title: Identification of a reactive cysteine in the flavin-binding domain of Rhodotorula gracilis D-amino acid oxidase.
abstract: The holoenzyme form of Rhodotorula gracilis D-amino acid oxidase, an 80-kDa homodimer, reacted only to a limited extent with general thiol reagents (2,2'-dithiodipyridine, 5,5'-dithiobis(2-nitrobenzoic acid), and N-[7-(dimethylamino)-4-methylcoumarinyl]maleimide) (60% residual activity), whereas the monomeric apoprotein was completely inactivated and denatured by these reagents. To investigate the presence of thiol residue(s) in the active site of the enzyme, the apoprotein was reconstituted with the 8-(methylsulfonyl)-FAD chemical-affinity probe. Competitive inhibition between this analogue and FAD for apoprotein binding was observed. The covalent attachment of the flavin analogue to the apoprotein was complete after approximately 20 h of incubation and the flavinylated enzyme, containing 8-(cysteinyl)-FAD, was monomeric and inactive. After HPLC isolation of the flavin-labeled tryptic peptides, Cys208 was identified as the only cysteine to react with the FAD analogue. These results show that a single cysteine of R. gracilis D-amino acid oxidase reacts with the flavin analogue and that this is located near or at the FAD-binding domain.
authors.affiliation: Department of Structural and Functional Biology, University of Milano, Varese, Italy.
authors.forename: L
authors.initials: L
authors.lastname: Pollegioni
authors.forename: S
authors.initials: S
authors.lastname: Campaner
authors.forename: A A
authors.initials: AA
authors.lastname: Raibekas
authors.forename: M S
authors.initials: MS
authors.lastname: Pilone
mesh_terms: D000345:Affinity Labels; D003545:Cysteine / Q000737:chemistry*; D003605:D-Amino-Acid Oxidase / Q000737:chemistry* / Q000378:metabolism; D005415:Flavins / Q000378:metabolism*; D012248:Rhodotorula / Q000201:enzymology*; D013439:Sulfhydryl Reagents / Q000737:chemistry
chemical_list: D000345:Affinity Labels; D005415:Flavins; D013439:Sulfhydryl Reagents; D003605:D-Amino-Acid Oxidase; D003545:Cysteine