Publications

• Crystal structure of human D-amino acid oxidase: context-dependent variability of the backbone conformation of the VAAGL hydrophobic stretch located at the si-face of the flavin ring.

  Kawazoe T., Tsuge H., Pilone M.S., Fukui K.

  In the brain, the extensively studied FAD-dependent enzyme D-amino acid oxidase (DAO) degrades the gliotransmitter D-serine, a potent activator of N-methyl-D-aspartate type glutamate receptors, and evidence suggests that DAO, together with its activator G72 protein, may play a key role in the path ... >> More

  In the brain, the extensively studied FAD-dependent enzyme D-amino acid oxidase (DAO) degrades the gliotransmitter D-serine, a potent activator of N-methyl-D-aspartate type glutamate receptors, and evidence suggests that DAO, together with its activator G72 protein, may play a key role in the pathophysiology of schizophrenia. Indeed, its potential clinical importance highlights the need for structural and functional analyses of human DAO. We recently succeeded in purifying human DAO, and found that it weakly binds FAD and shows a significant slower rate of flavin reduction compared with porcine DAO. However, the molecular basis for the different kinetic features remains unclear because the active site of human DAO was considered to be virtually identical to that of porcine DAO, as would be expected from the 85% sequence identity. To address this issue, we determined the crystal structure of human DAO in complex with a competitive inhibitor benzoate, at a resolution of 2.5 Angstrom. The overall dimeric structure of human DAO is similar to porcine DAO, and the catalytic residues are fully conserved at the re-face of the flavin ring. However, at the si-face of the flavin ring, despite the strict sequence identity, a hydrophobic stretch (residues 47-51, VAAGL) exists in a significantly different conformation compared with both of the independently determined porcine DAO-benzoate structures. This suggests that a context-dependent conformational variability of the hydrophobic stretch accounts for the low affinity for FAD as well as the slower rate of flavin reduction, thus highlighting the unique features of the human enzyme. << Less

  Protein Sci. 15:2708-2717(2006) [PubMed] [EuropePMC]

  This publication is cited by 4 other entries.

• A novel thermostable D-amino acid oxidase of the thermophilic fungus Rasamsonia emersonii strain YA.

  Shimekake Y., Furuichi T., Abe K., Kera Y., Takahashi S.

  D-Amino acid oxidase (DAAO) is a valuable flavoenzyme capable of being used in various practical applications, such as in determining D-amino acids and producing a material for semisynthetic cephalosporins, requiring higher thermal stability, higher catalytic activity, and broad substrate specific ... >> More

  D-Amino acid oxidase (DAAO) is a valuable flavoenzyme capable of being used in various practical applications, such as in determining D-amino acids and producing a material for semisynthetic cephalosporins, requiring higher thermal stability, higher catalytic activity, and broad substrate specificity. In this study, we isolated the thermophilic fungus Rasamsonia emersonii strain YA, which can grow on several D-amino acids as the sole nitrogen source, from a compost and characterized DAAO (ReDAAO) of the fungus. ReDAAO expressed in Escherichia coli exhibited significant oxidase activity against various neutral and basic D-amino acids, in particular hydrophobic D-amino acids. In addition, the enzyme also significantly acted on cephalosporin C, a starting material for semisynthetic antibiotics, and D-Glu, a general substrate for D-aspartate oxidase but not for DAAO, showing its unique and practically useful substrate specificity. The apparent k_cat and K_m values of the enzyme toward good substrates were comparable to those of higher catalytic fungal DAAOs, and the thermal stability (T₅₀ value of ~60 °C) was comparable to that of a thermophilic bacterial DAAO and significantly higher than that of other eukaryotic DAAOs. These results highlight the great potential of ReDAAO for use in practical applications. << Less

  Sci. Rep. 9:11948-11948(2019) [PubMed] [EuropePMC]

  This publication is cited by 17 other entries.

• Engineering the substrate specificity of porcine kidney D-amino acid oxidase by mutagenesis of the 'active-site lid'.

  Setoyama C., Nishina Y., Mizutani H., Miyahara I., Hirotsu K., Kamiya N., Shiga K., Miura R.

