title: Contribution of the dimeric state to the thermal stability of the flavoprotein D-amino acid oxidase.
abstract: 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 Deltaloop mutant, in which 14 residues belonging to a loop connecting strands betaF5-betaF6 have been deleted, and a monomer obtained by treating the native holoenzyme with 0.5 M NH(4)SCN. Thiocyanate specifically and reversibly affects monomer association in wild-type DAAO by acting on hydrophobic residues and on ionic pairs between the betaF5-betaF6 loop of one monomer and the alphaI3' and alphaI3" 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 Deltaloop mutant is thermodynamically less stable than dimeric wild-type DAAO (with melting temperatures (T(m)s) of 48 degrees C and 54 degrees C, respectively). The absence of complications ensuing from association equilibria in the mutant Deltaloop 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.
authors.affiliation: Department of Structural and Functional Biology, University of Insubria, 21100 Varese, Italy.
authors.forename: Loredano
authors.initials: L
authors.lastname: Pollegioni
authors.forename: Stefania
authors.initials: S
authors.lastname: Iametti
authors.forename: Dimitrios
authors.initials: D
authors.lastname: Fessas
authors.forename: Laura
authors.initials: L
authors.lastname: Caldinelli
authors.forename: Luciano
authors.initials: L
authors.lastname: Piubelli
authors.forename: Alberto
authors.initials: A
authors.lastname: Barbiroli
authors.forename: Mirella S
authors.initials: MS
authors.lastname: Pilone
authors.forename: Francesco
authors.initials: F
authors.lastname: Bonomi
mesh_terms: D003605:D-Amino-Acid Oxidase / Q000737:chemistry*; D019281:Dimerization; D004795:Enzyme Stability; D005420:Flavoproteins / Q000737:chemistry; D005656:Fungal Proteins / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011489:Protein Denaturation; D012248:Rhodotorula / Q000737:chemistry; D013696:Temperature; D013861:Thiocyanates / Q000494:pharmacology
chemical_list: D005420:Flavoproteins; D005656:Fungal Proteins; D013861:Thiocyanates; D003605:D-Amino-Acid Oxidase; C031760:thiocyanate