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, 'alpha beta domain' and 'pseudo-barrel domain'. The coenzyme FAD is in an elongated conformation and is bound at the N-terminal beta alpha beta 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 alpha -position to flavin N(5) which acts as a catalytic base. (C) 2001 Elsevier Science B.V. All rights reserved.