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Protein Science (2003), 12:1018-1029.
Copyright © 2003 The Protein Society

Contribution of the dimeric state to the thermal stability of the flavoprotein D-amino acid oxidase

Loredano Pollegioni1, Stefania Iametti2, Dimitrios Fessas3, Laura Caldinelli1, Luciano Piubelli1, Alberto Barbiroli3, Mirella S. Pilone1 and Francesco Bonomi2

1 Department of Structural and Functional Biology, University of Insubria, 21100 Varese, Italy
2 Dipartimento di scienze molecolari agroalimentari (DISMA) and
3 Dipartimento di scienze e tecnologie alimentari e microbiologiche (DISTAM), University of Milan, 20133 Milan, Italy

Reprint requests to: Francesco Bonomi, DISMA, University of Milan, via Celoria 2, 20133 Milan, Italy; e-mail: francesco.bonomi{at}unimi.it; fax: 39-02-50316801.

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 {Delta}loop mutant, in which 14 residues belonging to a loop connecting strands ßF5–ßF6 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 ßF5–ßF6 loop of one monomer and the {alpha}I3' and {alpha}I3'' 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 {Delta}loop 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 {Delta}loop 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.

Keywords: Flavoprotein; lipophilic ions; folding; dimerization; energetic domains; structural domains; quaternary structure; thermal stability

Abbreviations: DAAO, D-amino acid oxidase (EC 1.4.3.3) • RgDAAO, Rhodotorula gracilis D-amino acid oxidase • DSC, differential scanning calorimetry • GR, glutathione reductase


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