The flavoenzyme DAAO from Rhodotorula gracilis, a structural paradigm of the glutathione-reductase family of flavoproteins, is a stable homodimer with a flavin adenine dinucleotide (FAD) molecule tightly bound to each 40-kD subunit. In this work, the thermal unfolding of dimeric DAAO was compared with that of two monomeric forms of the same protein: a Δloop mutant, in which 14 residues belonging to a loop connecting strands βF5-βF6 have been deleted, and a monomer obtained by treating the native holoenzyme with 0.5 M NH4SCN. Thiocyanate specifically and reversibly affects monomer association in wild-type DAAO by acting on hydrophobic residues and on ionic pairs between the βF5-βF6 loop of one monomer and the αI3′ and αI3″ helices of the symmetry-related monomer. By using circular dichroism spectroscopy, protein and flavin fluorescence, activity assays, and DSC, we demonstrated that thermal unfolding involves (in order of increasing temperatures) loss of tertiary structure, followed by loss of some elements of secondary structure, and by general unfolding of the protein structure that was concomitant to FAD release. Temperature stability of wild-type DAAO is related to the presence of a dimeric structure that affects the stability of independent structural domains. The monomeric Δloop mutant is thermodynamically less stable than dimeric wild-type DAAO (with melting temperatures (Tms) of 48°C and 54°C, respectively). The absence of complications ensuing from association equilibria in the mutant Aloop DAAO allowed identification of two energetic domains: a low-temperature energetic domain related to unfolding of tertiary structure, and a high-temperature energetic domain related to loss of secondary structure elements and to flavin release.
|Titolo:||Contribution of the dimeric state to the thermal stability of the flavoprotein D-amino acid oxidase|
|Autori interni:||BONOMI, FRANCESCO (Ultimo)|
IAMETTI, STEFANIA (Secondo)
BARBIROLI, ALBERTO GIUSEPPE
|Settore Scientifico Disciplinare:||Settore CHIM/02 - Chimica Fisica|
Settore BIO/10 - Biochimica
|Data di pubblicazione:||2003|
|Appare nelle tipologie:||01 - Articolo su periodico|
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