The flavor of strawberry (Fragaria 3 ananassa) fruit is dominated by an uncommon group of aroma compounds with a 2,5-dimethyl-3(H)-furanone structure. We report the characterization of an enzyme involved in the biosynthesis of 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF; Furaneol), the key flavor compound in strawberries. Protein extracts were partially purified, and the observed distribution of enzymatic activity correlated with the presence of a single polypeptide of ;37 kD. Sequence analysis of two peptide fragments showed total identity with the protein sequence of a strongly ripeninginduced, auxin-dependent putative quinone oxidoreductase, Fragaria 3 ananassa quinone oxidoreductase (FaQR). The open reading frame of the FaQR cDNA consists of 969 bp encoding a 322-amino acid protein with a calculated molecular mass of 34.3 kD. Laser capture microdissection followed by RNA extraction and amplification demonstrated the presence of FaQR mRNA in parenchyma tissue of the strawberry fruit. The FaQR protein was functionally expressed in Escherichia coli, and the monomer catalyzed the formation of HDMF. After chemical synthesis and liquid chromatography-tandem mass spectrometry analysis, 4-hydroxy-5-methyl-2-methylene-3(2H)-furanone was confirmed as a substrate of FaQR and the natural precursor of HDMF. This study demonstrates the function of the FaQR enzyme in the biosynthesis of HDMF as enone oxidoreductase and provides a foundation for the improvement of strawberry flavor and the biotechnological production of HDMF.

Fragaria x ananassa enone oxidoreductase (FaEO), earlier putatively assigned as quinone oxidoreductase, is a ripening-induced, negatively auxin-regulated enzyme that catalyzes the formation of 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF), the key flavor compound in strawberry fruit by the reduction of the alpha,beta-unsaturated bond of the highly reactive precursor 4-hydroxy-5-methyl-2-methylene-3(2H)-furanone (HMMF). Here we show that recombinant FaEO does not reduce the double bond of straight-chain 2-alkenals or 2-alkenones but rather hydrogenates previously unknown HMMF derivatives substituted at the methylene functional group. The furanones were prepared from 4-hydroxy-5-methyl-3(2H)-furanone with a number of aldehydes and a ketone. The kinetic data for the newly synthesized aroma-active substrates and products are similar to the values obtained for an enone oxidoreductase from Arabidopsis thaliana catalyzing the alpha,beta-hydrogenation of 2-alkenals. HMMF, the substrate of FaEO that is formed during strawberry fruit ripening, was also detected in tomato and pineapple fruit by HPLC-ESI-MSn and became 13C-labeled when d-[6-13C]-glucose was applied to the fruits, which suggested that a similar HDMF biosynthetic pathway occurs in the different plant species. With a database search (http://ted.bti.cornell.edu/ and http://genet.imb.uq.edu.au/Pineapple/), we identified a tomato and pineapple expressed sequence tag that shows significant homology to FaEO. Solanum lycopersicon EO (SlEO) was cloned from cDNA, and the protein was expressed in Escherichia coli and purified. Biochemical studies confirmed the involvement of SlEO in the biosynthesis of HDMF in tomato fruit.

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