D-amino acid oxidase is the prototype of the FAD-dependent oxidases. It catalyses the oxidation of D-amino acids to the corresponding alpha-ketoacids. The reducing equivalents are transferred to molecular oxygen with production of hydrogen peroxide. We have solved the crystal structure of the complex of D-amino acid oxidase with benzoate, a competitive inhibitor of the substrate, by single isomorphous replacement and eightfold averaging. Each monomer is formed by two domains with an overall topology similar to that of p-hydroxybenzoate hydroxylase. The benzoate molecule lays parallel to the flavin ring and is held in position by a salt bridge with Arg-283. Analysis of the active site shows that no side chains are properly positioned to act as the postulated base required for the catalytic carboanion mechanism. On the contrary, the benzoate binding mode suggests a direct transfer of the substrate alpha-hydrogen to the flavin during the enzyme reductive half-reaction.The active site Of D-amino acid oxidase exhibits a striking similarity with that of flavocytochrome b2, a structurally unrelated FMN-dependent flavoenzyme. The active site groups (if these two enzymes are in fact superimposable once the mirror-image of the flavocytochrome b2 active site is generated with respect to the flavin plane. Therefore, the catalytic sites of D-amino acid oxidase and flavocytochrome b2 appear to have converged to a highly similar but enantiomeric architecture in order to catalvze similar reactions (oxidation of alpha-amino acids or alpha-hydroxy acids), although with opposite stereochemistry.

Human histone demethylase LSD1 is a flavin-dependent amine oxidase that catalyzes the specific removal of methyl groups from mono- and dimethylated Lys⁴ of histone H3. The N-terminal tail of H3 is subject to various covalent modifications, and a fundamental question in LSD1 biology is how these epigenetic marks affect the demethylase activity. We show that LSD1 does not have a strong preference for mono- or dimethylated Lys⁴ of H3. Substrate recognition is not confined to the residues neighboring Lys⁴, but it requires a sufficiently long peptide segment consisting of the N-terminal 20 amino acids of H3. Electrostatic interactions are an important factor in protein-substrate recognition, as indicated by the high sensitivity of K_m to ionic strength. We have probed LSD1 for its ability to demethylate Lys⁴ in presence of a second modification on the same peptide substrate. Methylation of Lys⁹ does not affect enzyme catalysis. Conversely, Lys⁹ acetylation causes an almost 6-fold increase in the K_m value, whereas phosphorylation of Ser¹⁰ totally abolishes activity. LSD1 is inhibited by a demethylated peptide with an inhibition constant of 1.8 μm, suggesting that LSD1 can bind to H3 independently of Lys⁴ methylation. LSD1 is a chromatin-modifying enzyme, which is able to read different epigenetic marks on the histone N-terminal tail and can serve as a docking module for the stabilization of the associated corepressor complex(es) on chromatin.