TY  - JOUR
AU  - Kachalova, G.
AU  - Decker, K.
AU  - Holt, A.
AU  - Bartunik, H. D.
AU  - DESY
TI  - Crystallographic snapshots of the complete reaction cycle of nicotine degradation by an amine oxidase of the monoamine oxidase (MAO) family
JO  - Proceedings of the National Academy of Sciences of the United States of America
VL  - 108
SN  - 1091-6490
CY  - Washington, DC
PB  - Academy
M1  - PHPPUBDB-18289
SP  - 4800-4805
PY  - 2011
AB  - 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.
KW  - Arthrobacter: enzymology
KW  - Bacterial Proteins: chemistry
KW  - Catalytic Domain
KW  - Crystallography, X-Ray
KW  - Kinetics
KW  - Monoamine Oxidase: chemistry
KW  - Nicotine: chemistry
KW  - Oxidation-Reduction
KW  - Oxidoreductases Acting on CH-NH Group Donors: chemistry
KW  - Structure-Activity Relationship
KW  - Bacterial Proteins (NLM Chemicals)
KW  - Nicotine (NLM Chemicals)
KW  - Monoamine Oxidase (NLM Chemicals)
KW  - Oxidoreductases Acting on CH-NH Group Donors (NLM Chemicals)
KW  - 6-hydroxy-L-nicotine oxidase (NLM Chemicals)
LB  - PUB:(DE-HGF)16
C6  - pmid:21383134
C2  - pmc:PMC3064382
UR  - <Go to ISI:>//WOS:000288712200025
DO  - DOI:10.1073/pnas.1016684108
UR  - https://bib-pubdb1.desy.de/record/94652
ER  -