STRUCTURE OF THE FLAVOENZYME D-AMINO ACID OXIDASE. M. Bolognesi^1,3, A. Mattevi¹, M.A. Vanoni², F. Todone¹, B. Curti², ¹Dip. Geneticae Microbiol. Univ.Pavia, via Abbiategrasso 207, 27100 Pavia, ²Dip. Biochimica e Fisiologia Gen. Universita' di Milano, via Celoria 26, 20133 Milano; ³Dip.Fisica Centro Biotec.Avanzate, Univ.Genova, L.go R.Benzi 10, 16132 Genova, Italy

D-amino acid oxidase is the prototype of the oxidase class of FAD-dependent enzymes. The protein catalyses the oxidative degradation of D-amino acids to the corresponding keto acids with the release of ammonia and hydrogen peroxide. The enzyme displays a broad substrate specificity and is capable of oxidising several D-amino acids. We have determined the three-dimensional crystal structure of pig kidney D-amino acid oxidase by multiple isomorphous replacement and eight-fold averaging.

The overall structure of D-amino acid oxidase encompasses two well characterised domains, which define at their interface the flavin ring binding site. The position of the competitive inhibitor benzoate allows the identification of the residues likely to take part in catalysis. Inspection of the active site reveals that there are no residues properly positioned to act as the active site base required for the carboanion mechanism, which has been postulated by most investigators. On the contrary, the crystallographic analysis suggests that the reaction proceeds by direct hydride transfer from the substrate Ca atom to the flavin N5 atom. The active site of D-amino acid oxidase closely resembles that of flavocytochrome b2, a structurally unrelated FMN-dependent enzyme. The catalytic groups of the two enzymes are well superimposable once the mirror-image of flavocytochrome b2 is generated with respect to the flavin. This fact finds a precise explanation in the opposite stereospecificity of the two proteins, thus suggesting that flavocytochrome b2 and D-amino acid oxidase represent a striking example of mirror image convergent evolution.