Crystal structure of nitrile hydratase from a thermophilic Bacillus smithii

Hourai, Shinji; Miki, Masayuki; Takashima, Yuki; Mitsuda, Satoshi; Yanagi, Kazunori

doi:10.1016/j.bbrc.2003.10.124

Cited by 60 publications

(56 citation statements)

References 17 publications

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“…1 C and D (4,28)] of L-NHase derived from nhlBAE was found to be 0.88 Ϯ 0.04 mol/mol of ␣␤, whereas those of the heterodimer and heterotetramer derived from nhlBA were very low (Table 1). Whereas the active enzyme is presumed to contain the two oxidized cysteine ligands (8,9,13,14), the apoprotein is likely to be nonmodified, judging from previous studies of NHase (12) and the related enzyme thiocyanate hydrolase (SCNase) (29). These findings suggest that the gene products derived from nhlBA are apo-L-NHases (apo-␣␤ and apo-␣ 2 ␤ 2 , respectively), in contrast to the holo-L-NHase (holo-␣ 2 ␤ 2 ) derived from nhlBAE.…”

Section: Resultsmentioning

confidence: 99%

“…Posttranslationally modified Cys-SO 2 H and Cys-SOH have deprotonated Cys-SO 2 Ϫ and Cys-SO Ϫ structures, respectively (7), and the deprotonated Cys-SO 2 Ϫ and Cys-SO Ϫ in the holo-␣-subunit form salt bridges with two arginines of the ␤-subunit (which are conserved in all known Co-type and Fe-type NHases) in the holo-enzyme (8,9,13,14). The saltbridge mechanism postulates that the electrostatic network around a cobalt ion including the two negative modified cysteines (Cys-112 and Cys-114) in the ␣-subunit and the two arginines (R52 and R157) in the ␤-subunit play a key role in stabilizing the subunit interface (Fig.…”

Section: Driving Force For ␣-Subunit Exchange Between Apo-l-nhase Andmentioning

confidence: 99%

“…Metalloproteins have been characterized intensively for decades, yet investigators only recently have focused on the mechanisms of biological metallocenter assembly (1,3). Nitrile hydratase (NHase; EC4.2.1.84) (4,5), which is composed of ␣-and ␤-subunits, contains either a nonheme iron (6)(7)(8)(9) or noncorrin cobalt ion (10)(11)(12) in a ligand environment that includes two oxidized cysteine residues [CXLC(SO 2 H)SC(SOH)] (8,9,13,14). The enzyme catalyzes the hydration of a nitrile to the corresponding amide, followed by consecutive reactions: amide 3 acid 3 acylCoA by amidase (15) and acyl-CoA synthetase (16,17), respectively.…”

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confidence: 99%

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Discovery of posttranslational maturation by self-subunit swapping

Zhou

Hashimoto²,

Shiraki³

et al. 2008

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

127

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Several general mechanisms of metallocenter biosynthesis have been reported and reviewed, and in all cases, the components or subunits of an apoprotein remain in the final holoprotein. Here, we first discovered that one subunit of an apoenzyme did not remain in the functional holoenzyme. The cobalt-containing low-molecular-mass nitrile hydratase (L-NHase) of Rhodococcus rhodochrous J1 consists of ␤-and ␣-subunits encoded by the nhlBA genes, respectively. An ORF, nhlE, just downstream of nhlBA, was found to be necessary for L-NHase activation. In contrast to the cobaltcontaining L-NHase (holo-L-NHase containing Cys-SO 2 ؊ and Cys-SO ؊ metal ligands) derived from nhlBAE, the gene products derived from nhlBA were cobalt-free L-NHase (apo-L-NHase lacking oxidized cysteine residues). We discovered an L-NHase maturation mediator, NhlAE, consisting of NhlE and the cobalt-and oxidized cysteine-containing ␣-subunit of L-NHase. The incorporation of cobalt into L-NHase was shown to depend on the exchange of the nonmodified cobalt-free ␣-subunit of apo-L-NHase with the cobaltcontaining cysteine-modified ␣-subunit of NhlAE. This is a posttranslational maturation process different from general mechanisms of metallocenter biosynthesis known so far: the unexpected behavior of a protein in a protein complex, which we named ''self-subunit swapping.'' metalloenzyme ͉ modification ͉ sulfinic acid ͉ sulfenic acid ͉ chaperone

