title: Relevance of weak flavin binding in human D-amino acid oxidase.
abstract: In the brain, the human flavoprotein D-amino acid oxidase (hDAAO) is involved in the degradation of the gliotransmitter D-serine, an important modulator of NMDA-receptor-mediated neurotransmission; an increase in hDAAO activity (that yields a decrease in D-serine concentration) was recently proposed to be among the molecular mechanisms leading to the onset of schizophrenia susceptibility. This human flavoenzyme is a stable homodimer (even in the apoprotein form) that distinguishes from known D-amino acid oxidases because it shows the weakest interaction with the flavin cofactor in the free form. Instead, cofactor binding is significantly tighter in the presence of an active site ligand. In order to understand how hDAAO activity is modulated, we investigated the FAD binding process to the apoprotein moiety and compared the folding and stability properties of the holoenzyme and the apoprotein forms. The apoprotein of hDAAO can be distinguished from the holoenzyme form by the more "open" tertiary structure, higher protein fluorescence, larger exposure of hydrophobic surfaces, and higher sensitivity to proteolysis. Interestingly, the FAD binding only slightly increases the stability of hDAAO to denaturation by urea or temperature. Taken together, these results indicate that the weak cofactor binding is not related to protein (de)stabilization or oligomerization (as instead observed for the homologous enzyme from yeast) but rather should represent a means of modulating the activity of hDAAO. We propose that the absence in vivo of an active site ligand/substrate weakens the cofactor binding, yielding the inactive apoprotein form and thus avoiding excessive D-serine degradation.
authors.affiliation: Department of Biotechnology and Molecular Sciences, University of Insubria, via J.H. Dunant 3, 21100 Varese, Italy.
authors.forename: Laura
authors.initials: L
authors.lastname: Caldinelli
authors.forename: Gianluca
authors.initials: G
authors.lastname: Molla
authors.forename: Silvia
authors.initials: S
authors.lastname: Sacchi
authors.forename: Mirella S
authors.initials: MS
authors.lastname: Pilone
authors.forename: Loredano
authors.initials: L
authors.lastname: Pollegioni
mesh_terms: D001059:Apoproteins / Q000737:chemistry / Q000378:metabolism; D003605:D-Amino-Acid Oxidase / Q000737:chemistry / Q000378:metabolism*; D004795:Enzyme Stability; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D057927:Hydrophobic and Hydrophilic Interactions; D011485:Protein Binding; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D013696:Temperature; D014357:Trypsin / Q000378:metabolism; D014508:Urea / Q000378:metabolism
chemical_list: D001059:Apoproteins; D005182:Flavin-Adenine Dinucleotide; D014508:Urea; D003605:D-Amino-Acid Oxidase; D014357:Trypsin