Structural Basis for the Enzymatic Formation of the Key Strawberry Flavor Compound 4-Hydroxy-2,5-dimethyl-3(2H)-furanone

Background: Fragaria x ananassa enone oxidoreductase catalyzes the ripening-induced formation of 4-hydroxy-2,5-dimethyl-3(2H)-furanone in strawberries. Results: By determining six x-ray structures of different substrate complexes the enzymatic mechanism was elucidated and experimentally confirmed by deuterium labeling. Conclusion: The 4R-hydride of NAD(P)H is transferred to an exo-cyclic carbon double bond. Significance: Enzymatic 4-hydroxy-2,5-dimethyl-3(2H)-furanone synthesis reveals a new reaction mechanism and advances understanding of a biotechnologically relevant biosynthetic pathway. 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-2H]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.


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-5methyl-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.
Incorporation experiments with radiolabeled precursors and substrates labeled with stable isotopes indicated D-fructose-1,6-bisphosphate as an effective progenitor of HDMF and provided initial evidence for the enzymatic formation of this important aroma compound in strawberries (12)(13)(14). D-Fructose-1,6-bisphosphate is presumably converted, by as yet The atomic coordinates and structure factors ( unknown enzymes, into 4-hydroxy-5-methyl-2-methylene-3(2H)-furanone (HMMF), which serves as substrate for an oxidoreductase that catalyzes the final biosynthetic step (15,16). The corresponding ripening-induced, negatively auxin-regulated enzyme was originally assigned as Fragaria x ananassa quinone oxidoreductase (FaQR), based on its sequence similarity to known quinone oxidoreductases and catalytic activity on 9,10-phenanthrenequinone (16), but subsequent enzymatic studies showed that this plant protein efficiently catalyzes the reduction of the exocyclic unsaturated bond of the highly reactive precursor HMMF, as well as derivatives thereof, utilizing NADPH as preferred cofactor (Fig. 1). Consequently, on the basis of this physiological reaction the enzyme was renamed to Fragaria x ananassa enone oxidoreductase (FaEO) (15). Notably, the kinetic data of FaEO, and also of the orthologous protein from Solanum lycopersicon (SlEO), for the aroma-active substrate HMMF and its chemical homologs resemble those of an earlier characterized enone oxidoreductase from Arabidopsis thaliana that was described to catalyze the hydrogenation of 2-alkenals, despite low sequence homology (15).
In the present study, we report the crystallization and x-ray structural analysis of FaEO in complex with different 4-hydroxy-3(2H)-furanone-derived substrates or products, thus providing hints on the catalytic mechanism and how hydride ion transfer from NAD(P)H is initiated. Isotope labeling experiments using stereospecifically deuterated [4R-2 H]NADH and chiral-phase analysis of the products served to experimentally confirm the proposed reaction mechanism.
Enzymatic Synthesis and Purification of Deuterated NADH-[4R-2 H]NADH was synthesized following a previously described procedure (17). Briefly, a 6-ml solution of 50 mM sodium carbonate, 0.1 M deuterated formic acid, and 15 mM NAD ϩ was titrated to pH 8.5 with 1 M NaOH. 20 Units of S. cerevisiae formate dehydrogenase were then added and the course of NAD ϩ reduction was spectrophotometrically monitored at 340 nm. After about 3 h at room temperature the reaction was complete. The solution was diluted to 15 ml with water and the product was purified on an ÄKTA purifier system (GE Healthcare, Munich, Germany (NH 4 )HCO 3 gradient (0 -0.4 M) in 300 ml. Fractions containing [4R-2 H]NADH were pooled and lyophilized. Purity and degree of deuteration were determined by LC-UV/ESI-MS n and NMR spectroscopy (see below).
Liquid Chromatography Electrospray Ionization Mass Spectrometry (LC-UV/ESI-MS n )-The purified [4R-2 H]NADH was dissolved at 2.5 mg/ml in water. For LC-UV/ESI-MS n analysis 5 l of the solution was injected into a 1100 HPLC system (Agilent, Waldbronn, Germany) with a Luna 3u C18(2) 100 Å column (15 cm ϫ 2 mm; Phenomenex, Torrance, CA), which was connected to an Agilent 6340 Ion Trap LC/MS mass spectrometer. The LC solvents were 0.1% (v/v) formic acid in water (A) and 0.1% (v/v) formic acid in methanol (B). For elution of [4R-2 H]NADH a gradient from 100% A/0% B to 0% A/100% B was applied at a flow rate of 0.1 ml/min during 20 min, kept for a further 20 min at the latter conditions, and then reset to 100% A/0% B within 1 min. The voltage of the capillary was set to Ϫ4000 V and that of the end plate was Ϫ500 V. The dry gas (N 2 ) was heated to 330°C and applied at a flow rate of 9 liters/min. Full scan mass spectra were measured from m/z 100 to 2200 for up to 200 ms until the ICC target reached 100,000 for positive ions or 70,000 for negative ions, whichever came first. Tandem mass spectrometry was performed using helium as collision gas, and the collision energy was set at 1.0 V. The target mass for MS 2 spectra was set to m/z 666 or 667. Mass spectra were acquired in the negative and positive ionization mode. Autotandem mass spectrometry was used to break down the most abundant [M ϩ H] ϩ or [M Ϫ H] Ϫ ion. Data analysis was performed using the 6300 Series Ion Trap LC/MS Version 4.0 software (Bruker Daltonics, Bremen, Germany).
