A CONSERVED ASPARTATE IS ESSENTIAL FOR FAD BINDING AND CATALYSIS IN THE D-AMINO-ACID OXIDASE FROM TRIGONOPSIS-VARIABILIS

To evaluate the possible contribution of Asp(206) of Trigonopsis varia bilis D-amino acid oxidase (DAO) to its flavin adenine dinucleotide (F AD) binding and catalytic function, six mutant enzymes were constructe d by site-directed mutagenesis. Western immunoblot analysis revealed t hat a protein with an apparent molecular mass of about 39.2 kDa was pr esent in the cell-free extracts of wild-type and mutant strains. Repla cement of Asp(206) with Leu, Gly, and Asn resulted in the loss of DAO activity and characteristic absorption spectrum for flavoenzyme, while the other mutant DAOs, Asp(206)Glu, Asp(206)Ser, and Asp(206)Ala, exh ibited a similar spectral profile to that of wildtype enzyme and retai ned about 6-90% of the enzyme activity. These results suggested that A sp(206) of T. variabilis DAO might play an important role in the bindi ng of FAD. (C) 1998 Federation of European Biochemical Societies.