chemistry