Nuclear Magnetic Resonance (NMR) Spectrometry-Samples were dissolved in tetradeuteromethanol (D 4 -MeOH) containing 0.03% (v/v) tetramethylsilane. One-dimensional ( 1 H and 13 C) NMR spectra were recorded on a Bruker DMX-400 spectrometer (Bruker, Rheinstetten, Germany). The spectrometer frequencies were 500 and 125 Hz for the determination of 1 H and 13 C chemical shifts, respectively, using tetramethylsilane as internal standard in the proton dimension and D 4 -MeOH in the carbon dimension. NMR spectral data were analyzed using the MestReNova software (Mestrelab Research, Escondido, CA).
Synthesis and Purification of EDHMF-HMF (2.6 mmol), copper(II) acetate (1.2 mmol), and sodium acetate (1.6 mmol) were dissolved in 4.5 ml of acetic acid. Acetaldehyde (2.7 mmol) was added and the mixture was heated to 60°C with stirring for 1 h, followed by dilution with 9 ml of water. Products were extracted with n-pentane/diethyl ether (1:1), which was dried over sodium sulfate and concentrated by rotary evaporation. The residue was dissolved in 1 ml of water, purified by preparative RP18-HPLC-UV/ESI-MS n , extracted with n-pentane/diethyl ether (1:1), and analyzed by GC/MS and LC-UV/ ESI-MS n . Preparative purification was achieved by RP18-HPLC-UV/ESI-MS n using a HPLC system (Jasco, Groß-Umstadt, Germany) equipped with a Synergi 4u Fusion-RP 80 column (25 cm ϫ 21.5 mm; Phenomenex) connected to an Agilent LC/MSD Trap XCT mass spectrometer. The HPLC solvents were 0.1% (v/v) formic acid in water (A) and 0.1% (v/v) formic acid in methanol (B). For purification of EDHMF, 100% A/0% B was applied at a flow rate of 9.5 ml/min for 2 min, then continued to 30% A/70% B during 28 min, followed by 0% A/100% B for 5 min, finally returning to 100% A/0% B during 5 min. About 4% of the eluent was diverted to the mass spectrometer, whereas fractions of each 9.1 ml were collected. The ionization parameters and inert gas flow were set as described above. The full scan mass spectra were measured from m/z 100 to 800 for up to 200 ms until the ICC target reached 100,000 for positive ions or 70,000 for negative ions, whichever first. Tandem mass spectrometry was performed using helium as the collision gas with the collision energy set to 1.0 V. The target mass for MS 2 spectra was set to m/z 400. Mass spectra were acquired in the negative and positive ionization mode. Autotandem mass spectrometry was used to break down the most abundant [M ϩ H] ϩ or [M Ϫ H] Ϫ ion. Data analysis was performed using the Jasco ChromPass version 1.9.302.1124 software and the 6300 Series Trap Control Version 6.2 software (Bruker Daltonics).
Recombinant Strep-FaEO was produced in the Escherichia coli K-12 strain JM83 (19) grown at 22°C in 2 liters of LB medium (20) supplemented with 100 mg/liter of ampicillin using a 5-liter shake flask. Gene expression was induced at a cell density A 550 ϭ 0.6 by adding 0.2 mg/liter of anhydrotetracycline (21). After further shaking overnight the cells were harvested by centrifugation, suspended in 20 ml of lysis buffer (100 mM Tris/HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA) supplemented with 0.5 mg/ml of lysozyme, and incubated for 4 h at 4°C prior to sonification in an S-250D Cell Disrupter (Branson, Danbury, CT). The soluble fraction of the cell extract was prepared by centrifugation (10,000 ϫ g) and sterile filtration (0.45 m). Strep-FaEO was purified by mutant streptavidin affinity chromatography (22) in lysis buffer. After elution with 5 mM biotin in the lysis buffer, the enzyme was concentrated and subjected to size exclusion chromatography in the same buffer using a Superdex 200 16/60 column (GE Healthcare), eluting with an apparent molecular size of 35.2 kDa. For final polishing by anion exchange chromatography, Strep-FaEO was dialyzed against 20 mM Tris/HCl, pH 8.0, 1 mM EDTA, applied to a Resource Q column (GE Healthcare), and eluted with a linear concentration gradient of NaCl in the same buffer. Purified Strep-FaEO was finally dialyzed against 10 mM Tris/HCl, pH 8.0, 50 mM NaCl, 0.02% (w/v) NaN 3 and concentrated to 10 mg/ml (283 M) for further experiments. The yield of the purified enzyme was ϳ3 mg per liter of E. coli culture.
