A comparison of the main biochemical properties of mammalian DAAOs is reported in Sacchi et al. (2012). Recombinant hDAAO is produced in fairly large amounts in E. coli cells (Kawazoe et al., 2006; Molla et al., 2006; Romano et al., 2009). It is purified as active holoenzyme by adding exogenous FAD to the purification buffers: hDAAO shows the classical properties of flavoprotein oxidases, such as a quick reaction with O₂ in the reduced form and stabilization of the anionic red flavin semiquinone.

In the 6–10 pH range, hDAAO shows a good activity and stability (Murtas et al., 2017b). From the fitting of the activity values determined at different pH values, two dissociations were apparent: a pK_a of 2.5 and 11.1, respectively. Notably, the enzyme is fully stable after 60 min of incubation at 4°C at pH values ≥3.0 and ≤ 10.0. The flavoenzyme is stable up to 45°C, a temperature corresponding to the optimum for the enzymatic activity. The melting temperature determined following the loss of activity was ~55°C (Murtas et al., 2017b), higher than the values determined using the changes in protein fluorescence intensity (Caldinelli et al., 2010): this result suggests that the alteration in protein conformation brings forward the loss of enzymatic activity.

hDAAO activity is not affected by the presence of divalent ions (Ca²⁺ and Mg²⁺) and/or nucleotides. Similarly, the reducing agent N-acetyl-cysteine, a derivative of L-cysteine, acting as antioxidant and anti-inflammatory agent and that is able to modulate NMDAR activity (Kumar, 2015), does not affect the activity of hDAAO (Murtas et al., 2017b).

hDAAO shows a wide substrate acceptance: the best substrates are hydrophobic and bulky D-amino acids (D-DOPA > D-Tyr > D-Phe > D-Trp, Figure 2). The highest k_cat value was determined for D-3,4-dihydroxy-phenylalanine (D-DOPA) (Kawazoe et al., 2007a,b; Murtas et al., 2017a,b), also showing a high affinity due to two additional H-bonds between the OH-groups of the substrate and His217 and Gln53 (Kawazoe et al., 2007a). However, the oxidation of D-DOPA is hindered by the substrate inhibition effect, K_i of 0.5 (Murtas et al., 2017b) or 41mM (Kawazoe et al., 2007b).

hDAAO is also active on small, uncharged D-amino acids (D-Cys > D-Ala > D-Pro > D-Ser) (Molla et al., 2006; Kawazoe et al., 2007b; Frattini et al., 2011; Murtas et al., 2017b). Purified recombinant hDAAO shows a low catalytic efficiency on what is known as the main physiological substrate, D-serine. Whether in vivo (and especially in glial cells) an increase in kinetic efficiency is achieved by the binding with a cellular compound (i.e., a protein or a small size ligand) or by a post-translational modification is still unknown: this issue deserves further investigations. The highest catalytic efficiency was determined for D-cysteine, a compound involved in H₂S generation (see above) (Shibuya et al., 2013).

hDAAO is not active on glycine and acidic D-amino acids (NMDA and D-Glu) while the activity on D-Asp is hampered by the high apparent K_m (in the molar range) (Molla et al., 2006; Murtas et al., 2017b). hDAAO also oxidizes D-kynurenine with an apparent K_m value (0.7mM) resembling that determined for D-cysteine. Kynurenic acid, the product of D-kynurenine oxidation, binds to the modulatory glycine site of the NMDAR resulting in an inhibitory effect. Furthermore, hDAAO is also active on D-cycloserine, an NMDAR modulator (Kumar, 2015).

The substrate promiscuity of hDAAO supports the hypothesis that this flavoenzyme might play a role in different tissues and cells.

The activity of hDAAO on D-serine is partially inhibited by the L-enantiomer (Murtas et al., 2017b). L-Serine acts as competitive inhibitor (K_i of 26.2 mM). Under anaerobic conditions L-serine, as well as L-alanine or L-valine, are able to reduce FAD. However, a physiological concentration of L-serine (corresponding to ≤2 mM in brain tissues and in blood) (Weatherly et al., 2017) should not modify the oxidation of D-serine by hDAAO.

