Identification of a Reactive Cysteine in the Flavin-Binding Domain ofRhodotorula gracilisd-Amino Acid Oxidase (original) (raw)

1997, Archives of Biochemistry and Biophysics

corresponding 2-oxo-acids, hydrogen peroxide and am-The holoenzyme form of Rhodotorula gracilis D-monia. The native DAAO from the yeast Rhodotorula amino acid oxidase, an 80-kDa homodimer, reacted gracilis is an 80-kDa homodimer, containing one FAD only to a limited extent with general thiol reagents molecule per 40 kDa subunit (1, 2). The apoprotein of (2,2-dithiodipyridine, 5,5-dithiobis(2-nitrobenzoic acid), R. gracilis DAAO, obtained with high yield in a fully and N-[7-(dimethylamino)-4-methylcoumarinyl]maleireconstitutable form, is a 40-kDa monomer under all mide) (60% residual activity), whereas the monomeric conditions (2). The sequence of the 368 amino acids in apoprotein was completely inactivated and denatured the enzyme, in which 6 cysteines are present, has been by these reagents. To investigate the presence of thiol determined, and recently, limited trypsinolysis studies residue(s) in the active site of the enzyme, the apoprodemonstrated that the 38.3-kDa enzyme monomer is tein was reconstituted with the 8-(methylsulfonyl)catalytically competent (3, 4). DAAO from R. gracilis FAD chemical-affinity probe. Competitive inhibition shows some characteristics which distinguish it from between this analogue and FAD for apoprotein bindthe enzyme purified from pig kidney, e.g., a low degree ing was observed. The covalent attachment of the flaof primary structure similarity (27% of sequence idenvin analogue to the apoprotein was complete after É20 tity), a tight coenzyme binding (K d of 2 1 10 08 M for h of incubation and the flavinylated enzyme, con-FAD), and the rate-limiting step in the kinetic mechataining 8-(cysteinyl)-FAD, was monomeric and inacnism corresponding to the reductive half-reaction, in tive. After HPLC isolation of the flavin-labeled tryptic contrast with the mammalian enzyme, where this step peptides, Cys208 was identified as the only cysteine to is represented by the product released from the oxireact with the FAD analogue. These results show that dized enzyme (2, 3, 5). The properties of the yeast ena single cysteine of R. gracilis D-amino acid oxidase zyme are of interest in view of its biotechnological and reacts with the flavin analogue and that this is located industrial applications. near or at the FAD-binding domain. ᭧ 1997 Academic Press