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 center dot 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 center dot substrate charge transfer complex likely to contain the electron-rich P2C enamine. Calculated rates of formation and dissociation of the nikD center dot P2C complex are similar to those observed for the enzyme center dot 1-cyclohexenoate complex. Formation of a reduced enzyme center dot DHP complex, (EH2 center dot 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, (EH2 center dot 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.