The flavoenzyme D-amino acid oxidase (DAAO) from Rhodotorula gracilis is a peroxisomal enzyme and a prototypical member of the glutathione reductase family of flavoproteins. DAAO is a stable homodimer with a FAD molecule tightly bound to each 40-kDa subunit. In this work, the urea-induced unfolding of dimeric DAAO was compared with that of a monomeric form of the same protein, a deleted dimerization loop mutant. By using circular dichroism spectroscopy, protein and flavin fluorescence, 1,8-anilinonaphtalene sulfonic acid binding and activity assays, we demonstrated that the urea-induced unfolding of DAAO is a three-state process, yielding an intermediate, and that this process is reversible. The intermediate species lacks the catalytic activity and the characteristic tertiary structure of native DAAO but has significant secondary structure and retains flavin binding. Unfolding of DAAO proceeds through formation of an expanded, partially unfolded inactive intermediate, characterized by low solubility, by increased exposure of hydrophobic surfaces, and by increased sensitivity to trypsin of the β-strand F5 belonging to the FAD binding domain. The oligomeric state does not modify the inferred folding process. The strand F5 is in contact with the C-terminal α-helix containing the Ser-Lys-Leu sequence corresponding to the type 1 peroxisomal targeting signal, and this structural element interacts with the N-terminal βαβ flavin binding motif (Rossmann fold). The expanded conformation of the folding intermediate (and in particular the higher disorder of the mentioned secondary structure elements) could match the structure of the inactive holoenzyme required for in vivo trafficking of DAAO through the peroxisomal membrane.
|Titolo:||Unfolding intermediate in the peroxisomal flavoprotein D-amino acid oxidase|
IAMETTI, STEFANIA (Secondo)
|Settore Scientifico Disciplinare:||Settore BIO/10 - Biochimica|
|Data di pubblicazione:||2004|
|Digital Object Identifier (DOI):||10.1074/jbc.M403489200|
|Appare nelle tipologie:||01 - Articolo su periodico|