Crystallization and Structure Determination of FaEO-Crystals of FaEO in its apo form as well as in complex with the cofactor NADP(H) and substrate/product or analogs were grown by vapor diffusion, initially from combinatorial screens in 400-nl sitting drops using a Freedom Evo robotic system (Tecan, Crailsheim, Germany). Subsequent fine screens were manually performed in hanging drops by mixing 1 l of protein solution with 1 l of reservoir solution and equilibrating against a 1-ml reservoir. Crystal manipulation and harvesting was carried out with LithoLoops (Molecular Dimensions, Suffolk, UK).
X-ray diffraction data were collected on beamlines BL14.1 or BL14.2 operated by the Helmholtz-Zentrum Berlin at the BESSY electron storage ring (Berlin-Adlershof, Germany (23)) and processed with the XDS package (24) ( Table 1). The structure of FaEO⅐NADPH/HDMF was solved by molecular replacement using PHASER (25) with the quinone oxidoreductase HB8 from Thermus thermophilus (PDB entry 1IYZ (26)) as the search model. The crystal structure was built and refined in iterative cycles with COOT (27) and REFMAC5 (28). The other five FaEO structures were solved by molecular replacement using the FaEO⅐NADPH/HDMF complex.
To account for domain motions and flexibilities, Translation, Libration and Screw (TLS) groups were determined with TLSMD (29) and used for a final TLS and restraint refinement with REFMAC5. The six refined FaEO structures were finally validated with COOT and MolProbity (30). Molecular graphics were prepared with PyMOL (Schroedinger, Portland, OR). The atomic coordinates and structure factors have been deposited in the Protein Data Bank under accession codes 4IDA, 4IDB, 4IDC, 4IDD, 4IDE, and 4IDF.
FaEO Enzyme Assay-FaEO assays with EDHMF as substrate and NADH or [4R-2 H]NADH as cosubstrate were performed as previously described (15). 24 g of the purified recombinant enzyme from above was incubated at 30°C in the presence of 250 M EDHMF and 350 M NADH or [4R-2 H]NADH in a total volume of 1 ml of 0.1 M K 2 HPO 4 /KH 2 PO 4 , pH 5.0 or 7.0, for 30 min under agitation. The product EHMF was then extracted with diethyl ether for GC/MS analysis and with ethyl acetate for chiral-phase HPLC/UV analysis. The product solution was dried over sodium sulfate, concentrated under steady nitrogen flow, and was either directly injected into GC/MS or diluted in n-hexane and analyzed by HPLC/UV. Control reactions were performed without the addition of enzyme.
Gas Chromatography-coupled Mass Spectrometry (GC/ MS)-Isotope-labeled and unlabeled EHMF were analyzed by GC/MS using an Agilent 6890N gas chromatograph equipped with an Agilent 5975 mass selective detector. A 2-l aliquot of the extract from the FaEO enzyme assay was injected at a port temperature of 250°C with the purge valve on (split mode), using a split ratio 15:1 and split flow of 16.3 ml/min. Separation of tautomers was achieved on an Agilent VF-5 ms column (10 m, 0.25 mm, 0.25 m) using helium as carrier gas with a flow of 1.1 ml/min and an average velocity of 38 cm/s. The GC oven temperature was initially 40°C, then ramped to 200°C at 5°C/ min, and held there for 10 min. The total run time was 47 min, including a post-run time of 5 min at 320°C, whereas the GCmass spectrometer interface was kept at 310°C. Mass spectra were collected in the scan mode within a m/z 45-350 range using a threshold of 150 and gain factor of 2. Ionization was performed by electron impact at 70 eV with calibration by autotuning. Data were analyzed with the MSD ChemStation E.02.00.493 software (Agilent Technologies).
Chiral-phase High Performance Liquid Chromatography (Chiral-phase HPLC/UV)-Separation of EHMF isomers and enantiomers was achieved with a HPLC/UV system according to a published procedure (31). The samples, which were EHMF produced from EDHMF by FaEO as described above or synthetic EHMF (Sigma), were applied to a MaxiStar HPLC system (Knauer, Berlin, Germany) connected to a variable wavelength detector set to 288 nm and a Chiralpak IA column (250 ϫ 4.6 mm; Daicel Chemical Industries, Illkirch, France) using n-hexane/ethyl acetate (90:10) as isocratic solvent system at a flow rate of 1 ml/min. Data were analyzed with the EuroChrom 2000 software (Knauer).

Sequence, Bacterial Expression, and Crystallization of FaEO-
For biochemical characterization and protein crystallization FaEO was subcloned from the plasmid pET29a-FaEO (16) onto pASK-IBA5plus (22), thus encoding the gene product MAS-StrepII-G-FaEO(2-321) equipped with an N-terminal affinity tag. DNA sequencing of the resulting expression plasmid and also of the pET29a-FaEO precursor revealed three amino acid exchanges when compared with the published FaEO sequence (16): the substitutions T113P and Y125D and the deletion of Ala-210. Except for Pro-113, this FaEO sequence was identical with the one of Fragaria vesca (gene 28406) (32).
