| Abstract
| - d-Amino acid oxidase (DAAO) is the prototype of theflavin-containing oxidases. It catalyzesthe oxidative deamination of various d-amino acids, rangingfrom d-Ala to d-Trp. We have carriedoutthe X-ray analysis of reduced DAAO in complex with the reaction productimino tryptophan (iTrp) andof the covalent adduct generated by the photoinduced reaction of theflavin with 3-methyl-2-oxobutyricacid (kVal). These structures were solved by combination of 8-folddensity averaging and least-squaresrefinement techniques. The FAD redox state of DAAO crystals wasassessed by single-crystal polarizedabsorption microspectrophotometry. iTrp binds to the reducedenzyme with the N, Cα, C, and Cβ atomspositioned 3.8 Å from the re side of the flavin. Theindole side chain points away from the cofactor andis bound in the active site through a rotation of Tyr224. Thisresidue plays a crucial role in that it adaptsits conformation to the size of the active site ligand, providing theenzyme with the plasticity required forbinding a broad range of substrates. The iTrp binding mode isfully consistent with the proposal, inferredfrom the analysis of the native DAAO structure, that substrateoxidation occurs via direct hydride transferfrom the Cα to the flavin N5 atom. In this regard, it isremarkable that, even in the presence of the bulkyiTrp ligand, the active center is made solvent inaccessible by loop216−228. This loop is thought toswitch between the “closed” conformation observed in the crystalstructures and an “open” state requiredfor substrate binding and product release. Loop closure is likelyto have a role in catalysis by increasingthe hydrophobicity of the active site, thus making the hydride transferreaction more effective. Bindingof kVal leads to keto acid decarboxylation and formation of a covalentbond between the keto acid Cαand the flavin N5 atoms. Formation of this acyl adduct results ina nonplanar flavin, characterized by a22° angle between the pyrimidine and benzene rings. Thus, inaddition to an adaptable substrate bindingsite, DAAO has the ability to bind a highly distorted cofactor.This ability is relevant for the enzyme'sfunction as a highly efficient oxidase.
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