For all known DAAOs, the oxidative deamination of D-amino acids follows a ternary-complex mechanism (Pollegioni et al., 1993; Umhau et al., 2000; Molla et al., 2006). The substrate dehydrogenation ensues by the direct hydride transfer of the α-H from the α-C of the D-amino acid to the flavin N(5): please see below and Figure 3D. The distance between these atoms is 3.6 Å in the hDAAO-imino serine complex: owing to the tetrahedral geometry of the substrate α-C, the mentioned atoms should be closer in the Michaelis complex (~3.2 Å). Following hydride transfer, the reduced flavin is negatively charged: the positive charge of the product imino group electrostatically stabilizes the reduced cofactor.

For mammalian DAAOs, and especially for hDAAO, the first half of the reaction (the reductive half-reaction), namely, the conversion of the tetrahedral D-amino acid into the planar imino acid coupled to the flavin reduction is fast (117 ± 6 s⁻¹ on D-serine), significantly faster than turnover (6.3 ± 1.4 s⁻¹). The rate-limiting step in hDAAO catalysis is the product release (Molla et al., 2006; Molla, 2017). The rate of imino acid release from the reduced enzyme is < 1 s⁻¹, too slow to allow the reoxidation step to start from the free, reduced enzyme. Accordingly, reoxidation must start from the corresponding reduced enzyme-imino acid complex: the second-order reaction corresponds to 1.25 × 10⁵ M⁻¹s⁻¹.

Each hDAAO protomer contains 347 amino acids (40.3 kDa), harbors one molecule of FAD, and is composed of 11 α-helices and 14 β-strands. hDAAO is constituted by two interconnected regions: an FAD-binding domain containing the dinucleotide binding motif (Rossman fold) and a substrate-binding domain in which a large, twisted, antiparallel β-sheet forms the active-site roof and part of the oligomerization interface (Figures 3A,B). hDAAO is a stable homodimer: the two monomers interact via a head-to-head geometry (Figure 3C; Kawazoe et al., 2006).

In the active site, the substrate is located above the re-side of the isoalloxazine ring of FAD, in a cavity of ~220 ų (Figure 3B). The substrate is bound via several hydrogen bonds in the correct orientation with respect to the flavin N(5) position for catalysis to proceed: the α-carboxylic group of the substrate electrostatically interacts with Arg283 and Tyr228, whereas the α-amino group interacts with Gly313 and the C(4) = O of FAD. The side chain of the substrate is placed in a pocket made up of hydrophobic residues (Leu51, Gln53, Leu215, and Ile230), named the substrate-specificity pocket (Kawazoe et al., 2006). The active-site “roof” is shaped by the side chain of Tyr224, a residue belonging to a mobile loop (216–228): the product/substrate exchange during catalysis is facilitated by the switch of this residue from a closed to an open conformation. This conformational change significantly influences the enzyme properties: limiting the turnover, increasing the hydrophobicity of the active site, and allowing hDAAO to bind large substrates (Molla et al., 2006; Kawazoe et al., 2007b).

The strict stereoselectivity of DAAO for the D-enantiomer of the amino acids is rationalized by the four-location model for enantioselectivity (Mesecar et al., 2000; Umhau et al., 2000; Mörtl et al., 2004). According to this model, the substrate establishes three interactions—using the α-carboxylic group, the α-amino group and the side chain—with the active site residues indicated above: the exact binding produces a “functional direction” exemplified by the alignment of the α-H of the substrate and the N(5) of FAD, which allows hydride transfer (Figure 3D).

Different from other DAAOs (Mattevi et al., 1996; Pollegioni et al., 2007; Frattini et al., 2011), an 80 kDa homodimer is generated by both the holo- and the apoprotein form of hDAAO (Molla et al., 2006). This results from a distinguishing charge distribution at the dimer interface (a region corresponding to ~1,500 Ų, i.e., the 9.8% of the overall solvent accessible surface, Figure 3C), where a significantly higher amino acidic substitution frequency was observed than for the overall protein (33 vs. 15%, respectively) (Kawazoe et al., 2006). Notably, the urea-induced dissociation of dimeric hDAAO generates protein conformers prone to aggregation (Caldinelli et al., 2009).

