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. Study holds ProTherm entries: 16143, 16144, 16145, 16146, 16147, 16148, 16149, 16150, 16151 Extra Details: 2 mM EDTA and 10% glycerol were added in the experiment Flavoprotein; lipophilic ions; folding; dimerization; energetic domains; structural domains; quaternary structure; thermal stability

Submission Details

ID: thM2MUjN4

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:47 p.m.

Version: 1

Publication Details
Pollegioni L;Iametti S;Fessas D;Caldinelli L;Piubelli L;Barbiroli A;Pilone MS;Bonomi F,Protein Sci. (2003) Contribution of the dimeric state to the thermal stability of the flavoprotein D-amino acid oxidase. PMID:12717024
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
1C0P 2000-11-22 1.2 D-AMINO ACIC OXIDASE IN COMPLEX WITH D-ALANINE AND A PARTIALLY OCCUPIED BIATOMIC SPECIES
1C0K 2000-11-22 1.46 CRYSTAL STRUCTURE ANALYSIS OF D-AMINO ACID OXIDASE IN COMPLEX WITH L-LACTATE
1C0L 2000-11-22 1.73 D-AMINO ACID OXIDASE: STRUCTURE OF SUBSTRATE COMPLEXES AT VERY HIGH RESOLUTION REVEAL THE CHEMICAL REACTTION MECHANISM OF FLAVIN DEHYDROGENATION
1C0I 2002-02-27 1.9 CRYSTAL STRUCTURE OF D-AMINO ACID OXIDASE IN COMPLEX WITH TWO ANTHRANYLATE MOLECULES

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 D-amino-acid oxidase P80324 OXDA_RHOTO