Spectral and kinetic characterization of intermediates in the aromatization reaction catalyzed by NikD, an unusual amino acid oxidase.
The flavoenzyme nikD, a 2-electron acceptor, catalyzes a remarkable aromatization of piperideine-2-carboxylate (P2C) to picolinate, an essential component of nikkomycin antibiotics. Steady-state kinetic data are indicative of a sequential mechanism where oxygen reacts with a reduced enzyme.dihydropicolinate (DHP) complex. The kinetics observed for complex formation with competitive inhibitors are consistent with a one-step binding mechanism. The anaerobic reaction with P2C involves three steps. The first step yields an enzyme.substrate charge transfer complex likely to contain the electron-rich P2C enamine. Calculated rates of formation and dissociation of the nikD.P2C complex are similar to those observed for the enzyme.1-cyclohexenoate complex. Formation of a reduced enzyme.DHP complex, (EH(2).DHP)(ini), occurs in a second step that exhibits a hyperbolic dependence on substrate concentration. The limiting rate of nikD reduction is at least 10-fold faster than the turnover rate observed with unlabeled or [4,4,5,5,6,6-D(6)]-P2C and exhibits a kinetic isotope effect (KIE = 6.4). The observed KIE on K(d apparent) (4.7) indicates that P2C is a sticky substrate. Formation of a final reduced species, (EH(2).DHP)(fin), occurs in a third step that is independent of P2C concentration and equal to the observed turnover rate. The observed KIE (3.3) indicates that the final step involves cleavage of at least one C-H bond. Tautomerization, followed by isomerization, of the initial DHP intermediate can produce an isomer that could be oxidized to picolinate in a reaction that satisfies known steric constraints of flavoenzyme reactions without the need to reposition a covalently tethered flavin or tightly bound intermediate.
Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, Pennsylvania 19102, USA.
Robert C
RC
Bruckner
Marilyn Schuman
MS
Jorns
D000594:Amino Acid Oxidoreductases / Q000378:metabolism*; D000818:Animals; D055162:Biocatalysis; D002264:Carboxylic Acids / Q000737:chemistry / Q000378:metabolism; D003435:Crotalid Venoms / Q000201:enzymology; D017839:Crotalus; D003903:Deuterium / Q000737:chemistry; D004058:Diffusion; D004579:Electron Transport; D007097:Imines / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D010100:Oxygen / Q000378:metabolism; D010848:Picolinic Acids / Q000737:chemistry / Q000378:metabolism; D013057:Spectrum Analysis; D013194:Staining and Labeling
D002264:Carboxylic Acids; D003435:Crotalid Venoms; D007097:Imines; D008024:Ligands; D010848:Picolinic Acids; D003903:Deuterium; D000594:Amino Acid Oxidoreductases; C030614:picolinic acid; D010100:Oxygen