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 Asp206 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 Asp206 with Leu, Gly, and Asn resulted in the loss of DAO activity and characteristic absorption spectrum for flavoenzyme, while the other mutant DAOs, Asp206Glu, Asp206Ser, and Asp206Ala, exhibited a similar spectral profile to that of wild-type enzyme and retained about 6-90% of the enzyme activity. These results suggested that Asp206 of T. variahilis DAO might play an important role in the binding of FAD.