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

Abstract

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 Å . 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.Keywords: D-amino acid oxidase; Homo sapiens; X-ray crystallography; structurally ambivalent peptides; conformational variability D-amino acid oxidase (DAO) (EC 1.4.3.3) was first identified by Hans Krebs in 1935 and was later recognized to be the first enzyme known to use FAD as a cofactor (Krebs 1935). DAO noncovalently binds FAD as a prosthetic group and catalyzes the oxidative deamination of D-amino acids to their corresponding imino acids with concomitant reduction of FAD. The reduced flavin is subsequently reoxidized by molecular oxygen generating H 2 O 2 , and the imino acid is released into the solvent where it nonenzymatically hydrolyzes, yielding the corresponding a-keto acid and ammonia. DAO exhibits optimal activity toward neutral amino acids and marginal activity toward basic ones; acidic D-amino acids are oxidized by another flavoprotein, D-aspartate oxidase.Reprint requests to: Kiyoshi Fukui, The Institute for Enzyme Research, The University of Tokushima, Japan; Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi

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

“…Indeed, these two ligands modify to a different extent the protein conformation (as made apparent by protein fluorescence): this difference might be ascribed to the two alternative positions occupied by Tyr224 side chain in the 3D structure of hDAAO in complex with benzoate or with iminoserine (PDB ID 2DU8 and 2E49). 1,16 Furthermore, the large change in protein fluorescence of the apoprotein in the presence of D-serine or CF 3 -DAla and the thermal stabilization following the binding of the pseudo-substrate clearly shows that the The change in hDAAO concentration with respect to the control was demonstrated to be statistically significant for CPZ-treated samples at 48 hours (P ¼ 0.017, see asterisk) and not statistically significant for all the remaining samples (P > 0.05). (C) Same analysis as reported in panel B performed on U87 glioblastoma cells stably expressing EGFP-hDAAO and transiently transfected with pLG72.…”

“…15 In this work we analyze the consequences of ligand binding on conformation and stability of hDAAO alone and in complex with pLG72. To reach this goal, the substrate D-serine or the pseudo-substrate trifluoro-D-alanine (CF 3 -D-Ala), benzoate (the most studied substrate-competitive inhibitor of hDAAO, K d ¼ 7 lM), 1,9,16 and the drug chlorpromazine (CPZ) were employed. The aliphatic phenothiazine CPZ is a dopamine D2 receptor antagonist that was introduced in the treatment of schizophrenia in the early 1950s; it also acts as a FAD-competitive inhibitor of hDAAO (K d ¼ 5 lM).…”

O2 Reactivity of Flavoproteins

“…Several Paths Lead O 2 to the Active Center-The three-dimensional structures of DAAO from yeast (19), human (26), and pig (27) have in common a narrow water-filled channel connecting the solvent and the Si-side of the flavin (see Figs. 1 and 2 and supplemental Fig.…”

“…The putative O 2 channel on the flavin Si-side is flexible, and the H 2 O molecules inside are not fixed but in constant exchange with the bulk water phase. No H 2 O is observed at site A in any of the four different DAAO x-ray structures (18,19,26,27) Obstructing O 2 Access, the G52X DAAO Variants-The O 2 access hypothesis was tested computationally as well as experimentally. Based on the three-dimensional structure (19), it can be predicted that mutation of the residue Gly-52 would interfere with the high affinity site A (Fig.…”

Chlorpromazine oligomer is a potentially active substance that inhibits humanD-amino acid oxidase, product of a susceptibility gene for schizophrenia

“…Human DAO was expressed and purified as described [24]. The preparation of the apoenzyme of human DAO was prepared by extensive dialysis of the holoenzyme against 1 M potassium bromide according to the method of Massey and Curti [25].…”