In hDAAO the FAD cofactor shows an elongated conformation and it is buried in the protein core: the isoalloxazine ring is located at the interface between the two subdomains with the re-side facing the interior of the active site (Kawazoe et al., 2006; Molla, 2017). At this side of the flavin ring, the conformation of the surrounding residues is highly conserved among mammalian DAAOs. Conversely, at the si-face, the conformation of the hydrophobic stretch (47-VAAGL-51, a structurally ambivalent peptide) differs between the human and porcine enzymes, causing loss of the H-bond between Ala49 and N(5) of the cofactor and likely decreasing the strength of the interaction of the flavin cofactor and the rate of flavin reduction (Kawazoe et al., 2006).

hDAAO possesses the weakest binding of the FAD cofactor (K_d = 8.0μM) among known DAAOs (K_d = 0.2 and 0.02μM for pig and yeast DAAOs, respectively). Accordingly, hDAAO exists in solution as an equilibrium of holo- and apoprotein forms (Caldinelli et al., 2010). The presence of an active-site ligand increases the affinity of the flavin to the protein moiety, K_d = 0.3μM (Molla et al., 2006; Caldinelli et al., 2010). Quenching of protein fluorescence intensity during titration of the apoprotein with the cofactor, in the presence or absence of sodium benzoate, is a biphasic process (Murtas et al., 2017b), suggesting that the apoprotein form exists in two conformations with differing cofactor binding affinity: the higher intensity amplitude associated with the first phase observed in the presence of benzoate indicates that binding of an active-site ligand favors the protein conformation with the higher avidity for FAD (Murtas et al., 2017b). A second possibility is the presence of two binding sites. Here, a recent investigation based on computational and labeling analyses suggests that an additional ligand-binding site is located at the monomer-monomer interface (Figure 3A; Kohiki et al., 2017).

The holoenzyme reconstitution is a sequential process: in the first step, FAD binds the apoprotein moiety and recovers the catalytic activity; in the second step, a slow, secondary conformational change generates the final holoenzyme conformation (Caldinelli et al., 2009). Notably, the first step is 20-fold faster when benzoate is present (Caldinelli et al., 2010).

The melting temperature for the unfolding of the holoenzyme is 6–9°C higher than for the corresponding apoprotein (Caldinelli et al., 2009).

The observed increase in cofactor binding affinity in the presence of benzoate suggested that, in addition to the different conformation of the hydrophobic VAAGL sequence observed in the hDAAO-benzoate complex (Kawazoe et al., 2006), an alternative conformation of the substrate-free enzyme should exist that binds the cofactor less efficiently (Murtas et al., 2017b). In any case, the structure of the free enzyme form (PDB 2e48) overlaps that of the hDAAO-benzoate complex (PDB 2du8).

Based on the in vivo concentration of FAD (~5 μM), it is conceivable that in the cell an equilibrium between the hDAAO holoenzyme (active) and the apoprotein (inactive) form exists, with the latter one being predominant in the absence of an active-site ligand.

hDAAO inhibitors can essentially be grouped into substrate-competitive and cofactor-competitive inhibitors (Molla et al., 2006; Sacchi et al., 2012; Terry-Lorenzo et al., 2014; Molla, 2017). Among the active-site ligands, small aromatic (aryl) carboxylic acids or acid isosteres are powerful hDAAO inhibitors used as scaffolds for developing novel drugs (see below). These compounds, such as benzoate, o-aminobenzoate, substituted quinilinones, or 4H-furo[3,2-b]pyrrole-5-carboxylic acid (Figure 4), bind the flavoenzyme similarly to the substrate: the inhibitor COOH group (or the corresponding C = O or OH substituents) interacts with Arg283 and Tyr228, an H-bond donor binds Gly313, and the remaining part interacts with the hydrophobic region of the active site (that can accommodate molecules containing 12–13 atoms) (Duplantier et al., 2009). In the hDAAO-inhibitor complex, the side chain of Tyr224 is shifted toward the inner part of the active site and forms a strong π-π stacking interaction, “sandwich,” between its aryl chain and the re-side of the isoalloxazine ring of the cofactor. The strongest interaction is observed when the aromatic rings are slightly displaced [i.e., with 3-hydroxyquinolin-2(1H)-one] (Figure 4) and not perfectly stacked (i.e., with benzoate). When saturated analogs of these compounds are used, a drop in the binding affinity is apparent, indicating the relevance of the π-π stacking interaction.

