ANALYSIS OF INTERACTION BETWEEN THE ARTHROBACTER SARCOSINE OXIDASE AND THE COENZYME FLAVIN ADENINE-DINUCLEOTIDE BY SITE-DIRECTED MUTAGENESIS

Sarcosine oxidase from Arthrobacter sp, TE1826 (SoxA) tightly binds wi th the coenzyme flavin adenine dinucleotide (FAD). The amino-terminal region of this enzyme,vas recognized as a part of the FAD-binding doma in by homology search analysis, Comparison with other structurally cel l-known flavoproteins suggested that the aspartate residue at position 35 (D-35) and the motif sequence (six residues at positions 12 to 17) were important for the interaction with FAD, Site-directed mutagenesi s of each position was performed, and mutant SoxAs were purified and c haracterized, When D-35 was substituted with glutamate, asparagine, an d alanine, it was indicated that the carboxyl group of the side chain interacted with FAD, Changes in the enzyme-bound FAD were also observe d from the altered spectral profiles. Thirteen mutant SoxAs were obtai ned by replacing amino acids in the motif sequence. Most of them showe d inhibited or remarkably decreased sarcosine oxidase activity, and th eir spectral profiles were altered, However, some of them were reactiv ated by chloride ion. Their spectral profiles also became close to tha t of wild type in the presence of chloride ion. These results strongly suggest that the inhibition of interaction of enzyme with FAD was cau sed by the substitution in the moth and that it could be recovered und er different conditions.