X-ray structure of a blue copper nitrite reductase at high pH and in copper-free form at 1.9 Å resolution

Ellis, Mark J.; Dodd, F.E.; Strange, R.W.; Prudêncio, Miguel; Sawers, R. Gary; Eady, Robert R.; Hasnain, S. Samar

doi:10.1107/s0907444901008654

Cited by 30 publications

(36 citation statements)

References 36 publications

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“…C-terminal residues 355 to 363, which were not modeled in the previous AniA structure (27), form an additional ␤-strand that reaches over to the neighboring subunit and engages in intersubunit ␤-strand complementation. Interestingly, similar intersubunit interactions have been observed in the structures of other bacterial nitrite reductases, for example, those from Alcaligenes faecalis (37), Achromobacter xylosoxidans (38), and Achromobacter cycloclastes (39). Otherwise, our AniA structures are gonorrhoeae survival under anoxia.…”

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confidence: 72%

Peptide Inhibitors Targeting the Neisseria gonorrhoeae Pivotal Anaerobic Respiration Factor AniA

Sikora

Mills

Weber

et al. 2017

Antimicrob Agents Chemother

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Neisseria gonorrhoeae causes the sexually transmitted infection gonorrhea, which is highly prevalent worldwide and has a major impact on reproductive and neonatal health. The superbug status of N. gonorrhoeae necessitates the development of drugs with different mechanisms of action. Here, we focused on targeting the nitrite reductase AniA, which is a pivotal component of N. gonorrhoeae anaerobic respiration and biofilm formation. Our studies showed that gonococci expressing AniA containing the altered catalytic residues D137A and H280A failed to grow under anaerobic conditions, demonstrating that the nitrite reductase function is essential. To facilitate the pharmacological targeting of AniA, new crystal structures of AniA were refined to 1.90-Å and 2.35-Å resolutions, and a phage display approach with libraries expressing randomized linear dodecameric peptides or heptameric peptides flanked by a pair of cysteine residues was utilized. Biopanning experiments led to the identification of 29 unique peptides, with 1 of them, C7-3, being identified multiple times. Evaluation of their ability to interact with AniA using enzyme-linked immunosorbent assay and computational docking studies revealed that C7-3 was the most promising inhibitor, binding near the type 2 copper site of the enzyme, which is responsible for interaction with nitrite. Subsequent enzymatic assays and biolayer interferometry with a synthetic C7-3 and its derivatives, C7-3m1 and C7-3m2, demonstrated potent inhibition of AniA. Finally, the MIC 50 value of C7-3 and C7-3m2 against anaerobically grown N. gonorrhoeae was 0.6 mM. We present the first peptide inhibitors of AniA, an enzyme that should be further exploited for antigonococcal drug development.

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confidence: 72%

Peptide Inhibitors Targeting the Neisseria gonorrhoeae Pivotal Anaerobic Respiration Factor AniA

Sikora

Mills

Weber

et al. 2017

Antimicrob Agents Chemother

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“…Below pH 6.5, we find that the B route is slower than the A route (indicated by k cat B /k cat A Ͻ 1) because the rate-limiting step of electron transfer to the type-2 site is slower with nitrite-bound than with H 2 O-bound, in agreement with literature reports that electron transfer from the type-1 to the type-2 site is slower in the presence of nitrite (43)(44)(45)(46) and that electron transfer is a rate-limiting step for turnover of the NiR (18,44,47). Above pH 6.5, the B route is faster than the A route (k cat B /k cat A Ͼ 1), because the type-2 H 2 O ligand (which is easily substituted by nitrite) is replaced by/converted to OH Ϫ (18,19), and electron transfer to the type-2 site is faster with nitrite bound than with OH Ϫ (Table 1, Fig. 3).…”

Section: Discussionmentioning

confidence: 99%

“…In the absence of nitrite a water molecule occupies the fourth ligand position. Deprotonation of this water molecule occurs between pH 6 and 7 (18) and results in OH Ϫ as the fourth ligand (19). NiR catalyzes the reduction of nitrite bidirectionally (NO 2 Ϫ ϩ e Ϫ ϩ 2H ϩ^N O ϩ H 2 O) (20).…”

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confidence: 99%

A Random-sequential Mechanism for Nitrite Binding and Active Site Reduction in Copper-containing Nitrite Reductase

