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

Brückner, R.; Jörns, Marilyn Schuman

doi:10.1021/bi900179j

Cited by 7 publications

(63 citation statements)

References 31 publications

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“…Our previous studies with wild-type nikD indicate that the slow step is likely to involve isomerization of an initially formed reduced enzyme•DHP complex (9). …”

Section: Resultsmentioning

confidence: 99%

“…Steady-state kinetic studies with Asp276Asn or Glu101Gln were conducted by monitoring picolinate formation at 264 nm (ε = 3980 M -1 cm -1 ) (5) in 100 mM potassium phosphate buffer, pH 8.0, equilibrated at 25 °C with water-saturated gas mixtures containing 10, 21, 44 or 100 % oxygen (balance nitrogen), as previously described (9). Steady-state kinetic parameters were estimated by fitting an equation for a sequential (equation 1) or a ping-pong (equation 2) mechanism to data obtained with Asp276Asn or Glu101Gln, respectively (A = P2C, B = O 2 ).…”

Section: Methodsmentioning

confidence: 99%

“…The ES complex is converted into a reduced enzyme•dihydropicolinate (DHP) complex in a second step that exhibits a hyperbolic dependence on substrate concentration (9). There are several possible paths for this initial 2-electron oxidation step (Scheme 2).…”

mentioning

confidence: 99%

“…It has been suggested that the second redox cycle may require prior conversion of the initially formed DHP intermediate to another isomer that can be oxidized to picolinate in a reaction that does not involve a change in regiospecificity as compared with the first redox cycle (9). …”

mentioning

confidence: 99%

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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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“…Our previous studies with wild-type nikD indicate that the slow step is likely to involve isomerization of an initially formed reduced enzyme•DHP complex (9). …”

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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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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“…In the proposed mechanism, the reaction is initiated with oxidation of a carbon-nitrogen bond. After tautomerisation and isomerisation of the formed imine, the same carbon-nitrogen bond is oxidised again resulting in the final, aromatic product (Bruckner and Jorns 2009).…”

Section: The Amine Oxidase Familymentioning

confidence: 99%

Flavoprotein oxidases: classification and applications

Dijkman

Gonzalo

Mattevi

et al. 2013

Appl Microbiol Biotechnol

132

106

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This review provides an overview of oxidases that utilise a flavin cofactor for catalysis. This class of oxidative flavoenzymes has shown to harbour a large number of biotechnologically interesting enzymes. Applications range from their use as biocatalysts for the synthesis of pharmaceutical compounds to the integration in biosensors. Through the recent developments in genome sequencing, the number of newly discovered oxidases is steadily growing. Recent progress in the field of flavoprotein oxidase discovery and the obtained biochemical knowledge on these enzymes are reviewed. Except for a structure-based classification of known flavoprotein oxidases, also their potential in recent biotechnological applications is discussed.

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Pleiotropic Impact of a Single Lysine Mutation on Biosynthesis of and Catalysis by N-Methyltryptophan Oxidase

2011

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N-Methyltryptophan oxidase (MTOX) contains covalently bound FAD. N-Methyltryptophan binds in a cavity above the re-face of the flavin ring. Lys259 is located above the opposite, si-face. Replacement of Lys259 by Gln, Ala, or Met blocks (>95%) covalent flavin incorporation in vivo. The mutant apoproteins can be reconstituted with FAD. Apparent turnover rates (kcat app) of the reconstituted enzymes are about 2500-fold slower than wild-type MTOX. Wild-type MTOX forms a charge-transfer Eox*S complex with the redox-active anionic form of NMT. The Eox*S complex formed with Lys259Gln does not exhibit a charge-transfer band and is converted to a reduced enzyme*imine complex (EH2*P) at a 60-fold slower rate than wild-type MTOX. The mutant EH2*P complex contains the imine zwitterion and exhibits a charge-transfer band, a feature not observed with the wild-type EH2*P complex. Reaction of reduced Lys259Gln with oxygen is 2500-fold slower than reduced wild-type MTOX. The latter reaction is unaffected by the presence of bound product. Dissociation of the wild-type EH2*P complex is 80-fold slower than kcat. The mutant EH2*P complex dissociates 15-fold faster than kcat app. Consequently, EH2*P and free EH2 are the species that react with oxygen during turnover of wild-type and mutant enzyme, respectively. The results show that: (i) Lys259 is the site of oxygen activation in MTOX and also plays a role in holenzyme biosynthesis and N-methyltryptophan oxidation; (ii) MTOX contains separate active sites for N-methyltryptophan oxidation and oxygen reduction on opposite faces of the flavin ring.

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

Cited by 7 publications

References 31 publications

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

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

Flavoprotein oxidases: classification and applications

Pleiotropic Impact of a Single Lysine Mutation on Biosynthesis of and Catalysis by N-Methyltryptophan Oxidase

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