D-Amino acid oxidase (DAO) is a biotechnologically attractive enzyme that can be used in a variety of applications, but its utility is limited by its relatively poor stability. A search of a bacterial genome database revealed a gene encoding a protein homologous to DAO in the thermophilic bacterium Rubrobacter xylanophilus (RxDAO). The recombinant protein expressed in Escherichia coli was a monomeric protein containing noncovalently bound flavin adenine dinucleotide as a cofactor. This protein exhibited oxidase activity against neutral and basic D-amino acids and was significantly inhibited by a DAO inhibitor, benzoate, but not by any of the tested D-aspartate oxidase (DDO) inhibitors, thus indicating that the protein is DAO. RxDAO exhibited higher activities and affinities toward branched-chain D-amino acids, with the highest specific activity toward D-valine and catalytic efficiency (k cat /K m ) toward D-leucine. Substrate inhibition was observed in the case of D-tyrosine. The enzyme had an optimum pH range and temperature of pH 7.5 to 10 and 65°C, respectively, and was stable between pH 5.0 and pH 8.0, with a T 50 (the temperature at which 50% of the initial enzymatic activity is lost) of 64°C. No loss of enzyme activity was observed after a 1-week incubation period at 30°C. This enzyme was markedly inactivated by phenylmethylsulfonyl fluoride but not by thiol-modifying reagents and diethyl pyrocarbonate, which are known to inhibit certain DAOs. These results demonstrated that RxDAO is a highly stable DAO and suggested that this enzyme may be valuable for practical applications, such as the determination and quantification of branched-chain D-amino acids, and as a scaffold to generate a novel DAO via protein engineering.T he flavoenzyme D-amino acid oxidase (DAO; EC 1.4.3.3) catalyzes the oxidative deamination of neutral and basic D-amino acids but not of acidic D-amino acids, which are substrates of D-aspartate oxidase (DDO). DAO was first identified in pig kidney (pkDAO) by Krebs in 1935 (1) and subsequently has been found mainly in eukaryotic organisms, ranging from fungi to humans (2). These eukaryotic DAOs have been extensively studied as a model of the oxidase-dehydrogenase class of flavoproteins. In fungi, the enzyme plays a role in the assimilation of D-amino acids for cell growth and also in the detoxification of D-amino acid toxicity (2, 3). In vertebrates, this enzyme is mainly localized in the liver and kidney, and it metabolizes both exogenous and endogenous D-amino acids (2). In addition, the enzyme plays a specific role in the regulation of D-serine, a coagonist for the N-methyl-Daspartate receptor, in the brain (2).In contrast, for a long time, it was thought that DAO did not exist in prokaryotic organisms. However, this enzyme was recently identified in Arthrobacter protophormiae (ApDAO) and Streptomyces coelicolor (ScDAO) (4, 5). In addition to these bacterial species, a search of a prokaryotic genome database revealed a wide distribution of DAO homologous proteins in bacteria, especially a...