Journal article
Spectral and kinetic characterization of intermediates in the aromatization reaction catalyzed by NikD, an unusual amino acid oxidase
Biochemistry (Easton), v 48(21), pp 4455-4465
02 Jun 2009
PMID: 19354202
Featured in Collection : UN Sustainable Development Goals @ Drexel
Abstract
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.
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Details
- Title
- Spectral and kinetic characterization of intermediates in the aromatization reaction catalyzed by NikD, an unusual amino acid oxidase
- Creators
- Robert C Bruckner - Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, Pennsylvania 19102, USAMarilyn Schuman Jorns
- Publication Details
- Biochemistry (Easton), v 48(21), pp 4455-4465
- Publisher
- American Chemical Society; Washington, DC
- Grant note
- R01 AI055590-04 / NIAID NIH HHS AI 55590 / NIAID NIH HHS R56 AI055590 / NIAID NIH HHS R01 AI055590 / NIAID NIH HHS
- Resource Type
- Journal article
- Language
- English
- Academic Unit
- Biochemistry and Molecular Biology
- Web of Science ID
- WOS:000266412300004
- Scopus ID
- 2-s2.0-66349094817
- Other Identifier
- 991014878319704721
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- Web of Science research areas
- Biochemistry & Molecular Biology