Structure and action of a C-C bond cleaving alpha/beta-hydrolase involved in nicotine degradation.
The enzyme 2,6-dihydroxy-pseudo-oxynicotine hydrolase from the nicotine-degradation pathway of Arthrobacter nicotinovorans was crystallized and the structure was determined by an X-ray diffraction analysis at 2.1 A resolution. The enzyme belongs to the alpha/beta-hydrolase family as derived from the chain-fold and from the presence of a catalytic triad with its oxyanion hole at the common position. This relationship assigns a pocket lined by the catalytic triad as the active center. The asymmetric unit contains two C(2)-symmetric dimer molecules, each adopting a specific conformation. One dimer forms a more spacious active center pocket and the other a smaller one, suggesting an induced-fit. All of the currently established C-C bond cleaving alpha/beta-hydrolases are from bacterial meta-cleavage pathways for the degradation of aromatic compounds and cover their active center with a 40 residue lid placed between two adjacent strands of the beta-sheet. In contrast, the reported enzyme shields its active center with a 110 residue N-terminal domain, which is absent in the meta-cleavage hydrolases. Since neither the substrate nor an analogue could be bound in the crystals, the substrate was modeled into the active center using the oxyanion hole as a geometric constraint. The model was supported by enzymatic activity data of 11 point mutants and by the two dimer conformations suggesting an induced-fit. Moreover, the model assigned a major role for the large N-terminal domain that is specific to the reported enzyme. The proposal is consistent with the known data for the meta-cleavage hydrolases although it differs in that the reaction does not release alkenes but a hetero-aromatic compound in a retro-Friedel-Crafts acylation. Because the hydrolytic water molecule can be assigned to a geometrically suitable site that can be occupied in the presence of the substrate, the catalytic triad may not form a covalent acyl-enzyme intermediate but merely support a direct hydrolysis.
Schleberger
Christian
C
Institut für Organische Chemie und Biochemie, Albert-Ludwigs-Universität, Albertstr. 21, D-79104 Freiburg im Breisgau, Germany.
Sachelaru
Paula
P
Brandsch
Roderich
R
Schulz
Georg E
GE
D000595:Amino Acid Sequence; D001173:Arthrobacter / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D019281:Dimerization; D006867:Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009538:Nicotine / Q000378:metabolism*; D017433:Protein Structure, Secondary; D014867:Water / Q000737:chemistry
D001426:Bacterial Proteins; D014867:Water; D009538:Nicotine; D006867:Hydrolases