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 variabilis D-amino acid oxidase (DAO) to its flavin adenine dinucleotide (FAD) binding and catalytic function, six mutant enzymes were constructed by site-directed mutagenesis. Western immunoblot analysis revealed that a protein with an apparent molecular mass of about 39.2 kDa was present in the cell-free extracts of wild-type and mutant strains. Replacement 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, exhibited a similar spectral profile to that of wildtype enzyme and retained about 6-90% of the enzyme activity. These results suggested that Asp(206) of T. variabilis DAO might play an important role in the binding of FAD. (C) 1998 Federation of European Biochemical Societies.