FAD-linked oxidases constitute a class of enzymes which catalyze dehydrogenation as a fundamental biochemical reaction, followed by reoxidation of reduced flavin. Here, we present high-resolution crystal structures showing the flavoenzyme 6-hydroxy-L-nicotine oxidase in action. This enzyme was trapped during catalytic degradation of the native substrate in a sequence of discrete reaction states corresponding to the substrate-reduced enzyme, a complex of the enzyme with the intermediate enamine product and formation of the final aminoketone product. The inactive D-stereoisomer binds in mirror symmetry with respect to the catalytic axis, revealing absolute stereospecificity of hydrogen transfer to the flavin. The structural data suggest deprotonation of the substrate when bound at the active site, an overall binary complex mechanism and oxidation by direct hydride transfer. The amine nitrogen has a critical role in the dehydrogenation step and may activate carbocation formation at the α-carbon via delocalization from the lone pair to σ* C α -H. Enzymatically assisted hydrolysis of the intermediate product occurs at a remote (P site) cavity. Substrate entry and product exit follow different paths. Structural and kinetic data suggest that substrate can also bind to the reduced enzyme, associated with slower reoxidation as compared to the rate of reoxidation of free enzyme. The results are of general relevance for the mechanisms of flavin amine oxidases.catalytic mechanism | chiral stereospecificity | cryocrystallography | reaction intermediate | structural kinetics F lavin-dependent amine oxidases take part in a number of cellular processes ranging from degradative metabolism to chromatin remodeling. A large body of work has examined the common kinetic mechanism of flavoprotein oxidases, which includes a reductive half-reaction yielding the oxidized substrate bound to the reduced enzyme and subsequent reoxidation of the enzyme by oxygen in a binary or ternary complex scheme. The mechanism of individual steps remains controversial as discussed in a number of reviews (1-5). Hypotheses put forward for the dehydrogenation step of the catalytic reaction include radical mechanisms involving single-electron transfer from the substrate to the flavin as a first step (6), nucleophilic mechanisms involving an intermediate substrate-flavin adduct (7, 8), a carbanion mechanism (9), and a direct hydride transfer mechanism, which has been established for D-amino-acid oxidase (DAAO) (4, 10, 11). For a number of flavin amine oxidases including DAAO (11), L-amino-acid oxidase (LAAO) (12), monoamine oxidase (MAO) (1,13,14), and 6-hydroxy-L-nicotine oxidase (6HLNO) (15), a carbanion mechanism may be excluded because the active sites do not contain residues capable of and properly placed for acidbase catalysis. Most of the investigations of structural mechanisms involved enzymes in unproductive complexes with substrate analogues or inhibitors. Crystallographic studies of amine oxidases under productive oxidizing conditions were reported...