Porcine kidney d-amino acid oxidase: the three-dimensional structure and its catalytic mechanism based on the enzyme–substrate complex model

The three-dimensional structure of porcine kidney d-amino acid oxidase (DAO), an FAD-dependent oxidase, has been solved by X-ray crystallography. The overall structure is a dimer, subunits of which are correlated by a non-crystallographic two-fold axis. Each subunit comprises two domains, ‘αβ domain’ and ‘pseudo-barrel domain’. The coenzyme FAD is in an elongated conformation and is bound at the N-terminal βαβ dinucleotide binding motif. The active site is located in the boundary region between the two domains. The crystal structure of DAO in complex with a substrate analog, o-aminobenzoate, was also solved and is used for modeling the DAO-d-leucine complex, i.e. Michaelis complex, by means of molecular mechanics simulation. The Michaelis-complex model provided structural information leading to two alternative hypothetical mechanisms for the reductive half-reaction of DAO. These two hypotheses characterize themselves by electron transfer from the lone-pair orbital of the substrate amino nitrogen to flavin C(4a) and by proton transfer from the substrate α-position to flavin N(5) which acts as a catalytic base.