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D-Amino acid oxidase (EC 1.4.3.3, DAAO) catalyzes the dehydrogenation of D-isomers of amino acids to the corresponding α-imino acids, which after subsequent hydrolysis yield α- keto acids and ammonia (Scheme 1.7). The kinetic mechanism of DAAO, as is common in flavoprotein oxidases, is divided into a reductive half-reaction, in which the amino acid is oxidized with the concomitant reduction of the bound flavin, and an oxidative half-reaction, in which the reduced flavin is oxidized by molecular oxygen with the subsequent release of the product (Scheme 1.8). This FAD-dependent enzyme was shown to play a vital role in the modulation of serine levels in mammalian brain (157, 158).

C R O O- H H₃N FAD H₂O₂ O₂ FADH- C R O O- 2HN H₂O NH4+ C R O O- O

D-Amino acid α-Imino acid α-Keto acid

Scheme 1.7. Reaction catalyzed by D-amino acid oxidase.

Scheme 1.8. Kinetic mechanism of D-amino acid oxidase. Taken without permission from ref. (140).

Although the enzyme is well studied, no unequivocal mechanism for the dehydrogenation of the substrate has yet been established (28, 140, 159). Over the years, two different mechanisms have been proposed for the reaction catalyzed by this flavoprotein oxidase: the hydride transfer mechanism and the carbanion mechanism. Evidence that favors a direct hydride transfer mechanism comes from the transfer of the α-hydrogen of the substrate to the C(5) position of the enzyme reconstituted with 5-deaza-FAD (160). Similarly, the elimination of halide from β-chloro-D-alanine validates the hypothesis that the reductive half-reaction of DAAO involves the initial formation of a carbanion intermediate after the abstraction of the α- hydrogen of the substrate as a proton (161). Finally, a direct hydride transfer mechanism was considered in which α-H+ _{abstraction is coupled with the hydride transfer (162).}

Two different research groups have described the X-ray crystallographic structure of DAAO from pig kidney with the inhibitor benzoate bound (37, 163). The X-ray structure of the enzyme showed that there is no active site residue properly placed to act as a base for carbanion formation. The only notable interaction of the inhibitor with the active site residue includes the carboxylic group of benzoate with Arg283 and Tyr228 (37, 163). A similar interaction was observed between the carboxylic group of D-3,3,3-F3-alanine and the homologous arginine and

tyrosine residues (Arg285 and Tyr223) in a recent structure of DAAO from Rhodotorula gracilis (Figure 1.20) (164). In addition, the amino group of D-3,3,3-F3-alanine was also observed to

form a hydrogen bond with a water molecule and the carbonyl oxygen of Ser335 (164). In all structures the carbon atom of the inhibitor, which corresponds to the α-carbon of the amino acid substrate, was found to be located at a distance of ~3.4 Å from the N(5) of the isoalloxazine ring of the bound flavin (37, 163, 164). These structural data clearly suggest a direct hydride transfer mechanism as well as argue against the carbanion mechanism that was proposed earlier.

Figure 1.20. Active site of R. gracilis DAAO with D-3,3,3-F3-alanine bound (PDB code 1C0L).

FLA, D-3,3,3-F3-alanine.

The currently available structures of DAAO from different sources in complex with ligands have showed that Arg285 interacts with the carboxylate group of the ligand and that the negative charge that develops at the vicinity of N(1)-C(2)=O locus of the bound flavin is stabilized by the dipole of α-helix F5 (37, 163). This result argued against the previously proposed role of Arg285 in stabilizing the negative charge that develops in proximity of N(1) locus of the bound flavin. Consequently, this result disagreed with the involvement of Arg285 in the stabilization of the anionic flavin semiquinone and hydroquinone, and for the formation of tight flavin-N(5)-sulfite adduct as well as in the modulation of the electrophilicity of the bound flavin by increasing the oxidation-reduction potentials of the bound FAD (165). Recently, Pollegioni et al., through their biochemical and spectroscopic analyses of different mutant forms of DAAO in which Arg285 was replaced by selected residues (R285A, R285K, and R285D), concluded that in the free enzyme, Arg285 is still involved in the stabilization of the negative charge on the N(1)-C(2)=O locus of the flavin (159). This conclusion was based on the assumption that although the guanidinium group of Arg285 was found to be located at a distance

of ~7 Å from the N(1)-C(2)=O locus of the flavin in the structures of the enzyme in complex with substrate or ligands, in the absence of substrate or ligands the side chain of Arg285 would be able rotate to a distance of ~3 Å from the N(1)-C(2)=O locus of flavin (159). The role of Arg285 in stabilizing the negative charge at the vicinity of the N(1)-C(2)=O locus of the bound flavin in DAAO is still ambiguous due to the unavailability of the X-ray crystal structure of the unliganded enzyme

Fitzpatrick and his co-workers in the last few years have performed a thorough kinetics characterization of DAAO by using substrate and solvent kinetic isotope effects approaches to probe the order of CH bond cleavage and the C=N bond formation (140, 166-168). The results obtained from the analysis of isotope effects studies agree well with the structural studies allowed Fittzpatrick to conclude that the mechanism of DAAO is a direct hydride mechanism, in which both the CH bond cleavage and the C=N bond formation occur in the same transition state (Scheme 1.9). In agreement with this conclusion, a recent computational studies also suggested a direct hydride transfer mechanism involving the anionic form of the amino acid substrate (169).

N N N NH O O R R H C NH₂ N N NH N O O R H O O R C NH₂O O