D-amino acid oxidase catalyzes the oxidative deamination of D-amino acids. In the brain, the NMDA receptor coagonist D-serine has been proposed as its physiological substrate. In order to shed light on the mechanisms regulating D-serine concentration at the cellular level, we biochemically characterized human DAAO (hDAAO) in greater depth. In addition to clarify the physical-chemical properties of the enzyme, we demonstrated that divalent ions and nucleotides do not affect flavoenzyme function. Moreover, the definition of hDAAO substrate specificity demonstrated that D-cysteine is the best substrate, which made it possible to propose it as a putative physiological substrate in selected tissues. Indeed, the flavoenzyme shows a preference for hydrophobic amino acids, some of which are molecules relevant in neurotransmission, i.e., D-kynurenine, D-DOPA, and D-tryptophan. hDAAO shows a very low affinity for the flavin cofactor. The apoprotein form exists in solution in equilibrium between two alternative conformations: the one at higher affinity for FAD is favored in the presence of an active site ligand. This may represent a mechanism to finely modulate hDAAO activity by substrate/inhibitor presence. Taken together, the peculiar properties of hDAAO seem to have evolved in order to use this flavoenzyme in different tissues to meet different physiological needs related to D-amino acids.

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.

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The human enzyme D-3-phosphoglycerate dehydrogenase (hPHGDH) catalyzes the reversible dehydrogenation of 3-phosphoglycerate (3PG) into 3-phosphohydroxypyruvate (PHP) using the NAD+/NADH redox cofactor, the first step in the phosphorylated pathway producing L-serine. We focused on the full-length enzyme that was produced in fairly large amounts in E. coli cells; the effect of pH, temperature and ligands on hPHGDH activity was studied. The forward reaction was investigated on 3PG and alternative carboxylic acids by employing two coupled assays, both removing the product PHP; 3PG was by far the best substrate in the forward direction. Both PHP and α-ketoglutarate were efficiently reduced by hPHGDH and NADH in the reverse direction, indicating substrate competition under physiological conditions. Notably, neither PHP nor L-serine inhibited hPHGDH, nor did glycine and D-serine, the coagonists of NMDA receptors related to L-serine metabolism. The investigation of NADH and phosphate binding highlights the presence in solution of different conformations and/or oligomeric states of the enzyme. Elucidating the biochemical properties of hPHGDH will enable the identification of novel approaches to modulate L-serine levels and thus to reduce cancer progression and treat neurological disorders.