title: Structural basis for the enzymatic formation of the key strawberry flavor compound 4-hydroxy-2,5-dimethyl-3(2H)-furanone.
abstract: The last step in the biosynthetic route to the key strawberry flavor compound 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF) is catalyzed by Fragaria x ananassa enone oxidoreductase (FaEO), earlier putatively assigned as quinone oxidoreductase (FaQR). The ripening-induced enzyme catalyzes the reduction of the exocyclic double bond of the highly reactive precursor 4-hydroxy-5-methyl-2-methylene-3(2H)-furanone (HMMF) in a NAD(P)H-dependent manner. To elucidate the molecular mechanism of this peculiar reaction, we determined the crystal structure of FaEO in six different states or complexes at resolutions of ≤1.6 Å, including those with HDMF as well as three distinct substrate analogs. Our crystallographic analysis revealed a monomeric enzyme whose active site is largely determined by the bound NAD(P)H cofactor, which is embedded in a Rossmann-fold. Considering that the quasi-symmetric enolic reaction product HDMF is prone to extensive tautomerization, whereas its precursor HMMF is chemically labile in aqueous solution, we used the asymmetric and more stable surrogate product 2-ethyl-4-hydroxy-5-methyl-3(2H)-furanone (EHMF) and the corresponding substrate (2E)-ethylidene-4-hydroxy-5-methyl-3(2H)-furanone (EDHMF) to study their enzyme complexes as well. Together with deuterium-labeling experiments of EDHMF reduction by [4R-(2)H]NADH and chiral-phase analysis of the reaction product EHMF, our data show that the 4R-hydride of NAD(P)H is transferred to the unsaturated exocyclic C6 carbon of HMMF, resulting in a cyclic achiral enolate intermediate that subsequently becomes protonated, eventually leading to HDMF. Apart from elucidating this important reaction of the plant secondary metabolism our study provides a foundation for protein engineering of enone oxidoreductases and their application in biocatalytic processes.
authors.affiliation: Munich Center for Integrated Protein Science (CIPS-M) and Lehrstuhl für Biologische Chemie, 85350 Freising-Weihenstephan, Germany.
authors.forename: André
authors.initials: A
authors.lastname: Schiefner
authors.affiliation: Biotechnologie der Naturstoffe, Technische Universität München, 85350 Freising-Weihenstephan, Germany.
authors.forename: Quirin
authors.initials: Q
authors.lastname: Sinz
authors.affiliation: Munich Center for Integrated Protein Science (CIPS-M) and Lehrstuhl für Biologische Chemie, 85350 Freising-Weihenstephan, Germany.
authors.forename: Irmgard
authors.initials: I
authors.lastname: Neumaier
authors.affiliation: Biotechnologie der Naturstoffe, Technische Universität München, 85350 Freising-Weihenstephan, Germany. Electronic address: schwab@wzw.tum.de.
authors.forename: Wilfried
authors.initials: W
authors.lastname: Schwab
authors.affiliation: Munich Center for Integrated Protein Science (CIPS-M) and Lehrstuhl für Biologische Chemie, 85350 Freising-Weihenstephan, Germany.
authors.forename: Arne
authors.initials: A
authors.lastname: Skerra
mesh_terms: D002384:Catalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D031985:Fragaria / Q000201:enzymology*; D005663:Furans / Q000737:chemistry* / Q000378:metabolism; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000737:chemistry* / Q000378:metabolism; D009249:NADP / Q000737:chemistry* / Q000378:metabolism; D010940:Plant Proteins; D017510:Protein Folding
chemical_list: D005663:Furans; D010940:Plant Proteins; C058427:furaneol; D009249:NADP; D016660:NAD(P)H Dehydrogenase (Quinone)
keywords: Dehydrogenase; Enone Oxidoreductase; Enzyme Mechanisms; Enzyme Structure; Furaneol; NADPH; Secondary Metabolism; X-ray Crystallography