The binding of aromatic carboxylic acids to the hDAAO holoenzyme inhibits the flavoenzyme and perturbs its absorbance spectrum in the visible region. For example, benzoate yields a shoulder at ~497nm (K_d = 7μM and K_i = 9 7μM) (Kawazoe et al., 2006; Molla et al., 2006); anthranilate binding generates a spread classical charge transfer band at ~580nm (K_d = 40μM) (Molla et al., 2006). The ligand-binding site is present in the apoprotein form, too, as made apparent by the alteration in protein fluorescence and in thermostability of the latter hDAAO form when the substrate D-serine or the substrate analog tri-fluoro-D-alanine is added (Caldinelli et al., 2009, 2010).

Conversely to benzoate, for the binding of the inhibitor 6-chloro-benzo[d]isoxazol-3-ol (CBIO) (Figure 4) to hDAAO a single-step binding process is evident and the K_d value estimated following the quenching of protein fluorescence intensity corresponds well to the K_d, IC₅₀, and K_i values determined using different methods.

ADP and CPZ (Figure 4) (IC₅₀ of 580 and 5μM, respectively) behave as FAD-competitive inhibitors for binding to hDAAO (Iwana et al., 2008; Sacchi et al., 2008; Terry-Lorenzo et al., 2014). In particular, CPZ binding generates a protein conformation more sensitive to proteolysis and thermal unfolding than the native holoenzyme (Caldinelli et al., 2010). The near-UV CD spectra show that the tertiary structure of hDAAO-CPZ complex differs from that of the hDAAO-FAD: the former more closely resembles that of the apoprotein (Caldinelli et al., 2010).

Notably, the ligands D-serine, FAD, benzoate, and CPZ did not affect the formation of the hDAAO-pLG72 complex (see below).

Human flavoenzyme function is modulated by interacting with various proteins. hDAAO, through the PTS1-type peroxisomal-targeting signal, interacts with the Pex5p receptor, a protein involved in protein import and in the assembly of peroxisomes (Ghosh and Berg, 2010).

Genome-wide association studies and meta-studies in different populations have linked polymorphisms in the gene encoding pLG72 protein with schizophrenia and other psychiatric diseases (Drews et al., 2013; Sacchi et al., 2016; Pollegioni et al., 2018). In particular, hDAAO specifically binds to the primate-specific protein pLG72: two hDAAO homodimers interact with two pLG72 molecules, yielding a 200-kDa protein complex (K_d = 0.08–0.53 μM); for a recent review (see Pollegioni et al., 2018). In vitro, the formation of the 200-kDa complex does not alter the kinetic parameters or the binding with the FAD cofactor of hDAAO, but rather induces a change in its overall tertiary structure, causing a time-dependent inactivation (Sacchi et al., 2008). By using low-resolution techniques (i.e., limited proteolysis coupled to mass spectroscopy and cross-linking experiments) structural elements involved in forming the interface surface in the hDAAO-pLG72 complex have been identified, highlighting the role of the N-terminal region of pLG72 in forming the oligomerization interface (Birolo et al., 2016; Sacchi et al., 2017). hDAAO in transiently transfected glial cells (i.e., the U87 human glioblastoma cell line) is largely localized in peroxisomes but also present in cytosol (Sacchi et al., 2011) while pLG72 shows a mitochondrial localization. We proposed that, in this model cell system, newly synthesized hDAAO interacts with pLG72 on the cytosolic side of the outer mitochondrial membrane (Sacchi et al., 2011). Such an interaction increases the D-serine/total serine ratio and decreases hDAAO activity and half-life, see above (Sacchi et al., 2008, 2011; Cappelletti et al., 2014). We recently proposed that pLG72 (itself or recruiting further proteins) might target the cytosolic form of hDAAO to the ubiquitin-proteasome system, thus starting its degradation (Cappelletti et al., 2014). This mechanism could represent a further process to regulate the D-serine levels in the hindbrain where the flavoenzyme is expressed in glial cells.

Analogously, the activity of hDAAO is negatively regulated by bassoon, a component of the cytoskeletal matrix, mainly located at the presynaptic active zone. The hDAAO-bassoon complex formation has been proposed to prevent D-serine depletion acting on the active, extraperoxisomal enzyme form located at presynaptic terminals (Popiolek et al., 2011). The inhibitory effect of bassoon may account for the difficulties in detecting hDAAO activity in the forebrain (Verrall et al., 2007), a region where the enzyme is mostly expressed in neurons.

Abnormal changes in hDAAO activity yielding locally decreased D-serine levels have been correlated with neurological disorders (e.g., schizophrenia); therefore, the identification of hDAAO inhibitors (to slowing down the neuromodulator degradation process) to be used as drugs has garnered growing interest. This treatment has beneficial effects on cognition and learning functions (Hopkins et al., 2013b).

