Abstract
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.
Highlights
D-amino acid oxidase (DAAO, EC 1.4.3.3) is the FAD-containing enzyme that catalyzes the strictly stereospecific oxidative deamination of the D-isomer of neutral amino acids
Concerning the pH effect on stability, the activity values determined after 30 or 60 min show that human DAAO (hDAAO) is fully stable in a pH range of 4–10 (Figure 1B); experimental values were fitted based on the equation for two dissociations: estimated pKa’s were 2.5 ± 0.1 and 11.1 ± 0.1
To investigate the temperature dependence of the initial rate of the reaction catalyzed by hDAAO, the enzymatic activity was assayed at different temperature values on 28 mM D-alanine, at pH 8.5, and in the presence of 200 μM FAD by measuring the O2 consumption
Summary
D-amino acid oxidase (DAAO, EC 1.4.3.3) is the FAD-containing enzyme that catalyzes the strictly stereospecific oxidative deamination of the D-isomer of neutral amino acids. DAAO plays various roles in different organisms: in microorganisms it catalyzes the catabolism of D-amino acids for cell metabolism, and specific functions have been proposed in nematodes, insects, and lower vertebrates (Pollegioni et al, 2007; Saitoh et al, 2012). In higher vertebrates, it Abbreviations: CBIO, 6-chloro-benzo(d)isoxazol-3-ol; CD, circular dichroism; DAAO, D-amino acid oxidase (EC 1.4.3.3); DOPA, 3,4-dihydroxyphenylalanine; hDAAO, human D-amino acid oxidase; L-NAC, N-acetyl-L-cysteine; NMDAR, Nmethyl-D-aspartate subtype of glutamate receptor It Abbreviations: CBIO, 6-chloro-benzo(d)isoxazol-3-ol; CD, circular dichroism; DAAO, D-amino acid oxidase (EC 1.4.3.3); DOPA, 3,4-dihydroxyphenylalanine; hDAAO, human D-amino acid oxidase; L-NAC, N-acetyl-L-cysteine; NMDAR, Nmethyl-D-aspartate subtype of glutamate receptor.
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