Crystallographic snapshots of the complete reaction cycle of nicotine degradation by an amine oxidase of the monoamine oxidase (MAO) family

Kachalova, G.; Decker, K.; Holt, A.; Bartunik, H. D.

doi:10.1073/pnas.1016684108

Journal Article

Crystallographic snapshots of the complete reaction cycle of nicotine degradation by an amine oxidase of the monoamine oxidase (MAO) family

Kachalova, G. ; Decker, K. ; Holt, A. ; Bartunik, H. D. ; DESY

2011
Academy Washington, DC

Proceedings of the National Academy of Sciences of the United States of America 108, 4800-4805 (2011) [10.1073/pnas.1016684108]

This record in other databases:

Please use a persistent id in citations: doi:10.1073/pnas.1016684108

Abstract: 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.

Keyword(s): Arthrobacter: enzymology (MeSH) ; Bacterial Proteins: chemistry (MeSH) ; Catalytic Domain (MeSH) ; Crystallography, X-Ray (MeSH) ; Kinetics (MeSH) ; Monoamine Oxidase: chemistry (MeSH) ; Nicotine: chemistry (MeSH) ; Oxidation-Reduction (MeSH) ; Oxidoreductases Acting on CH-NH Group Donors: chemistry (MeSH) ; Structure-Activity Relationship (MeSH) ; Bacterial Proteins ; Nicotine ; Monoamine Oxidase ; Oxidoreductases Acting on CH-NH Group Donors ; 6-hydroxy-L-nicotine oxidase