A Novel Inhibitor for Fe-type Nitrile Hydratase:  2-Cyano-2-propyl Hydroperoxide

Tsujimura, Maki; Odaka, Masafumi; Nakayama, Hiroshi; Dohmae, Naoshi; Koshino, Hiroyuki; Asami, Tadao; Hoshino, Mikio; Takio, Koji; Yoshida, Shigeo; Maeda, Mizuo; Endo, Itaru

doi:10.1021/ja035018z

Cited by 51 publications

(57 citation statements)

References 60 publications

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“…Considering these findings, we would like to propose that the oxygen atoms in the oxidized cysteine residues are derived from water molecules, although the oxygen atoms in the cysteine residues modified through oxidation using an inhibitor (2-cyano-2-propyl hydroperoxide) for Fe-NHase and spontaneous oxidation are proposed to come from water molecules (41) and dissolved oxygen (34), respectively.…”

Section: Discussionmentioning

confidence: 99%

Self-subunit Swapping Chaperone Needed for the Maturation of Multimeric Metalloenzyme Nitrile Hydratase by a Subunit Exchange Mechanism Also Carries Out the Oxidation of the Metal Ligand Cysteine Residues and Insertion of Cobalt

Zhou¹,

Hashimoto²,

Kobayashi

2009

Journal of Biological Chemistry

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The incorporation of cobalt into low molecular mass nitrile hydratase (L-NHase) of Rhodococcus rhodochrous J1 has been found to depend on the ␣-subunit exchange between cobalt-free L-NHase (apo-L-NHase lacking oxidized cysteine residues) and its cobalt-containing mediator (holo-NhlAE containing Cys-SO 2 ؊ and Cys-SO ؊ metal ligands), this novel mode of post-translational maturation having been named self-subunit swapping, and NhlE having been recognized as a self-subunit swapping chaperone (Zhou, Z., Hashimoto, Y., Shiraki, K., and Kobayashi, M. (2008) Proc. Natl. Acad. Sci. U. S. A. 105, 14849 -14854). We discovered here that cobalt was inserted into both the cobaltfree NhlAE (apo-NhlAE) and the cobalt-free ␣-subunit (apo-␣-subunit) in an NhlE-dependent manner in the presence of cobalt and dithiothreitol in vitro. Matrix-assisted laser desorption ionization time-of-flight mass spectroscopy analysis revealed that the non-oxidized cysteine residues in apo-NhlAE were posttranslationally oxidized after cobalt insertion. These findings suggested that NhlE has two activities, i.e. cobalt insertion and cysteine oxidation. NhlE not only functions as a self-subunit swapping chaperone but also a metallochaperone that includes a redox function. Cobalt insertion and cysteine oxidation occurred under both aerobic and anaerobic conditions when Co 3؉ was used as a cobalt donor, suggesting that the oxygen atoms in the oxidized cysteines were derived from water molecules but not from dissolved oxygen. Additionally, we isolated apo-NhlAE after the self-subunit swapping event and found that it was recycled for cobalt transfer into L-NHase.

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Section: Discussionmentioning

confidence: 99%

Self-subunit Swapping Chaperone Needed for the Maturation of Multimeric Metalloenzyme Nitrile Hydratase by a Subunit Exchange Mechanism Also Carries Out the Oxidation of the Metal Ligand Cysteine Residues and Insertion of Cobalt

Zhou¹,

Hashimoto²,

Kobayashi

2009

Journal of Biological Chemistry

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“…Oxidation of the equatorial Cys residues is required for catalytic activity. 10,11 Even though the structures of Fe-and Co-type NHases are very similar, Fe-type NHases are specific for Fe(III), whereas Co-type NHases are specific for Co (III). 8 Several open reading frames have been identified just downstream from the structural α-and β-subunit genes in NHases, and one of these genes has been proposed to function as an activator protein ( Figure 1).…”

