NikD, an Unusual Amino Acid Oxidase Essential for Nikkomycin Biosynthesis: Structures of Closed and Open Forms at 1.15 and 1.90 Å Resolution

Carrell, C.J.; Brückner, R.; Venci, David; Zhao, Gouhua; Jörns, Marilyn Schuman; Mathews, F.S.

doi:10.1016/j.str.2007.06.010

Cited by 20 publications

(36 citation statements)

References 34 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…There are several possible paths for this initial 2-electron oxidation step (Scheme 2). Oxidation of the bond between N(1) and C(6) in the enamine tautomer (Scheme 2 path A) is consistent with results obtained in structural and biochemical studies (4, 10). In addition, nikD exhibits homology with monomeric sarcosine oxidase (MSOX) and other members of a flavoenzyme family that oxidize carbon-nitrogen bonds in various amino acid substrates (11-16).…”

supporting

confidence: 89%

“…The loss of the charge-transfer band may reflect a pH-induced change from a coplanar to a perpendicular configuration of the flavin and indole rings, as observed in the open and closed crystal forms, respectively, of the nikD•picolinate complex (10). Protonation of the unknown residue is required for ligand binding, as judged by the observed effect of pH on the stability of enzyme•inhibitor complexes2.…”

Section: Discussionmentioning

confidence: 99%

“…Crystal structures have been determined for two forms of a complex of wild-type enzyme with picolinate (10). In the closed form, the active site is inaccessible to solvent.…”

mentioning

confidence: 99%

See 2 more Smart Citations

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

Self Cite

View full text Add to dashboard Cite

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.

show abstract

supporting

confidence: 89%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

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

Self Cite

View full text Add to dashboard Cite

show abstract

“…Puzzlingly, Lys265 is neither conserved nor conservatively substituted in nikD, an enzyme that exhibits significant sequence (22% identity) and structural (RMSD = 1.8 Å) similarity with MSOX. However, it is worth noting that nikD contains a mobile cation-binding loop that has no equivalent in other members of the MSOX family (15). Monoamine oxidase B, polyamine oxidase, monoamine oxidase A, and L-amino acid oxidase contain a lysine residue that is hydrogen bonded to flavin N(5) via a bridging water molecule (25-28), similar to that observed for Lys265 in MSOX.…”

Section: Discussionmentioning

confidence: 99%

Identification of the Oxygen Activation Site in Monomeric Sarcosine Oxidase: Role of Lys265 in Catalysis

2008

Self Cite

View full text Add to dashboard Cite

Monomeric sarcosine oxidase (MSOX) catalyzes the oxidation of N-methylglycine and contains covalently bound FAD that is hydrogen bonded at position N(5) to Lys265 via a bridging water. Lys265 is absent in the homologous but oxygen-unreactive FAD site in heterotetrameric sarcosine oxidase. Isolated preparations of Lys265 mutants contain little or no flavin but can be covalently reconstituted with FAD. Mutation of Lys265 to a neutral residue (Ala, Gln, Met) causes a 6000-to 9000-fold decrease in apparent turnover rate whereas a 170-fold decrease is found with Lys265Arg. Substitution of Lys265 with Met or Arg causes only a modest decrease in the rate of sarcosine oxidation (9.0-or 3.8-fold, respectively), as judged by reductive half-reaction studies which show that the reactions proceed via an initial enzyme•sarcosine charge transfer complex and a novel spectral intermediate not detected with wild-type MSOX. Oxidation of reduced wild-type MSOX (k = 2.83 × 10 5 M −1 s −1 ) is more than 1000-fold faster than observed for the reaction of oxygen with free reduced flavin. Mutation of Lys265 to a neutral residue causes a dramatic 8000-fold decrease in oxygen reactivity whereas a 250-fold decrease is observed with Lys265Arg. The results provide definitive evidence for Lys265 as the site of oxygen activation and show that a single positively charged amino acid residue is entirely responsible for the rate acceleration observed with wild-type enzyme. Significantly, the active sites for sarcosine oxidation and oxygen reduction are located on opposite faces of the flavin ring.The reduction of oxygen to hydrogen peroxide by free reduced flavin is thermodynamically favorable but kinetically slow because the 2-electron reduction of triplet oxygen by a diamagnetic organic molecule is spin-forbidden. In fact, the 2-electron reduction of oxygen by reduced flavin proceeds via an initial 1-electron transfer step that generates a flavin radical/ superoxide anion radical pair in a spin-allowed but energetically unfavorable, rate-determining reaction. The term oxygen activation is used in reference to the accelerated rates of oxygen reduction observed with flavoprotein oxidases and other enzymes that reduce molecular oxygen (1,2). A series of elegant studies by Klinman and Roth identified His516 as the site of oxygen activation in the flavoenzyme glucose oxidase and showed that the reaction required the protonated form of this residue (2-4). Surprisingly little is, however, currently known about the detailed mechanism of oxygen activation by other flavoprotein oxidases, especially regarding the specific role of active site residues in these reactions.

show abstract

“…The structure was therefore determined by molecular replacement with Phaser-MR (Bunkó czi et al, 2013). Initially, coordinates of putative homologues with PDB codes 2q6u (Carrell et al, 2007) and 2r4j (Yeh et al, 2008), both displaying less than 18% sequence identity to PA4991, were obtained from the PDB. Search models were derived from these coordinates by removing side chains and loop regions differing between the two structures, but no molecular-replacement solutions were found.…”

Section: Structure Determinationmentioning

confidence: 99%

Crystal structure of the flavoenzyme PA4991 fromPseudomonas aeruginosa

et al. 2016

View full text Add to dashboard Cite

The locus PA4991 in Pseudomonas aeruginosa encodes an open reading frame that has been identified as essential for the virulence and/or survival of this pathogenic organism in the infected host. Here, it is shown that this gene encodes a monomeric FAD-binding protein of molecular mass 42.2 kDa. The structure of PA4991 was determined by a combination of molecular replacement using a search model generated with Rosetta and phase improvement by a lowoccupancy heavy-metal derivative. PA4991 belongs to the GR 2 family of FADdependent oxidoreductases, comprising an FAD-binding domain typical of the glutathione reductase family and a second domain dominated by an eightstranded mixed -sheet. Most of the protein-FAD interactions are via the FADbinding domain, but the isoalloxazine ring is located at the domain interface and interacts with residues from both domains. A comparison with the structurally related glycine oxidase and glycerol-3-phosphate dehydrogenase shows that in spite of very low amino-acid sequence identity (<18%) several active-site residues involved in substrate binding in these enzymes are conserved in PA4991. However, enzymatic assays show that PA4991 does not display aminoacid oxidase or glycerol-3-phosphate dehydrogenase activities, suggesting that it requires different substrates for activity.

show abstract

NikD, an Unusual Amino Acid Oxidase Essential for Nikkomycin Biosynthesis: Structures of Closed and Open Forms at 1.15 and 1.90 Å Resolution

Cited by 20 publications

References 34 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

Identification of the Oxygen Activation Site in Monomeric Sarcosine Oxidase: Role of Lys265 in Catalysis

Crystal structure of the flavoenzyme PA4991 fromPseudomonas aeruginosa

Contact Info

Product

Resources

About