4IDC

Structure of the Fragaria x ananassa enone oxidoreductase in complex with NADPH and HDMF

Summary for 4IDC

• Entry DOI: 10.2210/pdb4idc/pdb
• Related: 4IDA 4IDB 4IDD 4IDE 4IDF
• Descriptor: Ripening-induced protein, NADPH DIHYDRO-NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE, (2R)-4-hydroxy-2,5-dimethylfuran-3(2H)-one, ... (6 entities in total)
• Functional Keywords: medium chain dehydrogenase/reductase family, zinc-independent, rossmann fold, enone oxidoreductase, furaneol, hydride transfer, nadph, nadh, hdmf, 4-hydroxy-2, 5-dimethyl-3(2h)-furanone, oxidoreductase
• Biological source: Fragaria vesca (woodland strawberry)
• Total number of polymer chains: 1
• Total formula weight: 36471.13

Authors

Schiefner, A.,Skerra, A. (deposition date: 2012-12-12, release date: 2013-04-17, Last modification date: 2023-11-08)

Primary citation

Schiefner, A.,Sinz, Q.,Neumaier, I.,Schwab, W.,Skerra, A.
Structural basis for the enzymatic formation of the key strawberry flavor compound 4-hydroxy-2,5-dimethyl-3(2H)-furanone
J.Biol.Chem., 288:16815-16826, 2013

PubMed 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.

PubMed: 23589283
DOI: 10.1074/jbc.M113.453852

Experimental method

X-RAY DIFFRACTION (1.4 Å)