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Section: Resultsmentioning

confidence: 99%

Section: Driving Force For ␣-Subunit Exchange Between Apo-l-nhase Andmentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

Discovery of posttranslational maturation by self-subunit swapping

Zhou

Hashimoto²,

Shiraki³

et al. 2008

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

127

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“…Complex 1-Na has a square-planar geometry with N 2 S 2 donor atoms as well as the equatorial coordination in the active site of NHase. [9][10][11][12] The bond lengths around the metal center [Co1-N1 = 1.919(3), Co1-N2 = 1.918(3), Co1-S1 = 2.141(1), Co1-S2 = 2.142(1) Å] are comparable to those of the Co III complexes with amide (Co-N = 1.86-1.90 Å) and thiolate coordinations (Co-S = 2.13-2.14 Å) that were previously reported. [18c,19c] Each carbonyl oxygen of the ligand L was significantly coordinated to three water molecules [O carbonyl ···O water = 2.84-2.91 Å] in the crystal.…”

Section: Introductionmentioning

confidence: 99%

Co^III Complexes with Square‐Planar N₂S₂‐ and N₂(SO₂)₂‐Type Ligands as An Active Site Structural Model for Nitrile Hydratase – Biological Implications of an Amidate Coordination

et al. 2006

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}(tBuNC) 2 ] (3) were synthesized and characterized on the basis of electronic absorption spectroscopy, IR spectroscopy, cyclic voltammetry, and X-ray structural analysis. Both of the crystal structures of complexes 1-Na and 1-PPh 4 revealed a square planar structure with N 2 S 2 donating atoms, and 2 exhibited an octahedral structure coordinated with two tert-butylisocyanide (tBuNC) molecules at the axial sites of complex 1-PPh 4 . Complex 3, which showed an octahedral structure with sulfinate sulfur atoms equatorially coordinated to the center, was synthesized by the treatment of 2 with a suitable oxidant. The reduction potential values from Co III to Co II for complex 3 in solution demonstrated a larger positive shift when compared with those of complexes 1-PPh 4 and 2, which indicates that the oxygenation of the sulfur atoms increased the Lewis acidity of the Co III center. Interestingly, the coordination equilib-

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“…1,6,7 The metal ion is ligated by three cysteine sulfur atoms, two backbone amide nitrogens, and a water molecule. [8][9][10][11] Two of the active site cysteine residues are post-translationally modified to cysteine-sulfinic acid (Cys-SO2H) and cysteine-sulfenic acid (Cys-SOH) yielding an unusual metal coordination geometry, termed the "claw-setting". 8,9 Recently, X-ray crystal structures of a Co-type NHase from Pseudonocardia thermophila JCM 3095(PtNHase) bound by butane boronic acid and phenylboronic acid revealed a covalent bond between the oxygen atom of the sulfenic acid ligand and the boron atom of the boronic acid.…”

Section: Introductionmentioning

confidence: 99%

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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Crystal structure of nitrile hydratase from a thermophilic Bacillus smithii

Cited by 60 publications

References 17 publications

Discovery of posttranslational maturation by self-subunit swapping

Discovery of posttranslational maturation by self-subunit swapping

Co^III Complexes with Square‐Planar N₂S₂‐ and N₂(SO₂)₂‐Type Ligands as An Active Site Structural Model for Nitrile Hydratase – Biological Implications of an Amidate Coordination

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

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Crystal structure of nitrile hydratase from a thermophilic Bacillus smithii

Cited by 60 publications

References 17 publications

Discovery of posttranslational maturation by self-subunit swapping

Discovery of posttranslational maturation by self-subunit swapping

CoIII Complexes with Square‐Planar N2S2‐ and N2(SO2)2‐Type Ligands as An Active Site Structural Model for Nitrile Hydratase – Biological Implications of an Amidate Coordination

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

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Co^III Complexes with Square‐Planar N₂S₂‐ and N₂(SO₂)₂‐Type Ligands as An Active Site Structural Model for Nitrile Hydratase – Biological Implications of an Amidate Coordination