As all other known enone oxidoreductase (EO) polypeptide sequences from various species, with Ն70% sequence identity as retrieved from UniProt, neither contain an aromatic residue at position 125 nor an additional Ala at position 210 and exhibit either Lys or Thr at position 113, we decided to mutate Pro-113 to Thr and otherwise utilize the sequence as cloned. Thus, except for the N terminally appended Strep-tag II (22), the final FaEO expression construct corresponds to the sequence of the F. vesca EO. The recombinant enzyme was produced in the cytoplasm of E. coli as soluble monomeric protein and purified to homogeneity from the total cell extract using Strep-Tactin affinity, size exclusion, and anion exchange chromatography.
Crystals of FaEO in the apo-state and in complex with various substrates and analogs grew as tetragonal bipyramids reaching a final size of 300 -500 m in their largest dimension, which corresponded to the 4-fold axis of the crystals. The FaEO⅐ NADPH/HDMF complex was crystallized at pH ϳ 7.5 using PEG3350 as precipitant. Crystals appeared overnight and reached their final size within a week. In contrast, FaEO⅐ NADPH/HMF crystals took about 4 weeks to grow to their final size. Crystals of apo-FaEO were obtained around pH 6.0, also with PEG3350 as precipitant, reaching their mature size after 3 weeks. To obtain crystals of the ligand complexes FaEO⅐ NADP ϩ , FaEO⅐NADP ϩ /EDHMF, and FaEO⅐NADPH/EHMF, crystals of FaEO⅐NADPH/HDMF were incubated in the presence of an excess of 9,10-phenanthrenequinone (to oxidize NADPH and deplete HDMF), EDHMF, and EHMF, respectively. The x-ray diffraction quality of all FaEO crystals was excellent with a diffraction limit ranging from 1.6 to 1.4 Å resolution ( Table 1).
The crystal structure of FaEO was solved by molecular replacement using the published x-ray structure of a quinone oxidoreductase from T. thermophilus (PDB code 1IYZ (26)) as a starting model, having an overall amino acid sequence identity of 30% as calculated by ClustalW2 (33). After refinement, all six FaEO crystal structures showed excellent statistics (Table 1). Except for the N terminally appended Strep-tag II, all residues of FaEO (i.e. amino acids 2-321 encoded on the natural gene), as well as the N-terminal Gly residue of the linker, were resolved in the electron density.
FaEO has approximate dimensions of 70 ϫ 40 ϫ 40 Å 3 and behaves as a monomeric enzyme in solution as judged by size exclusion chromatography during FaEO purification, in line with previous reports (16). Analysis of all crystal packing contacts with Protein Interfaces Surfaces and Assemblies (PISA) (36) revealed no biologically relevant assemblies within the crystal lattice. Notably, the most prominent crystal contact of FaEO differs significantly between the apo and the complex structures, with buried surface areas of 767 Å 2 and (on average) 912 Å 2 , respectively. A structural similarity search of FaEO⅐NADPH/HDMF with the protein structure comparison service "Fold at European Bioinformatics Institute" (ebi.ac.uk/msd-srv/ssm) revealed a putative quinone oxidoreductase from Coxiella burnetii with bound NADPH (PDB code 3TQH) 3 as the most similar structure with a root mean square deviation of 1.6 Å for 290 aligned C␣ positions, despite merely 36% amino acid sequence identity. As mentioned above, FaEO was also initially annotated as a quinone oxidoreductase and renamed after identification of its natural substrate.
Thr-113, which has been mutated (see above), mediates two hydrogen bonds, one with its side chain hydroxyl group from the amide nitrogen of Phe-115 and one with its amide nitrogen to the side chain carboxamide of Asn-105. Both of these hydrogen bonds cannot be formed by a Pro residue at position 113. In fact, the two hydrogen bonds apparently restrain the conformation of the 104 -114 loop, which participates in the substratebinding pocket (see below).
The apo-structure of FaEO shows an open active-site cleft (Fig. 2B) that is largely filled with water molecules. A sulfate ion from the crystallization buffer is bound in the cofactor-binding site via residues Ser-197, Lys-200, and Tyr-215 as part of the Rossmann-fold. Furthermore, an ethylene glycol molecule of the cryo-protectant is bound in the substrate pocket. The crystal structure of the FaEO⅐NADP ϩ complex shows NADP ϩ tightly bound in the cofactor-binding site, which is located between the two domains described above and stretches across the small dimension of FaEO (Fig. 2B). Again, an ethylene glycol molecule is bound in the substrate pocket.
