Tryptophan 243 Affects Interprotein Contacts, Cofactor Binding and Stability in D-amino Acid Oxidase From Rhodotorula Gracilis

FEBS J. 2006 Feb;273(3):504-12. doi: 10.1111/j.1742-4658.2005.05083.x.

Abstract

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 betaF5-betaF6 loop of one monomer and negatively charged residues belonging to the alpha-helices I3' and I3'' of the other monomer, and (b) the interaction of residues (e.g. 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 by efficiently excluding solvent.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • D-Amino-Acid Oxidase / chemistry*
  • D-Amino-Acid Oxidase / drug effects
  • Enzyme Activation
  • Enzyme Stability / drug effects
  • Kinetics
  • Models, Molecular
  • Mutation
  • Protein Conformation
  • Protein Folding
  • Protein Structure, Tertiary
  • Rhodotorula / enzymology*
  • Static Electricity
  • Structure-Activity Relationship
  • Temperature
  • Time Factors
  • Tryptophan / chemistry*
  • Urea / pharmacology

Substances

  • Tryptophan
  • Urea
  • D-Amino-Acid Oxidase