Aminoketocarbonsäuren VI. Über die Konstitution und Strukturformen der α-Ketoanaloga natürlicher Diaminosäuren

Macholán, Lumír; Svátek, E.

doi:10.1135/cccc19602564

Cited by 10 publications

(14 citation statements)

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“…An aliquot of each stock solution of P2C was diluted into 0.1 N NaOH. ∆ 1 -P2C is converted to the enamine (∆ 2 -P2C) in 0.1 N NaOH (13). The concentration of P2C was estimated using the extinction coefficient reported for ∆ 2 -P2C at 256 nm ( 256 ) 725 M -1 cm -1 ) (13,17).…”

Section: Methodsmentioning

confidence: 99%

“…The first step in peptidyl biosynthesis is catalyzed by nikC (12), an aminotransferase that converts L-lysine to the corresponding R-keto derivative. The R-keto derivative spontaneously cyclizes and dehydrates to yield ∆ 1 -or ∆ 2 -piperideine-2-carboxylate (P2C), 1 a compound that can exist in two tautomeric forms (13). The imine (∆ 1 -P2C) is the predominant tautomer at acid or neutral pH; the enamine (∆ 2 -P2C) is the major species at alkaline pH and is thought to be the more easily oxidized form (14).…”

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Nikkomycin Biosynthesis: Formation of a 4-Electron Oxidation Product during Turnover of NikD with Its Physiological Substrate

et al. 2004

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Nikkomycins are peptidyl nucleoside antibiotics that act as therapeutic antifungal agents in humans and easily degraded insecticides in agriculture. The nikkomycin peptidyl moiety contains a pyridyl residue derived from L-lysine. The first step in peptidyl biosynthesis is an aminotransferase-catalyzed reaction that converts L-lysine to Delta(1)- or Delta(2)-piperideine-2-carboxylate (P2C). Spectral, chromatographic, and kinetic analyses show that the aerobic reaction of nikD with P2C results in the stoichiometric formation of picolinate, accompanied by the reduction of 2 mol of oxygen to hydrogen peroxide. A high resolution HPLC method, capable of separating picolinate, nicotinate and isonicotinate, was developed and used in product identification. NikD contains 1 mol of covalently bound FAD and exists as a monomer in solution. Reductive and oxidative titrations with dithionite and potassium ferricyanide, respectively, show that FAD is the only redox-active group in nikD. Anaerobic reaction of nikD with 1 mol of P2C results in immediate reduction of enzyme-bound FAD. Because nikD is an obligate 2-electron acceptor, it is proposed that the observed 4-electron oxidation of P2C to picolinate occurs via a mechanism involving two successive nikD-catalyzed 2-electron oxidation steps. In addition to nikkomycins, a nikD-like reaction is implicated in the biosynthesis of an L-lysine-derived pyridyl moiety found in streptogramin group B antibiotics that are used as part of a last resort treatment for severe infections due to gram positive bacteria.

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Section: Methodsmentioning

confidence: 99%

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confidence: 99%

Nikkomycin Biosynthesis: Formation of a 4-Electron Oxidation Product during Turnover of NikD with Its Physiological Substrate

et al. 2004

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“…Synthesis of the pyridyl moiety is initiated by an α-aminotransferase that converts L-lysine to piperideine-2-carboxylate (P2C) (2), a compound that can exist in imine and enamine tautomeric forms (3–5). Picolinate is produced in a remarkable aromatization reaction catalyzed by nikD, involving an overall 4-electron oxidation of P2C (Scheme 1) (6).…”

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confidence: 99%

Spectral and Kinetic Characterization of Intermediates in the Aromatization Reaction Catalyzed by NikD, an Unusual Amino Acid Oxidase

