Crystal Structure of LAAO from Calloselasma rhodostoma with an l-Phenylalanine Substrate: Insights into Structure and Mechanism

Abstract

l-Amino acid oxidase is a dimeric glycosylated flavoenzyme, a major constituent of the venom-from the snake Calloselasma rhodostoma. The enzyme exhibits apoptosis inducing effects as well as antibacterial and anti-HIV activities. The structure of l-amino acid oxidase with its substrate ( l-phenylalanine) has been refined to a resolution of 1.8 Å. The complex structure reveals the substrate bound to the reduced flavin (FAD red). Alternative conformations for the key residues His223 and Arg322 are evident, suggesting a dynamic active site. Furthermore, conformational changes are apparent for the isoalloxazine ring; the three-ring system exhibits more bending around the N5—N10 axis compared to the oxidized flavin. The implications of the observed dynamics on the mechanism of catalysis are discussed. Inspection of buried surfaces in the enzyme reveals a Y-shaped channel system extending from the external surface of the protein to the active site. One portion of this channel may serve as the entry path for O 2 during the oxidative half-reaction. The second region, separated from the proposed O 2 channel by the N terminus (residues 8–16) of the protein, may play a role in H 2O 2 release. Interestingly, the latter portion of the channel would direct the H 2O 2 product to the exterior surface of the protein, near the glycan moiety, thought to anchor the enzyme to the host cell. This channel location may explain the ability of the enzyme to localize H 2O 2 to the targeted cell and thus induce the apoptotic effect.