CRYSTAL-STRUCTURE OF D-AMINO-ACID OXIDASE - A CASE OF ACTIVE-SITE MIRROR-IMAGE CONVERGENT EVOLUTION WITH FLAVOCYTOCHROME B(2)

D-amino acid oxidase is the prototype of the FAD-dependent oxidases. I t catalyses the oxidation of D-amino acids to the corresponding alpha- ketoacids. The reducing equivalents are transferred to molecular oxyge n with production of hydrogen peroxide. We have solved the crystal str ucture of the complex of D-amino acid oxidase with benzoate, a competi tive inhibitor of the substrate, by single isomorphous replacement and eightfold averaging, Each monomer is formed by two domains with an ov erall topology similar to that of p-hydroxybenzoate hydroxylase. The b enzoate molecule lays parallel to the flavin ring acid is held in posi tion by a salt bridge with Arg-283, Analysis of the active site shows that no side chains are properly positioned to act as the postulated b ase required for the catalytic carboanion mechanism, On the contrary, the benzoate binding mode suggests a direct transfer of the substrate alpha-hydrogen to the flavin during the enzyme reductive hail-reaction . The active site of D-amino acid oxidase exhibits a striking similari ty with that of flavocytochrome b(2), a structurally unrelated FMN-dep endent flavoenzyme. The active site groups of these two enzymes are in fact superimposable once the mirror-image of the flavocytochrome b(2) active site is generated with respect to the flavin plane, Therefore, the catalytic sites of D-amino acid oxidase and flavocytochrome b(2) appear to have converged to a highly similar but enantiomeric architec ture in order to catalyze similar reactions (oxidation of alpha-amino acids or alpha-hydroxy acids), although with opposite stereochemistry.