Brückner¹,

Jörns²

2009

Biochemistry

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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.NikD is a flavoprotein oxidase that plays an essential role in the biosynthesis of nikkomycins. Nikkomycins comprise a group of related peptidyl nucleoside antibiotics that resemble the natural substrate of chitin synthase. Chitin, the second most abundant polysaccharide in nature, is an integral component of the cell wall in fungi and the exoskeleton of invertebrates but is not found in mammals. Nikkomycins are potent antifungal agents that act by competitively inhibiting chitin synthase. Nikkomycins are therapeutically effective in treating human fungal infections that are especially prevalent in immunocompromised patients and are also useful in agriculture as easily degraded insecticides that are nontoxic for mammals (1).The nikkomycin peptide is synthesized by a nonribosomal pathway and contains an N-terminal pyridyl moiety, derived from L-lysine, that is essential for antibiotic function. Synthesis of the pyridyl moiety is initiated by an α-aminotransferase that converts L-lysine to piperideine-2-carboxylate (P2C) (2), a compound that can exist in imine and enamine tautomeric forms (3- † This work was supported in part by Grant AI 55590 (M. S. J.) from the National Institutes of Health.*To whom correspondence should be addressed. Phone: (215) 762-7495 FAX: (215) 762-4452. E-mail: E-mail:...

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“…Synthesis of the pyridyl moiety requires a L-lysine α-aminotransferase (3) and nikD, an unusual amino acid oxidase that contains one mol of covalently bound FAD (4) and acts as an obligate 2-electron acceptor (5). The α-aminotransferase converts L-lysine to piperideine-2-carboxylate (P2C), a compound that can exist in imine and enamine tautomeric forms (6-8). NikD catalyzes a remarkable aromatization reaction that converts P2C to picolinate, accompanied by the reduction of 2 mol of oxygen to hydrogen peroxide (Scheme 1) (5).…”

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Probing the Role of Active Site Residues in NikD, an Unusual Amino Acid Oxidase That Catalyzes an Aromatization Reaction Important in Nikkomycin Biosynthesis

et al. 2009

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NikD catalyzes a remarkable aromatization reaction that converts piperideine-2-carboxylate (P2C) to picolinate, a key component of the nonribosomal peptide in nikkomycin antibiotics. The enzyme exhibits a FAD-Trp355 charge-transfer band at weakly alkaline pH that is abolished upon protonation of an unknown ionizable residue that exhibits a pKa of 7.3. Stopped-flow studies of the reductive half-reaction with wild-type nikD and P2C show that the enzyme oxidizes the enamine tautomer of P2C but do not distinguish among several possible paths for the initial 2-electron oxidation step. Replacement of Glu101 or Asp276 by a neutral residue does not eliminate the ionizable group, although the observed pKa is 1 or 2 pH units higher, respectively, compared with wild-type nikD. Importantly, the mutations cause only a modest decrease (< 5-fold) in the observed rate of oxidation of P2C to dihydropicolinate. The results rule out the only possible candidates for a catalytic base in the initial 2-electron oxidation step. This outcome provides compelling evidence that nikD oxidizes the bond between N(1) and C(6) in the enamine tautomer of P2C, ruling out alternative paths that require an active site base to mediate the oxidation of a carbon-carbon bond. Because the same restraint applies to the second 2-electron oxidation step, the dihydropicolinate intermediate must be converted to an isomer that contains an oxidizable carbon-nitrogen bond. A novel role is proposed for reduced FAD as an acid-base catalyst in the isomerization of dihydropicolinate.

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Aminoketocarbonsäuren VI. Über die Konstitution und Strukturformen der α-Ketoanaloga natürlicher Diaminosäuren

Cited by 10 publications

References 0 publications

Nikkomycin Biosynthesis: Formation of a 4-Electron Oxidation Product during Turnover of NikD with Its Physiological Substrate

Nikkomycin Biosynthesis: Formation of a 4-Electron Oxidation Product during Turnover of NikD with Its Physiological Substrate

Spectral and Kinetic Characterization of Intermediates in the Aromatization Reaction Catalyzed by NikD, an Unusual Amino Acid Oxidase

Probing the Role of Active Site Residues in NikD, an Unusual Amino Acid Oxidase That Catalyzes an Aromatization Reaction Important in Nikkomycin Biosynthesis

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