ACTIVE-SITE PLASTICITY IN D-AMINO-ACID OXIDASE - A CRYSTALLOGRAPHIC ANALYSIS

D-Amino acid oxidase (DAAO) is the prototype of the flavin-containing oxidases. It catalyzes the oxidative deamination of various D-amino ac ids, ranging from D-Ala to D-Trp. We have carried out the X-ray analys is of reduced DAAO in complex with the reaction product imino tryptoph an (iTrp) and of the covalent adduct generated by the photoinduced rea ction of the flavin with 3-methyl-2-oxobutyric acid (kVal). These stru ctures were solved by combination of 8-fold density averaging and leas t-squares refinement techniques. The FAD redox state of DAAO crystals was assessed by single-crystal polarized absorption microspectrophotom etry. iTrp binds to the reduced enzyme with the N, C alpha, C, and C b eta atoms positioned 3.8 Angstrom from the re side of the flavin. The indole side chain points away from the cofactor and is bound in the ac tive site through a rotation of Tyr224. This residue plays a crucial r ole in that it adapts its conformation to the size of the active site ligand, providing the enzyme with the plasticity required for binding a broad range of substrates. The iTrp binding mode is fully consistent with the proposal, inferred from the analysis of the native DAAO stru cture, that substrate oxidation occurs via direct hydride transfer fro m the C alpha to the flavin N5 atom. In this regard, it is remarkable that, even in the presence of the bulky iTrp ligand, the active center is made solvent inaccessible by loop 216-228. This loop is thought to switch between the ''closed'' conformation observed in the crystal st ructures and an ''open'' state required for substrate binding and prod uct release. Loop closure is likely to have a role in catalysis by inc reasing the hydrophobicity of the active site, thus making the hydride transfer reaction more effective. Binding of kVal leads to keto acid decarboxylation and formation of a covalent bond between the keto acid Ca and the flavin N5 atoms. Formation of this acyl adduct results in a nonplanar flavin, characterized by a 22 degrees angle between the py rimidine and benzene rings. Thus, in addition to an adaptable substrat e binding site, DAAO has the ability to bind a highly distorted cofact or. This ability is relevant for the enzyme's function as a highly eff icient oxidase.