Compared with the apoenzyme, binding of NADP ϩ causes a small conformational change, resulting in a 5°rotation between the two domains of FaEO. NADP ϩ forms a total number of 168 contacts (within 4 Å) with FaEO, including 16 hydrogen bonds, 12 water-mediated hydrogen bonds, and 2 salt bridges. The salt bridges are formed between the 2Ј-phosphate group and resi-  dues Lys-200 and Arg-311. This position is occupied by the sulfate ion in the apo-structure described above. The large number of contacts seen between both domains of FaEO and NADP ϩ suggests that binding of NAD(H), which can replace NADP(H) as redox cofactor (16), might induce a similar conformational change despite lacking the 2Ј-phosphate group.

Complexes of FaEO with Substrates/Products and/or Analogs and Implications for the Catalytic Mechanism-
The substrate pocket of FaEO is lined by the side chains of amino acids Pro-55, Val-56, Lys-59, Phe-65, Ala-108, Leu-109, Leu-146, Val-265, and Leu-266 (Fig. 3). These residues are part of the substrate-binding domain and, with the exception of Lys-59, exclusively mediate hydrophobic contacts. Phe-65 adopts two distinct conformations of which only either one is observed in each of the complexes. However, there is no structural evidence that these different conformations play a role during catalysis. Apart from the protein residues, a significant portion of the substrate pocket is formed by the noncovalently bound redox cofactor NADP(H).
Also, two structurally conserved water molecules are bound within the substrate pocket. Water molecule 1 (Wat-1) is held in place by the main chain nitrogen of Val-56 (Fig. 3) and a second water molecule (Wat-2). Wat-2 is fixed by the side chain of Asn-54 and the 5Ј-phosphate of the nicotinamide nucleotide. The substrate EDHMF, the products HDMF and EHMF as well as the product analog HMF are all tightly bound within the substrate pocket; they are involved in numerous Van der Waals contacts and four hydrogen bonds, namely with the side chain of Lys-59, with Wat-1, and twice with the 2Ј-OH of the ribose moiety that carries the nicotinamide group (Figs. 3 and 4).
According to chemical principles, reduction of ␣,␤-unsaturated ketones such as in HMMF by way of hydride ion transfer from NAD(P)H, in contrast with FADH 2 , can either occur at the polarized carbonyl group (here at C3; Fig. 1) in a 1,2-hydrogen addition reaction or at the exocyclic double bond (here at C6) via a 1,4 addition. The reaction itself suggests a 1,4 addition mechanism as no alkene intermediates have been identified. Comparison of the different FaEO ligand complexes crystallized in this study provides insight into how the latter reaction is catalyzed.
The FaEO⅐NADPH/HDMF complex was obtained by cocrystallization of FaEO in the presence of 10-and 50-fold molar ratios of NADP ϩ and HDMF, respectively. Due to the large excess of HDMF over NADP ϩ it has to be assumed that the NADP ϩ cofactor became reduced upon mixing with the enzyme, with the excess HDMF remaining in its reduced state. Although hydrogen atoms cannot be resolved in the crystal structure, despite the high resolution obtained, we conclude that the complex represents an unproductive assembly of FaEO with bound NADPH and HDMF (Fig. 4). Although HDMF exists as a racemic mixture in aqueous solution, the clearly defined electron density shows that only its 2R-4,5-enolic-3keto form is bound in the substrate pocket. There, HDMF forms hydrophobic contacts with the side chains of Val-56, Lys-59, Phe-65, Leu-109, and Val-265, whereas Lys-59 is also involved in a hydrogen bond with the carbonyl oxygen of HDMF (at C3). Another hydrogen bond to the hydroxyl group of HDMF (at C4) is mediated via the tightly bound water molecule Wat-1. Furthermore, HDMF forms contacts with the nicotinamide group and the adjacent ribose of the cofactor NADPH, including two hydrogen bonds between the 2Ј-OH of the ribose and the hydroxyl as well as carbonyl groups of HDMF. Otherwise, the ring atoms O1, C2, C5, and the two  methyl substituents of HDMF exclusively form apolar contacts in the substrate pocket. In this way, the methyl group at C5 of HDMF points into the substrate pocket and is positioned in close proximity, with a distance of 3.4 Å, to the C4 atom of the nicotinamide ring of NADPH (Fig. 4). On the other hand, the methyl group at the chiral C2 of HDMF points out of the substrate pocket into solvent.
The FaEO⅐NADPH/EHMF complex was obtained by soaking crystals of FaEO⅐NADPH/HDMF (see above) with EHMF, hence leaving the bound NADPH presumably reduced. Due to its extended keto-enol tautomerism, EHMF can occur as several structural isomers in aqueous solution that show distinct stereochemistries at C2 and C5 (Fig. 1). The clearly defined electron density indicates that only the R-configuration at the chiral, ethyl-substituted C2 of EHMF is complexed by the enzyme (Fig. 4). The overall binding mode of EHMF is very similar to that of HDMF, again resembling an unproductive, doubly reduced complex. Similarly, EHMF forms contacts with FaEO side chains Val-56, Lys-59, Phe-65, and Leu-109 as well as with the nicotinamide and the adjacent ribose of the cofactor NADPH. Also, EHMF participates in four hydrogen bonds equivalent to the hydrogen bond pattern of HDMF. EHMF binds with the methyl group at C5 into the hydrophobic subpocket such that the methyl group is within 3.5 Å distance to the C4 atom of NADPH, whereas the ethyl group at C2 points out of the substrate pocket into solvent (Fig. 4). Thus, EHMF is clearly bound in an opposite orientation as would be necessary for the hydride ion transfer in the backward reaction leading to EDHMF (cf. Fig. 1 and the next panel in Fig. 4).
