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PDBsum entry 2zpi
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References listed in PDB file
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Key reference
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Title
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Catalytic mechanism of nitrile hydratase proposed by time-Resolved X-Ray crystallography using a novel substrate, Tert-Butylisonitrile.
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Authors
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K.Hashimoto,
H.Suzuki,
K.Taniguchi,
T.Noguchi,
M.Yohda,
M.Odaka.
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Ref.
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J Biol Chem, 2008,
283,
36617-36623.
[DOI no: ]
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PubMed id
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Abstract
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Nitrile hydratases (NHases) have an unusual iron or cobalt catalytic center with
two oxidized cysteine ligands, cysteine-sulfinic acid and cysteine-sulfenic
acid, catalyzing the hydration of nitriles to amides. Recently, we found that
the NHase of Rhodococcus erythropolis N771 exhibited an additional catalytic
activity, converting tert-butylisonitrile (tBuNC) to tert-butylamine. Taking
advantage of the slow reactivity of tBuNC and the photoreactivity of
nitrosylated NHase, we present the first structural evidence for the catalytic
mechanism of NHase with time-resolved x-ray crystallography. By monitoring the
reaction with attenuated total reflectance-Fourier transform infrared
spectroscopy, the product from the isonitrile carbon was identified as a CO
molecule. Crystals of nitrosylated inactive NHase were soaked with tBuNC. The
catalytic reaction was initiated by photo-induced denitrosylation and stopped by
flash cooling. tBuNC was first trapped at the hydrophobic pocket above the iron
center and then coordinated to the iron ion at 120 min. At 440 min, the electron
density of tBuNC was significantly altered, and a new electron density was
observed near the isonitrile carbon as well as the sulfenate oxygen of
alphaCys(114). These results demonstrate that the substrate was coordinated to
the iron and then attacked by a solvent molecule activated by alphaCys(114)-SOH.
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Figure 5.
The steric hindrance at Sγ of βMet40 caused by tBuNC. The
refined structure around βMet^40 in the nitrosylated NHase
without (A) and with (B) tBuNC. Yellow, blue, red, and green
spheres represent carbon, nitrogen, oxygen, and sulfur atoms,
respectively. The black and red dashed lines indicate the
distances between Sγ of βMet^40 and the isonitrile carbon and
between Sγ of βMet^40 and the amide oxygen of βMet^40.
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Figure 6.
Proposed catalytic mechanisms of NHase. A, isonitrile
hydrolysis. B, nitrile hydration.
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The above figures are
reprinted
from an Open Access publication published by the ASBMB:
J Biol Chem
(2008,
283,
36617-36623)
copyright 2008.
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