Enhancement in the catalytic efficiency of D-amino acid oxidase from Glutamicibacter protophormiae by multiple amino acid substitutions

D-Amino acid oxidase (DAAO) is an imperative oxidoreductase that oxidizes D-amino acids to corresponding keto acids, producing ammonia and hydrogen peroxide. Previously, based on the sequence alignment of DAAO from Glutamicibacter protophormiae (GpDAAO-1) and (GpDAAO-2), 4 residues (E115, N119, T256, T286) at the surface regions of GpDAAO-2, were subjected to site-directed mutagenesis and achieved 4 single-point mutants with enhanced catalytic efficiency (kcat/Km) compared to parental GpDAAO-2. In the present study, to further enhance the catalytic efficiency of GpDAAO-2, a total of 11 (6 double, 4 triple, and 1 quadruple-point) mutants were prepared by the different combinations of 4 single-point mutants. All mutants and wild types were over -expressed, purified and enzymatically characterized. A triple-point mutant E115A/N119D/T286A exhibited the most significant improvement in catalytic efficiency as compared to wild-type GpDAAO-1 and GpDAAO-2. Structural modeling analysis elucidated that residue Y213 in loop region C209-Y219 might act as the active -site lid for controlling substrate access, the residue K256 substituted by threonine (K256T) might change the hydrogen bonding interaction between residue Y213 and the surrounding residues, and switch the conformation of the active-site lid from the closed state to the open state, resulting in the enhancement in substrate accessibility and catalytic efficiency.

Automated summary

Mutations on D-amino acid oxidase can enhance its catalytic efficiency, and the triple-point mutant E115A/N119D/T286A showed the greatest improvement. Structural modeling analysis suggested that residue Y213 in loop region C209-Y219 might act as the active-site lid to control substrate access. The substitution of residue K256 by threonine (K256T) could change the hydrogen bonding interaction between residue Y213 and surrounding residues, leading to the conformational switch of the active-site lid from the closed state to the open state, thus enhancing substrate accessibility and catalytic efficiency.