Human D-Amino Acid Oxidase: Structure, Function, and Regulation

Abstract

D-Amino acid oxidase (DAAO) is an FAD-containing flavoenzyme that catalyzes with absolute stereoselectivity the oxidative deamination of all natural D-amino acids, the only exception being the acidic ones. This flavoenzyme plays different roles during evolution and in different tissues in humans. Its three-dimensional structure is well conserved during evolution: minute changes are responsible for the functional differences between enzymes from microorganism sources and those from humans. In recent years several investigations focused on human DAAO, mainly because of its role in degrading the neuromodulator D-serine in the central nervous system. D-Serine is the main coagonist of N-methyl D-aspartate receptors, i.e., excitatory amino acid receptors critically involved in main brain functions and pathologic conditions. Human DAAO possesses a weak interaction with the FAD cofactor; thus, in vivo it should be largely present in the inactive, apoprotein form. Binding of active-site ligands and the substrate stabilizes flavin binding, thus pushing the acquisition of catalytic competence. Interestingly, the kinetic efficiency of the enzyme on D-serine is very low. Human DAAO interacts with various proteins, in this way modulating its activity, targeting, and cell stability. The known properties of human DAAO suggest that its activity must be finely tuned to fulfill a main physiological function such as the control of D-serine levels in the brain. At present, studies are focusing on the epigenetic modulation of human DAAO expression and the role of post-translational modifications on its main biochemical properties at the cellular level.

Keywords: D-amino acid oxidase; D-serine; NMDA receptor; structure-function relationships; substrate specificity.

Figure 1

Reaction catalyzed by DAAO.

Figure 2

Substrate preference of hDAAO. The apparent kinetic properties have been determined at 21% oxygen saturation, pH 8.5, and 25°C. All values were from Molla et al. (2006) with the exception of (a) Murtas et al. (2017b), Frattini et al. (2011); (b) Kawazoe et al. (2007b).

Figure 3

hDAAO three-dimensional structure (pdb codes 2e49). (A) The hDAAO protomer is constituted by two domains: the substrate and the FAD-binding domain (in blue and red, respectively). The entrance to the active site is indicated by a large arrow. The thin arrow indicates the putative additional ligand-binding site (Kohiki et al., 2017). (B) The substrate is located above the re-side of the isoalloxazine ring of FAD, in a cavity of ~220 ų. (C) hDAAO is a stable homodimer, characterized by a head-to-head mode of monomer interaction (Kawazoe et al., 2006). (D) 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), see dotted line (Umhau et al., 2000). Following hydride transfer, a negative charge is generated on the reduced flavin, which is stabilized by the positive charge generated on the imino group of the product. This figure has been generated by modeling a D-Tyr molecule instead of the original ligand imino serine. Figure prepared with 3dproteinimaging.com.

Figure 4

Structural formula of selected hDAAO inhibitors classified based on their mechanism of enzyme inhibition.

Figure 5

Apparent kinetic parameters of wild-type and hDAAO variants, determined at air saturation (0.25 mM oxygen), 25°C, and pH 8.5 (Molla et al., ; Caldinelli et al., ; Cappelletti et al., 2015).

Figure 6

Effect on the cellular D/(D+L) serine ratio in U87 human glioblastoma cells stably expressing EYFP-hDAAO variants (Caldinelli et al., ; Cappelletti et al., ; Murtas et al., 2017b). The effect of W209R substitution was established on transiently transfected cells (not shown): a figure of 4.2 ± 0.1 vs. 5.8 ± 0.4 was determined at 24 h for the variant and wild-type hDAAO, respectively.