Biochemical Properties of Human D-amino Acid Oxidase Variants and Their Potential Significance in Pathologies

Abstract

The stereoselective flavoenzyme D-amino acid oxidase (DAAO) catalyzes the oxidative deamination of neutral and polar D-amino acids producing the corresponding α-keto acids, ammonia, and hydrogen peroxide. Despite its peculiar and atypical substrates, DAAO is widespread expressed in most eukaryotic organisms. In mammals (and humans in particular), DAAO is involved in relevant physiological processes ranging from D-amino acid detoxification in kidney to neurotransmission in the central nervous system, where DAAO is responsible of the catabolism of D-serine, a key endogenous co-agonist of N-methyl-D-aspartate receptors. Recently, structural and functional studies have brought to the fore the distinctive biochemical properties of human DAAO (hDAAO). It appears to have evolved to allow a strict regulation of its activity, so that the enzyme can finely control the concentration of substrates (such as D-serine in the brain) without yielding to an excessive production of hydrogen peroxide, a potentially toxic reactive oxygen species (ROS). Indeed, dysregulation in D-serine metabolism, likely resulting from altered levels of hDAAO expression and activity, has been implicated in several pathologies, ranging from renal disease to neurological, neurodegenerative, and psychiatric disorders. Only one mutation in DAO gene was unequivocally associated to a human disease. However, several single nucleotide polymorphisms (SNPs) are reported in the database and the biochemical characterization of the corresponding recombinant hDAAO variants is of great interest for investigating the effect of mutations. Here we reviewed recently published data focusing on the modifications of the structural and functional properties induced by amino acid substitutions encoded by confirmed SNPs and on their effect on D-serine cellular levels. The potential significance of the different hDAAO variants in human pathologies will be also discussed.

Keywords: D-amino acid oxidase; D-amino acids; D-serine levels; human pathologies; protein conformation; protein variants; structural-functional properties.

Figure 1

Scheme of the reaction catalyzed by DAAO on the substrate D-Ser.

Figure 2

Three-dimensional structure of hDAAO holoenzyme (PDB code 2DU8; Kawazoe et al., 2006). (A) Schematic view of the head-to-head mode of monomers interaction. α-helices, β-strands and coils are drawn in cartoon representation. The FAD cofactor and the inhibitor sodium benzoate are shown as sticks, colored in yellow and red, respectively. The protein surface is drawn in dark and light gray, and the second putative ligand binding pocket located at protein monomer interface, is highlighted. (B, C) Details of the active site of the enzyme in complex with (B) imino-serine (PDB 2E49; Kawazoe et al., 2007a), and (C) benzoate (PDB 2DU8; Kawazoe et al., 2006). FAD (yellow), iminoserine and benzoate (blue) as well as side chains of important residue in the active are shown as sticks. Figures were prepared using PyMol.

Figure 3

Frequency of sequence conservation among DAAOs from different sources. WebLogo representation of conserved residues identified by the alignment of DAAOs from mammals (Homo sapiens, Mus musculus, Rattus norvegicus, Sus scrofa) and yeast (Rhodotorula gracilis and Trigonopsis variabilis). The x-axis represents amino acid position (the annotated numbering refers to the human enzyme). The y-axis indicates the sequence conservation at that position (measured in bits), whereas the height of symbols is proportional to degree of conservations of single residues. Panels represent sequence stretches of 31 amino acids containing the residues that are substituted in the hDAAO variants discussed in this review (shown in red). Residues belonging only to the yeast sequences are shown in blue. Figure prepared using WebLogo 3.0 (Crooks et al., 2004).

Figure 4

Structural models of hDAAO inactive variants. (A) The position of the point mutations on the protein dimeric structure (PDB code: 2DU8; Kawazoe et al., 2006) is shown. Substituted residues encoded by SNPs are depicted as thick sticks in red, while FAD cofactor is reported in yellow. (B–D) Details of the protein microenvironment surrounding at the mutated residues in the different hDAAO variants. The substituted residues are shown as sticks in magenta. The surrounding residues within a distance of 4 Å are shown as sticks in gray. Loops, α-helices and β-sheets indicated in the text are shown as cartoon in red, blue and green, respectively. The substrate-competitive inhibitor benzoate and the FAD cofactor are shown as sticks in orange and yellow, respectively. H-bond interactions are reported as black dashed line. Negatively charged (blue), positively charged (red) and neutral (gray) surfaces of the C-terminal α-helix are shown in (D). Figure were prepared with PyMol. Adapted from Caldinelli et al. (2013); Cappelletti et al. (2015); Murtas et al. (2017b).

Figure 5

Comparison of the CD spectra of wild-type (black), G183R (blue), R199W (green) and R199Q (red) hDAAO variants. (A,C) Far-UV CD spectra of (A) the holoenzyme and (C) the apoprotein forms of the hDAAO variants (0.1 mg/mL protein concentration). (B,D) Near-UV CD spectra of (B) the holoenzyme and (D) the apoprotein forms of the hDAAO variants (0.4 mg/mL protein concentration). From (Caldinelli et al., ; Cappelletti et al., ; Murtas et al., 2017b).

Figure 6

Size-exclusion chromatography elution profiles of wild-type (black), R199Q (red) and R199W (green) hDAAO holoenzymes (5 mg /mL protein concentration). From (Cappelletti et al., 2015).

Figure 7

Structural models of hDAAO hyperactive variants. (A) Location of the point mutations on the protein dimeric structure (PDB code: 2DU8; Kawazoe et al., 2006). Substituted residues encoded by SNPs are depicted as thick sticks in green, while FAD cofactor is reported in yellow. (B–D) Details of the protein microenvironment surrounding at the mutated residues in the different hDAAO variants. The substituted residues are shown as sticks in magenta. The surrounding residues within a distance of 4 Å are shown as sticks in gray. Loops, α-helices, and β-sheets indicated in the text are shown as cartoon in red, blue, and green, respectively. The substrate-competitive inhibitor benzoate and the FAD cofactor are shown as sticks in orange and yellow, respectively. The VAAGL sequence is shown as a cartoon in light blue in (D). H-bond interactions are reported as black dashed line. Figure were prepared with PyMol. Adapted from Caldinelli et al. (2013); Cappelletti et al. (2015).

Figure 8

Comparison of CD spectra of wild-type (black), D31H (blue), R279A (green) and W209R (red) hDAAO variants. (A,C) Far-UV CD spectra of (A) the holoenzyme and (C) the apoprotein forms of hDAAO variants (0.1 mg/mL). (B,D) Near-UV CD spectra of (B) the holoenzyme and (D) the apoprotein forms of hDAAO variants (0.4 mg/mL). From (Caldinelli et al., ; Cappelletti et al., 2015).