Primera declaración de responsabilidad del Estado legislador en 1998

PaDADH is a 42 kDa monomer that binds one FAD molecule (Figure 1.2D).33 _PaDADH catalyzes the same chemical reaction as DAAO, except it employs an electron acceptor other than molecular oxygen to reoxidize the flavin (Scheme 1.1B). Phenazine methosulfate is used in vitro as the physiological electron acceptor remains unknown.79 _{The enzyme functions as the} first step in a novel two-enzyme coupled system in P. aeruginosa that racemizes D-Arg to L- Arg.26 _{The second enzyme is an NAD(P)H-dependent L-arginine dehydrogenase that combines} ammonia and the α-keto acid to produce L-Arg. The three-dimensional structures of PaDADH in both a ligand-free and product-bound conformation, and that of the enzyme with an N5- iminoleucine adduct, are available.33, 80

PaDADH has a broad substrate specificity with the ability to oxidize all of the 19 common D-amino acids except D-Asp and D-Glu.33 _{The cationic amino acids, D-Arg and D-Lys, are the} best substrates for the enzyme as judged by the high kcat/Km values shown in Table 1.2 (105-106 M-1s-1).33 The position of iminoarginine bound to PaDADH suggests that the side-chain of the D-Arg substrate forms an electrostatic interaction with E87. The presence of E87 rationalizes the lack of reactivity with D-Asp and D-Glu. Similar to pDAAO, a hydrophobic cage formed by the side-chains of Y53, M240, V242 and Y249 (Figure 1.3D) engages in favorable van der Waals and π-π interactions with aliphatic and aromatic side-chains of the substrate, consequently D-Met

and D-Tyr are oxidized in the 104 _M-1_s-1 _{range. The active site volume of PaDADH, which is} calculated to be ~320 Å with Caver Analyst 1.0 (unpublished results; J. Ball and G. Gadda), is large enough to provide the necessary space to accommodate bulky substrates, such as D-Arg, D- Tyr and D-Trp. Contrary to the single arginine used to bind the α-carboxylate of the substrate in DAAOs, PaDADH employs two arginine residues, R222 and R305, to bind the substrate α- carboxylate (Figure 1.3D).

Mutagenic work on PaDADH established the importance of E87 to be unprotonated for optimal binding of cationic substrates.81 _{Although the E87L variant did not lose the ability to} oxidize D-Arg, the kcat/Km value was down 20-fold compared to the wild-type enzyme.81 An H48F variant decreased the kcat/Km value with D-Arg only by 2-fold, indicating this residue does not significantly affect substrate specificity.81 _{Overall, the mutagenic work suggests there is no} catalytic base operative in the mechanism, and the catalytic pKa seen, irrespective of the substrate or variant, belongs to the substrate α-amino group.81, 82 _{A base not being required for} the chemical step of catalysis was also proposed for DAAO.63, 67 A recent and extensive review on PaDADH can be found here.83

1.1.7 D-aspartate oxidase: an enzyme with preference for anionic substrates

DASPO is a specialized enzyme of 39 kDa that oxidizes the anionic amino acids D-Asp and D-Glu.84, 85 The enzyme catalyzes the oxidative deamination of D-Asp to generate 2- ketoglutarate, ammonia, and hydrogen peroxide. In the brain, D-Asp functions as a neurotransmitter.86 _{DASPO has been biochemically and kinetically characterized from various} mammalian sources, including human brain, beef kidney, pig kidney, mouse, and octopus.85, 87-90 DASPO has also been characterized from the yeast Cryptococcus humicola.91 _{Purified bDASPO}

forms a homotetramer while purified pig kidney DASPO is monomeric.85, 88 _{The enzyme from} all sources has a higher catalytic efficiency with D-Asp as compared to D-Glu. The major difference between human brain, beef kidney, pig kidney, and mouse DASPO to octopus DASPO is that the latter exhibits a lower activity against NMDA than the former enzymes.88, 90 Increasing evidence has linked DASPO to schizophrenia in mammals, where enhanced activity of DASPO in the post-mortem dorsolateral prefrontal cortex of patients with schizophrenia contributes to dysfunctional NMDA transmission.92-94 _{DASPO is proposed to play a role in the} assimilation and detoxification of D-Asp in yeast.95

The three-dimensional structure of DASPO has yet to be solved, but a structural model of

bDASPO using SWISS-MODEL with the pDAAO (PDB: 1KIF; 43% sequence identity) as template is shown in Figure 1.2E and Figure 1.3E. The position of R216 in the model suggests it might form an electrostatic interaction with the side-chain of D-Asp (Figure 1.3E). It appears the α-amino group may interact with S308 or Y223 (Figure 1.3E). Biochemical studies on R237 from mouse DASPO, which corresponds to R278 of bDASPO, suggests it serves to bind the α- carboxylate group of D-Asp, and does not significantly affect the strict specificity for anionic amino acids.96 _{Lower levels of turnover and higher K}

m values were detected with D-Glu, indicating an active site framework that is finely tuned for the one-carbon shorter D-Asp.34

Mutations on DASPO have focused on R216 and S308, which are conserved between

bDASPO and mouse DASPO. An R216L variant of mouse DASPO acquired the ability to oxidize D-Phe and D-His, but at the cost of appreciably lower activity against D-Asp and NMDA than the wild-type enzyme.96 _{Replacement of S308 with glycine doubled the specific activity} (U/mg) of mouse DASPO against D-Asp and nearly tripled the specific activity against NMDA.97 _{H56 of yeast DASPO has been shown to be important for the substrate specificity of}

the enzyme as the H56A and H56N variants of the enzyme lost the ability to oxidize D-Asp, but gained the ability to oxidize neutral substrates, such as D-Met, D-Phe, and D-Gln.98