Likewise, the FaEO⅐NADP ϩ /EDHMF complex was obtained by soaking crystals of FaEO⅐NADPH/HDMF with EDHMF to replace the bound HDMF. Due to the large excess of EDHMF, the enzyme-bound NADPH probably became oxidized, resulting in the unproductive, this time doubly oxidized FaEO⅐ NADP ϩ /EDHMF complex (Fig. 4). Indeed, the (fully) unsaturated EDHMF clearly binds as a planar molecule to the substrate pocket and forms contacts with the side chains of Val-56, Lys-59, Ala-108, Leu-146, and Val-265 as well as with the nicotinamide and ribose of NADP ϩ . Compared with EHMF, the ring plane of EDHMF is flipped by 180°but still forms similar hydrogen bonds within the substrate pocket. The EDHMF hydroxyl group is hydrogen-bonded by both the side chain of Lys-59 and the 2Ј-OH of the NADP ϩ ribose, whereas its carbonyl group forms hydrogen bonds with the 2Ј-OH of the NADP ϩ ribose and Wat-1. In this way, the EDHMF ethylidene group at C2 points into the hydrophobic environment of the substrate pocket, resulting in a short distance of 3.4 Å between C6 of EDHMF and the C4 atom of NADP ϩ . In fact, this would be an almost ideal arrangement for the reduction reaction, provided that NADP ϩ was replaced by NADPH (Figs. 3 and 4).
Finally, the FaEO⅐NADPH/HMF complex was prepared by co-crystallization of FaEO with a 10-and 50-fold molar ratio of NADPH and HMF, respectively. HMF is a substrate analog of HDMF (in the backward reaction) that lacks the methyl substituent at C5. HMF binds in a very similar manner to FaEO as HDMF and EHMF via contacts with the side chains of Val-56, Lys-59, Leu-109, and Val-265. Again, HMF participates in four hydrogen bonds. The hydroxyl group of HMF forms hydrogen bonds with the 2Ј-OH of the NADPH ribose and with Wat-1, whereas the carbonyl group forms hydrogen bonds with the same 2Ј-OH of the NADPH and the side chain of Lys-59. Due to the missing methyl substituent at C5, HMF can form even tighter contacts with the nicotinamide ring of the NADPH coenzyme. The methyl group of HMF at C2 shows, with 3.4 Å, the same close distance to the C4 atom of NADPH as HDMF and EDHMF in their respective complexes (Fig. 4). Indeed, this arrangement corresponds to the quasi "productive" orientation observed for EDHMF above; however, in this case both the substrate/product ligand HMF and the cofactor are present in the reduced state.
Taken together, in all four FaEO complex structures with bound substrate, product, or substrate analog, the reactive exo carbon atoms are within close distance (3.4 -3.5 Å) to the C4 atom of the NADP(H) cofactor. This strongly suggests that transfer of the 4R-hydride from NADPH to the substrates HMMF or EDHMF occurs at the outward carbon atom of the exo-double bond, thus leading to the formal 1,4-hydrogen addition reaction as postulated above.
Regiospecificity of Hydride Ion Transfer to a Surrogate Substrate-To experimentally confirm the atomic position of hydride ion transfer from NAD(P)H to the substrate, the coenzyme was stereospecifically deuterated and subsequently used to enzymatically synthesize 2 H-labeled EHMF from EDHMF. Because NADH has a very similar K m value (361 M) as NADPH (K m ϭ 325 M), despite a lower specificity constant k cat /K m ϭ 0.02 s Ϫ1 M Ϫ1 versus 0.15 s Ϫ1 M Ϫ1 , respectively, 2 H-labeled NADH was employed for this experiment, which was accessible by means of an established synthesis procedure (37). To this end, enzymatic synthesis of [4R-2 H]NADH from NAD ϩ with [ 2 H]formic acid catalyzed by formate dehydrogenase was monitored spectrophotometrically at 340 nm until completion. Single deuteration in the anion exchange-purified [4R-2 H]NADH was confirmed by LC-UV/ESI-MS n (Fig. 5). Using this cosubstrate, regioselective deuteration of EHMF in the presence of FaEO was investigated via GC-MS analysis (Fig.  5). Comparison of the mass spectrometric fragmentation pattern of the enzymatic reaction product either in the presence of [4R-2 H]NADH or of unlabeled NADH (Fig. 5) with published data (38) clearly revealed that the deuterium was transferred to the exo-carbon of the ethylidene moiety (attached to C2 of the furanone ring; cf. Fig. 1).
