Abstract
D-amino acid oxidase (DAAO) is a well-known flavoenzyme that catalyzes the oxidative FAD-dependent deamination of D-amino acids. As a result of the absolute stereoselectivity and broad substrate specificity, microbial DAAOs have been employed as industrial biocatalysts in the production of semi-synthetic cephalosporins and enantiomerically pure amino acids. Moreover, in mammals, DAAO is present in specific brain areas and degrades D-serine, an endogenous coagonist of the N-methyl-D-aspartate receptors (NMDARs). Dysregulation of D-serine metabolism due to an altered DAAO functionality is related to pathological NMDARs dysfunctions such as in amyotrophic lateral sclerosis and schizophrenia. In this protocol paper, we describe a variety of direct assays based on the determination of molecular oxygen consumption, reduction of alternative electron acceptors, or α-keto acid production, of coupled assays to detect the hydrogen peroxide or the ammonium production, and an indirect assay of the α-keto acid production based on a chemical derivatization. These analytical assays allow the determination of DAAO activity both on recombinant enzyme preparations, in cells, and in tissue samples.
Highlights
D-Amino acid oxidase (DAAO, EC 1.4.3.3) is a flavoenzyme containing a non-covalently bound FAD molecule per 40 kDa monomer that belongs to the dehydrogenase/oxidase class of flavoproteins (Pollegioni et al, 2007a)
The reaction catalyzed by DAAO can be divided into (Pollegioni et al, 2008): 1) a reductive half-reaction, in which the enzyme catalyzes the dehydrogenation of α-D-amino acids to their imino acid counterparts with concomitant reduction of FAD to FADH2 [reaction (1) in Figure 1]; Abbreviations: 4-AAP, 4-aminoantipyrine; CNS, central nervous system; DAAO, D-amino acid oxidase; DCPIP, 2,6-Dichlorophenol-indophenol; DNaseI, desossiribonuclease I; DNP, 2,4-dinitrophenylhydrazine; EDTA, ethylenediaminetetraacetic acid; FAD/FADH2, flavin adenine dinucleotide oxidized/reduced form; GDH, glutamate dehydrogenase; hDAAO, human D-amino acid oxidase; HRP, horseradish peroxidase; MG, methylene green; NAD+/NADH, nicotinamide adenine dinucleotide oxidized/reduced form; NMDARs, N-methyl-D-aspartate receptors; o-DNS, o-dianisidine; RgDAAO, D-amino acid oxidase from Rhodotorula gracilis; Thi, thionine
The ammonium produced by DAAO reaction on a D-amino acid can be detected by a second enzyme-coupled reaction in which α-ketoglutarate is converted to L-glutamate by L-glutamate dehydrogenase from bovine liver
Summary
D-Amino acid oxidase (DAAO, EC 1.4.3.3) is a flavoenzyme containing a non-covalently bound FAD molecule per 40 kDa monomer that belongs to the dehydrogenase/oxidase class of flavoproteins (Pollegioni et al, 2007a). The main role of DAAO in mammals (including humans) is related to its presence in selected brain areas where it is devoted to the catabolism of D-serine This αD-amino acid is a neuromodulator acting as a coagonist of the N-methyl-D-aspartate receptors (NMDARs). Which a chromogenic (o-dianisidine or 4-aminoantipyrine; Job et al, 2002; Sacchi et al, 2004; Rosini et al, 2008) or a fluorogenic (Amplex R UltraRed, Invitrogen; Sacchi et al, 2011; Hopkins et al, 2013) substrate is used in combination with horseradish peroxidase, or in which a chromogenic reagent (Purpald R : 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole, Sigma-Aldrich) is employed in combination with catalase (Dickinson and Jacobsen, 1970; Watanabe et al, 1978; Sasabe et al, 2014); (iv) α-keto acid formation, directly detected by measuring the increase in absorbance in the ultraviolet range (Tedeschi et al, 2012) or indirectly detected following the reaction of the αketo acid produced with 2,4-dinitrophenylhydrazine, to give a chromogenic 2,4-dinitrophenylhydrazone derivative (Nagata et al, 1988); and (v) ammonium production, indirectly detected using an enzyme-coupled assay in which α-ketoglutarate is converted to L-glutamate by the NAD+-dependent L-glutamate dehydrogenase (Holme and Goldberg, 1975). The sample is continuously stirred in order to ensure that the dissolved oxygen is kept evenly distributed throughout the reaction vessel
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