Characterization of human d‐amino acid oxidase

Abstract

D D-Amino acid oxidase (DAAO) has been proposed to be involved in the oxidation of D D-serine, an allosteric activator of the NMDA-type glutamate receptor in the brain, and to be associated with the onset of schizophrenia. The recombinant human DAAO was expressed in Escherichia coli and was isolated as an active homodimeric flavoenzyme. It shows the properties of the dehydrogenase-oxidase class of flavoproteins, possesses a low kinetic efficiency, and follows a ternary complex (sequential) kinetic mechanism. In contrast to the other known DAAOs, the human enzyme is a stable homodimer even in the apoprotein form and weakly binds the cofactor in the free form.

Novel insights into renal d-amino acid oxidase accumulation: propiverine changes DAAO localization and peroxisomal size in vivo

“…Recombinant rDAAO and hDAAO were expressed in BL21(DE3)Star E. coli strain and purified as previously reported in (Frattini et al 2011) and (Molla et al 2006), respectively.…”

“…Thus, we first evaluated the ability of propiverine or propiverine-N-oxide to interact with recombinant rat DAAO (rDAAO). We performed spectral analyses using the recombinant rat and human (hDAAO) proteins, since it is well documented that the binding of ligands in the active site of the flavoenzyme yields to concentration-dependent perturbations of the visible absorbance spectrum (Harris et al 1999;Molla et al 2006;Frattini et al 2011). Notably, only minimal changes in the spectrum were observed following the titration with both propiverine and propiverine-N-oxide, suggesting that neither of the two compounds is an rDAAO ligand (Fig.…”

“…Mammalian DAAO is expressed in kidney, liver and brain (descending order of expression level), whereby mice represent an exception as no hepatic expression was detected (Pollegioni et al 2007;Konno et al 2009). Rat DAAO (rDAAO) shares 93 and 80 % sequence identity with mouse DAAO (mDAAO) and human (hDAAO), respectively (Konno 1998); nonetheless, rDAAO differs significantly from the human counterpart regarding structural, biochemical and kinetic properties: (1) rDAAO consists of 346 amino (38.8 kDa), whereas hDAAO consists of 347 amino acid residues (39.5 kDa) (Konno 1998), (2) rDAAO is a monomer in the concentration range 1-20 mg/mL (Frattini et al 2011) while hDAAO is a stable dimer (Molla et al 2006), (3) rDAAO shows lower kinetic efficiency and a different substrate specificity as compared to the human counterpart (Frattini et al 2011), and, most importantly, (4) rDAAO and hDAAO differ in affinity for the binding of classical inhibitors (Molla et al 2006;Caldinelli et al 2010;Frattini et al 2011).…”

Effect of ligand binding on human D‐amino acid oxidase: Implications for the development of new drugs for schizophrenia treatment

“…6 We recently demonstrated that in vitro hDAAO specifically interacts with pLG72, yielding a % 200-kDa complex constituted by 2 hDAAO homodimers and 2 pLG72 monomers. [7][8][9] This interaction results in a time-dependent loss of hDAAO activity which is mainly due to alteration of the tertiary structure of hDAAO. Furthermore, we confirmed in vivo the hDAAO-pLG72 interaction and demonstrated that the cellular concentration of D-serine decreases in U87 glioblastoma cells transfected with a plasmid encoding for hDAAO but is not modified in those simultaneously transfected with cDNAs encoding both pLG72 and hDAAO.…”

“…[10][11][12][13] Human DAAO shows the main properties of the dehydrogenase-oxidase class of flavoproteins and shares a low turnover number and a ternary complex (sequential) kinetic mechanism with pig kidney DAAO (pkDAAO, %85% sequence identity). 7 Indeed, this human flavoenzyme can be distinguished from the other known DAAOs because it is a stable homodimer even in the apoprotein form, because the binding of the FAD cofactor is the weakest among the known DAAOs (K d value is in the micromolar range) 1,7 and because it specifically interacts with pLG72 (which is expressed on primates only). 6 These features raise a question concerning the actual in vivo activity of hDAAO: based on the weak cofactor binding (FAD concentration in brain is estimated to be %5 lM) 14 hDAAO might be largely present in the inactive apoprotein form.…”