Functional Expression of Nitrile Hydratase in Escherichia coli: Requirement of a Nitrile Hydratase Activator and Post-Translational Modification of a Ligand Cysteine

Nojiri, Masaki; Yohda, Masafumi; Odaka, Masafumi; Matsushita, Yohsuke; Tsujimura, Maki; Yoshida, Takao; Dohmae, Naoshi; Takio, Koji; Endo, Itaru

doi:10.1093/oxfordjournals.jbchem.a022339

Cited by 97 publications

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“…The oxidant in this reaction, 4, has been used previously to oxygenate free thiols. 44 The structure of the product of reaction 1, [Fe III (ADIT)(ADIT-O)] + (5), was determined by (1) X-ray crystallography and found to contain a singly oxygenated thiolate sulfur (i.e., a sulfenate) coordinated to the metal ion. As shown in the ORTEP of Figure 2, the iron of [Fe III (5) is six-coordinate and ligated by one thiolate sulfur (S(2)), two imine nitrogens (N(2) and N(4)), and two amine nitrogens (N(1) and N(3)), in addition to the singly oxygenated sulfenate sulfur (S(1)).…”

Section: Synthesis and Structure Of Singly Oxygenated [Fe III -(Adit)mentioning

confidence: 99%

How Does Single Oxygen Atom Addition Affect the Properties of an Fe−Nitrile Hydratase Analogue? The Compensatory Role of the Unmodified Thiolate

et al. 2006

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Nitrile hydratase (NHase) is one of a growing number of enzymes shown to contain posttranslationally modified cysteine sulfenic acids (Cys-SOH). Cysteine sulfenic acids have been shown to play diverse roles in cellular processes, including transcriptional regulation, signal transduction, and the regulation of oxygen metabolism and oxidative stress responses. The function of the cysteine sulfenic acid coordinated to the iron active site of NHase is unknown. Herein we report the first example of a sulfenate-ligated iron complex, [Fe III (ADIT)(ADIT-O)] + (5), and compare its electronic and magnetic properties with those of structurally related complexes in which the sulfur oxidation state and protonation state have been systematically altered. Oxygen atom addition was found to decrease the unmodified thiolate Fe-S bond length and blue-shift the ligand-to-metal charge-transfer band (without loss of intensity). S K-edge X-ray absorption spectroscopy and density functional theory calculations show that, although the modified RS-O − fragment is incapable of forming a π bond with the Fe III center, the unmodified thiolate compensates for this loss of π bonding by increasing its covalent bond strength. The redox potential shifts only slightly (75 mV), and the magnetic properties are not affected (the S = ½ spin state is maintained). The coordinated sulfenate S-O bond is activated and fairly polarized (S + -O − ). Addition of strong acids at low temperatures results in the reversible protonation of sulfenateligated 5. An X-ray structure demonstrates that Zn 2+ binds to the sulfenate oxygen to afford [Fe III HHS Public Access Author Manuscript Author ManuscriptAuthor ManuscriptAuthor Manuscript unmodified NHase thiolate, involving its ability to "tune" the electronics in response to protonation of the sulfenate (RS-O − ) oxygen and/or substrate binding, is discussed.Nitrile hydratases (NHases) are non-heme iron enzymes that convert nitriles to less toxic amides cleanly, and rapidly, under mild conditions. [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16] It is unusual for a hydrolytic metalloenzyme to incorporate iron, as opposed to zinc, 17,18 presumably because, unlike other metal ions, Zn 2+ is not complicated by redox chemistry. Iron, on the other hand, can promote unwanted side reactions with dioxygen (i.e., Fenton chemistry involving OH · radicals) upon reduction to the Fe 2+ oxidation state. 19,20 The NHase iron site is, however, redox inactive and stabilized in the 3+ oxidation state. The stabilization of Fe 3+ is accomplished by placing the iron in an electron-rich environment consisting of five anionic ligands-two deprotonated peptide amides and three cysteinates (Figure 1). Two of the three coordinated cysteinate sulfurs are oxidized (post-translationally modified) in NHase -one to a sulfenic acid ( 114 Cys-S-(OH)) and the other to a sulfinate 112 CysSO 2 −.3,21 The third cysteinate sulfur, which is trans to the inhibitor/substrate binding site and less accessible to solvent, remains unmodifie...

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Section: Synthesis and Structure Of Singly Oxygenated [Fe III -(Adit)mentioning

confidence: 99%

How Does Single Oxygen Atom Addition Affect the Properties of an Fe−Nitrile Hydratase Analogue? The Compensatory Role of the Unmodified Thiolate

et al. 2006

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“…The NO-bound NHase protein was expressed and purified as described in ref 35. The protein solutions (in 100 mM phosphate buffer, pH 7.5) were pre-equilibrated in a water-saturated He atmosphere for ~1 h to minimize bubble formation in the sample cell.…”

Section: Experimental Details Sample Preparationmentioning

confidence: 99%

Sulfur K-Edge XAS and DFT Calculations on Nitrile Hydratase: Geometric and Electronic Structure of the Non-heme Iron Active Site

et al. 2005

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The geometric and electronic structure of the active site of the non-heme iron enzyme nitrile hydratase (NHase) is studied using sulfur K-edge XAS and DFT calculations. Using thiolate (RS − )-, sulfenate (RSO − )-, and sulfinate (RSO 2 − )-ligated model complexes to provide benchmark spectral parameters, the results show that the S K-edge XAS is sensitive to the oxidation state of S-containing ligands and that the spectrum of the RSO − species changes upon protonation as the S-O bond is elongated (by ~0.1 Å). These signature features are used to identify the three cysteine residues coordinated to the low-spin Fe III in the active site of NHase as CysS − , CysSOH, and CysSO 2 − both in the NO-bound inactive form and in the photolyzed active form. These results are correlated to geometry-optimized DFT calculations. The pre-edge region of the X-ray absorption spectrum is sensitive to the Z eff of the Fe and reveals that the Fe in [FeNO] 6 NHase species has a Z eff very similar to that of its photolyzed Fe III counterpart. DFT calculations reveal that this results from the strong π back-bonding into the π* antibonding orbital of NO, which shifts significant charge from the formally t 2 6 low-spin metal to the coordinated NO.

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“…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). [12][13][14] The prevailing dogma is that both Co-and Fe-type NHase enzymes require the co-expression of an activator protein to be fully metallated, post-translationally modified, and fully functional. [12][13][14] While Co-and Fe-type NHase enzymes share high sequence similarity, their respective activator proteins are different in size and share little to no sequence identity, The alignment shows (light green -Walker A motif; dark green -Walker B motif; blue -metal-binding motif; orange -guanine-binding motif) shows conserved regions common to P-loop GTPases that are probably responsible for GTPase activity of the proteins.…”

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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Functional Expression of Nitrile Hydratase in Escherichia coli: Requirement of a Nitrile Hydratase Activator and Post-Translational Modification of a Ligand Cysteine

Cited by 97 publications

References 0 publications

How Does Single Oxygen Atom Addition Affect the Properties of an Fe−Nitrile Hydratase Analogue? The Compensatory Role of the Unmodified Thiolate

How Does Single Oxygen Atom Addition Affect the Properties of an Fe−Nitrile Hydratase Analogue? The Compensatory Role of the Unmodified Thiolate

Sulfur K-Edge XAS and DFT Calculations on Nitrile Hydratase: Geometric and Electronic Structure of the Non-heme Iron Active Site

The iron-type nitrile hydratase activator protein is a GTPase

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