A conserved aspartate is essential for FAD binding and catalysis in the d‐amino acid oxidase from Trigonopsis variabilis

FEBS Letters

Ju, Sheau-Shya¹  Lin, Long-Liu³  Wang, Wen-Ching²  Hsu, Wen-Hwei¹ 

[1] Institute of Molecular Biology, National Chung Hsing University, Taichung 402, Taiwan;Department of Life Science, National Tsing Hua University, Hsinchu 300, Taiwan;Department of Food Science and Nutrition, Hung Kuang Institute of Technology, 34 Chungchie Road, Shalu, Taichung, Taiwan

关键词: d-Amino acid oxidase;    Site-directed mutagenesis;    Flavin adenine dinucleotide binding;    Trigonopsis variabilis;   

DOI  :  10.1016/S0014-5793(98)01108-9

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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. variabilis DAO might play an important role in the binding of FAD.

【 授权许可】

Unknown