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

• Published in: Journal of molecular catalysis. B, Enzymatic Enzymatic, 2001-02, Vol.12 (1), p.43-52
• Main Authors: Miura, Retsu, Setoyama, Chiaki, Nishina, Yasuzo, Shiga, Kiyoshi, Miyahara, Ikuko, Mizutani, Hisashi, Hirotsu, Ken
• Format: Article
• Language: English
• Subjects: Catalytic mechanism; d-amino acid oxidase; Michaelis-complex model; Reductive half-reaction; Three-dimensional structure
• ISSN: 1381-1177; 1873-3158
• DOI: 10.1016/S1381-1177(00)00202-2

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.