Considering the prochiral nature of EDHMF, one would expect the preferential enzymatic synthesis of one stereoisomer of EHMF by FaEO. However, in solution EHMF comprises a mixture of tautomers with distinct keto-enol structures similar to HDMF (Fig. 1), including four prevailing diastereomers that can be chromatographically resolved by chiral-phase HPLC (31). Commercially available EHMF is a synthetic racemic mixture of the constitutional isomers 5-EHMF and 2-EHMF in a ratio of about 1:3 to 1:5 ( Fig. 6) (31,39). In contrast, EHMF freshly produced by FaEO comprises a mixture of 5-EHMF and 2-EHMF in a significantly lower ratio of 1:1.75 (Fig. 6). This indicates that the enzymatically catalyzed reaction has regiospecific preference, which will be discussed in the light of the proposed mechanism below.

DISCUSSION
The elucidation of all six different crystal structures of FaEO in complex with various ligands has provided detailed insight into the catalytic mechanism of this novel kind of enzyme.
Binding of NADP(H) to FaEO leads to conformational changes that result in its tight encapsulation and proper positioning of the nicotinamide ring within the cofactor-binding site, also shaping the adjacent substrate pocket. The substrate pocket is    . Chiral analysis of enzymatically produced EHMF stereoisomers. The distribution of EHMF configurational isomers (tautomers) and stereoisomers enzymatically produced by FaEO (continuous line) was compared with a synthetic reference sample (dashed line). The thermodynamically more stable racemate of R-(ϩ)-2-EHMF and S-(Ϫ)-2-EHMF clearly prevails over the racemate of the 5-EHMF configurational isomers in the reference sample, whereas a higher level of the latter two compounds (R,S-5-ethyl-4-hydroxy-2-methyl-3(2H)-furanone racemate) results immediately after enzymatic synthesis, notably without apparent stereospecificity. Numbers inside the ring refer to the numbering scheme applied to all furanone compounds in this article (cf. Fig. 1), whereas IUPAC numbering is used for the chemical names.
lined by hydrophobic side chains on one side and provides the hydrogen-bonding partners Lys-59, Wat-1, and the 2Ј-OH of NADPH as well as its nicotinamide ring that acts as hydride ion donor on the other side. However, due to their high pK a values none of the former groups can act as a general acid/base during catalysis. There is also no other distant amino acid side chain that might assist proton transfer, e.g. via the structural water molecule Wat-1, suggesting that the catalytic mechanism of the redox reaction does not depend on enzyme-mediated proton transfer.
All four substrate-product complexes have in common that both the carbonyl group and the hydroxyl group of the hydroxyfuranone ring system are engaged in two hydrogen bonds, each with the 2Ј-OH of the NADP(H) ribose and either one with Lys-59 or Wat-1 (Fig. 4). All four ligands form similar Van der Waals contacts with FaEO and the nicotinamide group of NADP(H). Notably, the C6 exo-carbon atom of the furanone derivatives EDHMF and HMF is in Van der Waals distance (3.4 Å) to the C4 position of NADP(H), which clearly suggests that the hydride ion transfer occurs to the methylidene (or ethylidene) group of the oxidized substrate HMMF (or the surrogate substrate EDHMF) rather than to the neighboring carbonyl C atom.
The complex structures with the corresponding reduced compounds HDMF and EHMF clearly reveal that the reaction product cannot enter the substrate pocket with its sp 3 -hybridized C2 carbon ahead. In both complexes the chiral C2 ring carbon points out of the substrate pocket. By contrast, the oxidized surrogate substrate EDHMF, which adopts a planar sp 2 configuration at the unsaturated C2 carbon, can enter the substrate pocket with its ethylidene group ahead. Similarly, the product analog HMF in its enol form, which lacks the methyl group of HDMF at C5, can even better enter the substrate pocket and form closer contacts with NADPH than HDMF. This suggests that a planar configuration at the hydride acceptor site of the substrate plays a role during enzymatic catalysis.
Due to their dynamic keto/enol tautomerization both products, HDMF and EHMF, can also adopt a fully planar di-enol configuration (Fig. 1). However, in aqueous solution only mixed keto/enol tautomers of HDMF and EHMF are observed in significant quantities by NMR, which also seems to be the case if bound at the substrate pocket of FaEO in the crystals. As clearly visible in the electron density, FaEO binds the chiral compounds only in the R-configuration. Notably, the observed binding mode of the reduced entities HDMF and 2-EHMF represents an unproductive assembly that would not allow hydride abstraction via backward reaction in the presence of the oxidized cofactor NADP ϩ . In fact, this orientation suggests a possible mode of product inhibition of FaEO by HDMF and EHMF.