More than 500 substrate-competitive inhibitors have been identified so far (Gilson et al., 2016). Analogously to the substrate, their chemical structure contains a planar moiety which interacts with the active-site residues close to the FAD cofactor isoalloxazine ring and a second portion which is positioned in the substrate side-chain binding pocket. The “core” of the planar moiety is usually formed by one or two fused rings (one of which might be aromatic) and contains at least a carboxylic group to establish the H-bond interaction with Arg283. The second part of the inhibitor molecule corresponds to the side chain of the substrate: this portion, depending on the size and chemical features, forms further interactions with residues belonging to the substrate specificity pocket and/or to the active site entrance.

A comprehensive review about the details of inhibitor binding to hDAAO has been published recently (Molla, 2017). It ranks classical and novel compounds in four classes:

Based on biochemical properties, hDAAO variants corresponding to known single nucleotide polymorphisms or sequence conflicts have been grouped into two classes: hypoactive and hyperactive; for a recent review (see Sacchi et al., 2018). The conditions and levels of recombinant expression of seven variants of hDAAO are reported in Table 2 of Sacchi et al. (2018).

The G183R, R199W, and R199Q hDAAOs show significantly decreased enzymatic activity (or fully abolished for the latter variant, see Figure 5), and a perturbation of the conformation: (a) in G183R hDAAO, corresponding to the coding mutation occurring in the ddY/DAAO^−/− mice strain expressing the inactive G181R DAAO (Konno and Yasumura, 1983), alterations in secondary structure elements likely alter the conformation of the flavin binding domain and thus negatively affect the cofactor binding (Murtas et al., 2017a); (b) the tertiary structure of R199W (the variant associated with the onset of fALS) and R199Q variants is significantly altered: this favors aggregation propensity but does not modify the interaction with pLG72 (Cappelletti et al., 2015; Murtas et al., 2017a); (c) in G331V hDAAO the change in the C-terminal α-helix promotes protein aggregation, strongly affecting the variant solubility (Caldinelli et al., 2013). At the cellular level, both G183R and G331V variants were partly mistargeted: they formed cytosolic protein aggregates, which largely colocalized with ubiquitin, and resulted in increased apoptosis (Caldinelli et al., 2013; Murtas et al., 2017a).

On the other hand, the D31H, W209R, and R279A substitutions have the opposite effect on hDAAO activity, resulting in slightly or significantly improved catalytic efficiency (Figure 5) and FAD affinity. For example, a 2-fold increased turnover number was apparent for the W209R hDAAO, which was more active than the wild-type hDAAO using 0.3mM D-serine and 5μM FAD, i.e., concentrations resembling physiological conditions (Cappelletti et al., 2015).

Following overexpression in U87 cells, all the investigated hDAAO variants significantly altered the cellular levels of D-serine (Figure 6; Caldinelli et al., 2013; Cappelletti et al., 2015; Murtas et al., 2017a). The expression of inactive variants of hDAAO could produce susceptibility to neurodegenerative disorders due to augmented D-serine levels which, when paralleled by elevated glutamate levels, could lead to hyperactivation of NMDAR and thus to excitotoxicity. In contrast, a deficit in NMDAR-mediated transmission might be related to the expression of hyperactive variants due to an abnormal decrease in D-serine at the synapses, as proposed in schizophrenia onset. Furthermore, hDAAO hyperactive variants produce nonphysiological levels of H₂O₂: this process could contribute to the molecular mechanism of the central sensitization typical of chronic pain.

To delve into the structure-function relationships in mammalian DAAOs, alanine-scanning analysis of first and second shell residues of the enzyme from pig prompted the focus on active-site lid residues (region 221–225) and on the positions 55 and 56 in hDAAO (Subramanian et al., 2018). Molecular dynamics simulations identified a narrow tunnel that could provide access to the active site of hDAAO, named tunnel T1. The Y55 residue was suggested to be involved in anchoring the lid loop in the closed conformation (its dynamics are hampered by Y314), modulating the solvent access and substrate/product exchange at the active site and separating T1 from an additional, putative tunnel. The Y55A substitution facilitated accessibility of the active site: a 2-fold increase in specific activity on D-Trp was observed.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.