  Comparison of the primary structures of pig kidney D-amino acid oxidase (DAO) and human brain D-aspartate oxidase (DDO) revealed a notable difference at I215-N225 of DAO and the corresponding region, R216-G220, of DDO. A DAO mutant, in which I215-N225 is substituted by R216-G220 of DDO, showed D-a ... >> More

  Comparison of the primary structures of pig kidney D-amino acid oxidase (DAO) and human brain D-aspartate oxidase (DDO) revealed a notable difference at I215-N225 of DAO and the corresponding region, R216-G220, of DDO. A DAO mutant, in which I215-N225 is substituted by R216-G220 of DDO, showed D-aspartate-oxidizing activity that wild-type DAO does not exhibit, together with a considerable decrease in activity toward D-alanine. These findings indicate that I215-N225 of DAO contributes profoundly to its substrate specificity. Based on these results and the crystal structure of DAO, we systematically mutated the E220-Y224 region within the short stretch in question and obtained five mutants (220D224G, 221D224G, 222D224G, 223D224G, and 224D), in each of which an aspartate residue is mutated to E220-Y224. All of the mutants exhibited decreased apparent K(m) values toward D-arginine, i.e., to one-seventh to one-half that of wild type DAO. The specificity constant, k(cat app)/K(m app), for D-arginine increased by one order of magnitude for the 221D224G or 222D224G mutant, whereas that for D-alanine or D-serine decreased to marginal or nil. << Less

  J. Biochem. 139:873-879(2006) [PubMed] [EuropePMC]

  This publication is cited by 4 other entries.

• Structural basis of D-DOPA oxidation by D-amino acid oxidase: alternative pathway for dopamine biosynthesis.

  Kawazoe T., Tsuge H., Imagawa T., Aki K., Kuramitsu S., Fukui K.

  D-amino acid oxidase (DAO) degrades the gliotransmitter D-serine, a potent endogenous ligand of N-methyl-D-aspartate type glutamate receptors. It also has been suggested that D-DOPA, the stereoisomer of L-DOPA, is oxidized by DAO and then converted to dopamine via an alternative biosynthetic pathw ... >> More

  D-amino acid oxidase (DAO) degrades the gliotransmitter D-serine, a potent endogenous ligand of N-methyl-D-aspartate type glutamate receptors. It also has been suggested that D-DOPA, the stereoisomer of L-DOPA, is oxidized by DAO and then converted to dopamine via an alternative biosynthetic pathway. Here, we provide direct crystallographic evidence that D-DOPA is readily fitted into the active site of human DAO, where it is oxidized by the enzyme. Moreover, our kinetic data show that the maximal velocity for oxidation of D-DOPA is much greater than for D-serine, which strongly supports the proposed alternative pathway for dopamine biosynthesis in the treatment of Parkinson's disease. In addition, determination of the structures of human DAO in various states revealed that the conformation of the hydrophobic VAAGL stretch (residues 47-51) to be uniquely stable in the human enzyme, which provides a structural basis for the unique kinetic features of human DAO. << Less

  Biochem. Biophys. Res. Commun. 355:385-391(2007) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• Overproduction and characterization of a recombinant D-amino acid oxidase from Arthrobacter protophormiae.

  Geueke B., Weckbecker A., Hummel W.

  A screening of soil samples for D-amino acid oxidase (D-AAO) activity led to the isolation and identification of the gram-positive bacterium Arthrobacter protophormiae. After purification of the wild-type D-AAO, the gene sequence was determined and designated dao. An alignment of the deduced prima ... >> More

  A screening of soil samples for D-amino acid oxidase (D-AAO) activity led to the isolation and identification of the gram-positive bacterium Arthrobacter protophormiae. After purification of the wild-type D-AAO, the gene sequence was determined and designated dao. An alignment of the deduced primary structure with eukaryotic D-AAOs and D-aspartate oxidases showed that the D-AAO from A. protophormiae contains five of six conserved regions; the C-terminal type 1 peroxisomal targeting signal that is typical for D-AAOs from eukaryotic origin is missing. The dao gene was cloned and expressed in Escherichia coli. The purified recombinant D-AAO had a specific activity of 180 U mg protein(-1) for D-methionine and was slightly inhibited in the presence of L-methionine. Mainly, basic and hydrophobic D-amino acids were oxidized by the strictly enantioselective enzyme. After a high cell density fermentation, 2.29 x 10(6) U of D-AAO were obtained from 15 l of fermentation broth. << Less

  Appl. Microbiol. Biotechnol. 74:1240-1247(2007) [PubMed] [EuropePMC]

  This publication is cited by 5 other entries.