Section: Introductionmentioning

confidence: 99%

The iron-type nitrile hydratase activator protein is a GTPase

Gumataotao

Lankathilaka

Bennett

et al. 2017

Biochemical Journal

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Abstract:The Fe-type nitrile hydratase activator protein from Rhodococcus equi TG328-2 (ReNHase TG328-2) was successfully expressed and purified. Sequence analysis and homology modeling suggest that it is a G3E P-loop guanosine triphosphatase (GTPase) within the COG0523 subfamily. Kinetic studies revealed that the Fe-type activator protein is capable of hydrolyzing NOT THE PUBLISHED VERSION; this is the author's final, peer-reviewed manuscript. The published version may be accessed by following the link in the citation at the bottom of the page.Biochemical Journal, Vol 474, No. 2 (January 15, 2017): pg. 247-258. DOI. This article is © Portland Press Limited and permission has been granted for this version to appear in e-Publications@Marquette. Portland Press Limited does not grant permission for this article to be further copied/distributed or hosted elsewhere without the express permission from Portland Press Limited. 2GTP to GDP with a kcat value of 1.2 × 10 −3 s −1 and a Km value of 40 μM in the presence of 5 mM MgCl2 in 50 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid at a pH of 8.0. The addition of divalent metal ions, such as Co(II), which binds to the ReNHase TG328-2 activator protein with a Kd of 2.9 μM, accelerated the rate of GTP hydrolysis, suggesting that GTP hydrolysis is potentially connected to the proposed metal chaperone function of the ReNHase TG328-2 activator protein. Circular dichroism data reveal a significant conformational change upon the addition of GTP, which may be linked to the interconnectivity of the cofactor binding sites, resulting in an activator protein that can be recognized and can bind to the NHase α-subunit. A combination of these data establishes, for the first time, that the ReNHase TG328-2 activator protein falls into the COG0523 subfamily of G3E P-loop GTPases, many of which play a role in metal homeostasis processes.

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“…On the other hand, the αH80A/αH81A and αH80W/αH81W mutant enzymes exhibited a blueshift along with an increase in molar absorptivity (Table S2). The observed red-shifts in this LMCT band are typically observed when (i) the pH is increased, (ii), upon interaction with an inhibitor such as butyric acid, (iii), changes in hydrogen-bonding at or near the active site or (iv) oxidation of αCys-SOH to αCys-SO2H 1,36,37 On the other hand, blue-shifts are observed by the addition of substrate and also upon disruption of the hydrogen-bonding network between βArg56 and accessed by following the link in the citation at the bottom of the page. Vol 20, No.…”

Section: Resultsmentioning

confidence: 94%

“…Several studies on both Fe-and Co-Type NHases have shown the importance of the stability and integrity of the Cys-SOH ligand for catalytic activity in NHases; for example, further oxidation to Cys-SO2H leads to inactivation. 27,37 Most recently, in our studies with the Co-Type NHase from P. thermophila JCM 3095, we showed that Cys-SOH can function as a nucleophile in the hydration of nitrile to amides by NHase. 12 Therefore stabilization of αCys 104 -SOH in CtNHase through hydrogenbond formation with αR157 is important for a fully functional NHase enzyme.…”

Section: Discussionmentioning

confidence: 93%

Analyzing the catalytic role of active site residues in the Fe-type nitrile hydratase from Comamonas testosteroni Ni1

Martinez

Krzywda

et al. 2015

J Biol Inorg Chem

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A strictly conserved active site arginine residue (αR157) and two histidine residues (αH80 and αH81) located near the active site of the Fe-type nitrile hydratase from Comamonas testosteroni Ni1 (CtNHase), were mutated. These mutant enzymes were examined for their ability to bind iron and hydrate acrylonitrile. For the αR157A mutant, the residual activity (kcat = 10 ± 2 s −1 ) accounts for less than 1 % of the wild-type activity (kcat = 1100 ± 30 s −1 ) while the Km value is nearly unchanged at 205 ± 10 mM. On the other hand, mutation of the active site pocket αH80 and αH81 residues to alanine resulted in enzymes with kcat values of 220 ± 40 and 77 ± 13 s −1 , respectively, and Km values of 187 ± 11 and 179 ± 18 mM. The double mutant (αH80A/αH81A) was also prepared and provided an enzyme with a kcat value of 132 ± 3 s −1 and a Km value of 213 ± 61 mM. These data indicate that all three residues are catalytically important, but not essential. X-ray crystal structures of the αH80A/αH81A, αH80W/αH81W, and αR157A mutant CtNHase enzymes were solved to 2.0, 2.8, and 2.5 Å resolutions, respectively. In each mutant enzyme, hydrogen-bonding interactions crucial for the catalytic function of the αCys 104 -SOH ligand are disrupted. Disruption of these hydrogen bonding interactions likely alters the nucleophilicity of the sulfenic acid oxygen and the Lewis acidity of the active site Fe(III) ion.

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A Novel Inhibitor for Fe-type Nitrile Hydratase: 2-Cyano-2-propyl Hydroperoxide

Cited by 51 publications

References 60 publications

Self-subunit Swapping Chaperone Needed for the Maturation of Multimeric Metalloenzyme Nitrile Hydratase by a Subunit Exchange Mechanism Also Carries Out the Oxidation of the Metal Ligand Cysteine Residues and Insertion of Cobalt

Self-subunit Swapping Chaperone Needed for the Maturation of Multimeric Metalloenzyme Nitrile Hydratase by a Subunit Exchange Mechanism Also Carries Out the Oxidation of the Metal Ligand Cysteine Residues and Insertion of Cobalt

The iron-type nitrile hydratase activator protein is a GTPase

Analyzing the catalytic role of active site residues in the Fe-type nitrile hydratase from Comamonas testosteroni Ni1

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