TAILIEUCHUNG - Báo cáo khoa học: Tryptophan 243 affects interprotein contacts, cofactor binding and stability in D-amino acid oxidase from Rhodotorula gracilis

The flavoenzyme d-amino acid oxidase from Rhodotorula gracilisis a homodimeric protein whose dimeric state has been proposed to occur as a result of (a) the electrostatic interactions between positively charged resi-dues of thebF5–bF6 loop of one monomer and negatively charged residues belonging to thea-helices I3¢ and I3¢¢ of the other monomer, and (b) the interaction of residues (. Trp243) belonging to the two monomers at the mixed interface region. | iFEBS Journal Tryptophan 243 affects interprotein contacts cofactor binding and stability in D-amino acid oxidase from Rhodotorula gracilis Laura Caldinelli Gianluca Molla Mirella S. Pilone and Loredano Pollegioni Department of Biotechnology and Molecular Sciences University of Insubria Varese Italy Keywords cofactor binding flavoprotein oligomerization state rationaldesign structural stability Correspondence L. Pollegioni Dipartimento di Biotecnologie e Scienze Molecolari University degli Studi dell Insubria Via J. H. Dunant 3-21100 Varese Italy Fax 39 0332 421500 Tel 39 0332 421506 E-mail Received 10 October 2005 revised 18 November 2005 accepted 2 December 2005 doi The flavoenzyme D-amino acid oxidase from Rhodotorula gracilis is a homodimeric protein whose dimeric state has been proposed to occur as a result of a the electrostatic interactions between positively charged residues of the PF5-PF6 loop of one monomer and negatively charged residues belonging to the a-helices I3 and I3 of the other monomer and b the interaction of residues . Trp243 belonging to the two monomers at the mixed interface region. The role of Trp243 was investigated by substituting it with either tyrosine or isoleucine both substitutions were nondisruptive as confirmed by the absence of significant changes in catalytic activity but altered the tertiary structure yielding a looser conformation and decreased the stability towards temperature and denaturants. The change in conformation interferes both with the interaction of the coenzyme to the apoprotein moiety although the kinetics of the apoprotein-FAD complex reconstitution process are similar between wild-type and mutant D-amino acid oxidases and with the interaction between monomers. Our results indicate that in the folded holoenzyme Trp243 is situated at a position optimal for increasing the interactions between monomers by maximizing van der Waals interactions and

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