Wijma

Jeuken

Verbeet

et al. 2006

Journal of Biological Chemistry

128

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The homotrimeric copper-containing nitrite reductase (NiR) contains one type-1 and one type-2 copper center per monomer. Electrons enter through the type-1 site and are shuttled to the type-2 site where nitrite is reduced to nitric oxide. To investigate the catalytic mechanism of NiR the effects of pH and nitrite on the turnover rate in the presence of three different electron donors at saturating concentrations were measured. The activity of NiR was also measured electrochemically by exploiting direct electron transfer to the enzyme immobilized on a graphite rotating disk electrode. In all cases, the steady-state kinetics fitted excellently to a randomsequential mechanism in which electron transfer from the type-1 to the type-2 site is rate-limiting. At low [NO 2 ؊ ] reduction of the type-2 site precedes nitrite binding, at high [NO 2 ؊ ] the reverse occurs.Below pH 6.5, the catalytic activity diminished at higher nitrite concentrations, in agreement with electron transfer being slower to the nitrite-bound type-2 site than to the water-bound type-2 site. Above pH 6.5, substrate activation is observed, in agreement with electron transfer to the nitrite-bound type-2 site being faster than electron transfer to the hydroxyl-bound type-2 site. To study the effect of slower electron transfer between the type-1 and type-2 site, NiR M150T was used. It has a type-1 site with a 125-mV higher midpoint potential and a 0.3-eV higher reorganization energy leading to an ϳ50-fold slower intramolecular electron transfer to the type-2 site. The results confirm that NiR employs a random-sequential mechanism.

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“…They are part of a hydrogen-bonding network involving a number of water molecules, which are likely to play a role in the transport of the protons. [A second network of water molecules in the vicinity of the active site has been identified, but its function is uncertain (36).] The water molecules exhibit partial disorder, as does His-255.…”

Section: It Can Be Shown (See Si Appendix Si Text) That This Connectmentioning

confidence: 99%

The enzyme mechanism of nitrite reductase studied at single-molecule level

Кузнецова

Zauner

Aartsma

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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A generic method is described for the fluorescence ''readout'' of the activity of single redox enzyme molecules based on Fö rster resonance energy transfer from a fluorescent label to the enzyme cofactor. The method is applied to the study of copper-containing nitrite reductase from Alcaligenes faecalis S-6 immobilized on a glass surface. The parameters extracted from the single-molecule fluorescence time traces can be connected to and agree with the macroscopic ensemble averaged kinetic constants. The rates of the electron transfer from the type 1 to the type 2 center and back during turnover exhibit a distribution related to disorder in the catalytic site. The described approach opens the door to singlemolecule mechanistic studies of a wide range of redox enzymes and the precise investigation of their internal workings.electron transfer ͉ redox enzyme ͉ Fö rster transfer ͉ nitric oxide ͉ fluorescent label I n the past few years, single-enzyme studies have revealed numerous hidden aspects of enzyme behavior (1). The huge potential of these studies to unravel the intricate kinetics and precise workings of enzymes, which are often hidden within the ensemble properties, is somewhat restricted by current approaches. The majority of existing single-molecule enzymatic assays are based on fluorescence and have been limited to the flavoenzymes, which contain a fluorescent cofactor (2-5), or to enzymes for which a suitable fluorogenic substrate could be designed (6-9). Recently, it was shown how redox enzyme activity in the bulk can be studied by Förster resonance energy transfer (FRET) from an attached fluorescent label to the enzyme cofactor (10, 11). Here, we report, first, how this technique can be successfully applied to study the enzymatic turnover of single surface-confined copper-containing nitrite reductase (NiR) molecules labeled with ATTO 655 using scanning confocal fluorescence microscopy. Second, it is shown how the kinetics of the fluorescence time traces can be connected with the kinetic parameters that describe the ensemble behavior of the enzyme. Finally, the rate of the electron transfer between the types 1 and 2 Cu centers during turnover could be established. The observed distribution of rates is connected to the partial structural disorder in the catalytic site that has been observed in crystallographic studies. ResultsEnzyme Mechanism. Dissimilatory copper-containing nitrite reductase (NiR) from Alcaligenes faecalis S-6 converts nitrite into nitric oxide. The enzyme is a homotrimer, each monomer containing one type 1 and one type 2 copper site (Fig. 1). The type 1 copper accepts an electron from the physiological donor and transfers it to the type 2 copper, where nitrite is reduced to nitric oxide (NO). The midpoint potentials of the types 1 and 2 Cu centers are close, resulting in a redox equilibrium constant for the two sites that is close to 1 (12, 13). Regarding the enzyme mechanism, it has been stated (14, 15) that, after reduction of the type 1 Cu site, the electron is passed on to the type 2...

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X-ray structure of a blue copper nitrite reductase at high pH and in copper-free form at 1.9 Å resolution

Cited by 30 publications

References 36 publications

Peptide Inhibitors Targeting the Neisseria gonorrhoeae Pivotal Anaerobic Respiration Factor AniA

Peptide Inhibitors Targeting the Neisseria gonorrhoeae Pivotal Anaerobic Respiration Factor AniA

A Random-sequential Mechanism for Nitrite Binding and Active Site Reduction in Copper-containing Nitrite Reductase

The enzyme mechanism of nitrite reductase studied at single-molecule level

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