In contrast, the fully planar surrogate substrate EDHMF in principle can enter the substrate pocket of FaEO with either its ethylidene group at C2 or its methyl group at C5 ahead. However, only the orientation with C2 pointing into the substrate pocket was observed in the electron density (Fig. 3B). Presumably, the ethylidene group can form better hydrophobic contacts among the FaEO side chains that line the substrate pocket. Consequently, EDHMF binds in an orientation in which it could accept a hydride ion from NADPH at its C6 carbon of the exo-double bond. Despite being crystallized in the presence of NADP ϩ , this orientation should represent the substrate-binding mode during hydride transfer. This interpretation is supported by deuteration experiments that unambiguously identified the C6 carbon of EDHMF as the hydride ion acceptor (Fig.  5). Based on this data, we conclude that the chemically more labile natural furaneol precursor HMMF during catalysis most likely binds in the same orientation as EDHMF to the FaEO substrate pocket, such that it can accept the hydride ion at its C6 carbon in the same way.
The keto/enol forms of the reduced products HDMF and 2-EHMF that are populated in aqueous solution each have a C-H acidic sp 3 -hybridized C2 carbon with theoretical pK a values of 7.1 and 7.5, respectively (Fig. 1). This group should be predominantly deprotonated at the slightly alkaline physiological pH of the plant cytoplasm. However, as outlined above, even in a deprotonated state the sp 3 -hybridized C2 carbon is likely not able to enter the substrate pocket of FaEO in a productive manner, due to its non-planar geometry. In contrast, the planar di-enol(ate) forms of HDMF and EHMF, which show theoretical pK a values of 7.1 and 7.0 for their enolic groups, respectively (Fig. 1), can easily enter, or leave, the substrate pocket. According to the principle of microscopic reversibility, this means that the enzyme actually prefers a less populated tautomer considering the backward reaction. Indeed, the negative charge of the enolate anion of the reduced product can be compensated by the positive charges of both the basic Lys-59 side chain and the pyridinium ring of the oxidized NADP ϩ cofactor. Thus, in the backward reaction hydride ion transfer can occur from the C6 carbon of HDMF or EHMF to the cofactor, resulting in the unsaturated and fully planar FaEO substrates HMMF and EDHMF.
Taken together, the in planta substrate HMMF, as well as the surrogate substrate EDHMF, is a planar molecule that can enter the substrate pocket of FaEO with its methylidene, or ethylidene, moiety, ahead. The substrate forms several contacts with side chains in the active site of FaEO and, importantly, also with the NADPH cofactor, including four hydrogen bonds with Lys-59, Wat-1, and twice the 2Ј-OH of the NADPH ribose. In this way the unsaturated exo-carbon is oriented in optimal position to the C4 atom of the NADPH nicotinamide ring (Fig. 7). Transfer of the hydride ion leads to reduction via a formal 1,4hydrogen addition. This initially results in an enol/enolate product that corresponds to the aromatic furan structure of HDMF (or EHMF). The emerging positively charged oxidized nicotinamide, together with Lys-59, should favor this hydride transfer by electrostatically stabilizing the negative charge of the enolate anion (Fig. 7).
Subsequently, the proton of the enol group on the other side of the product molecule may be transferred onto the enolate, possibly mediated by the 2Ј-OH group of the NADP ϩ ribose sugar via a Grotthuss-like mechanism, resulting in the opposite enolate group positioned in proximity to the positively charged Lys-59, thus allowing formation of another ion pair. The product HDMF (or EHMF) then leaves the substrate pocket of FaEO and most likely becomes (partially) protonated by the solvent, which eventually completes the biocatalytic reaction cycle.
In principle, protonation of the product can occur by two different mechanisms: (i) the planar mixed enolate/enol form of HDMF (or EHMF) (Fig. 1) directly leaves the substrate pocket or (ii) it first tautomerizes to the corresponding keto/enol form with an sp 3 -hybridized deprotonated C5 carbon (Fig. 1), which can point out of the pocket in a sterically favorable manner, prior to dissociating from the substrate pocket. In the case of HDMF these alternative protonation scenarios cannot be distinguished due to the pseudo-symmetry of the molecule (cf. Fig.  1). However, the second scenario appears to be of importance for protonation of the surrogate product EHMF as the enzymatically synthesized compound shows an increased ratio between the 5-EHMF and 2-EHMF tautomers compared with a synthetic racemic mixture ( Fig. 6; note that the 2-EHMF stereoisomers are only formed after keto/enol tautomerization of the primary reaction product).
Only recently, NAD(P)H-dependent, non-flavin ene reductases have been investigated for their ability to reduce C ϭ C double bonds in a number of structurally diverse substrates (40 -43). In this regard, FaEO exhibits a narrow substrate spectrum and, beside its natural substrate HMMF, predominantly reduces ␣,␤-unsaturated diesters and nitroalkenes, apart from the earlier described quinones. This is in line with the reaction mechanism deduced here and the structural finding that only planar enones can enter the active site of FaEO. Therefore, our results not only provide insight into the peculiar catalytic cycle of this novel enone oxidoreductase but also should facilitate protein-engineering efforts for the development of improved biocatalysts for biotechnological processes.