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PDBsum entry 4jb4
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Oxidoreductase
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PDB id
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4jb4
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PDB id:
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| Name: |
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Oxidoreductase
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Title:
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Expression, purification, characterization, and solution nmr study of highly deuterated yeast cytochromE C peroxidase with enhanced solubility
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Structure:
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CytochromE C peroxidase, mitochondrial. Chain: a, c. Fragment: cytochromE-C peroxidase, unp residues 68-361. Synonym: ccp. Engineered: yes. Mutation: yes
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Source:
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Saccharomyces cerevisiae. Baker's yeast. Organism_taxid: 559292. Strain: atcc 204508 / s288c. Gene: ccp1, ccp, cpo, ykr066c. Expressed in: escherichia coli. Expression_system_taxid: 562
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Resolution:
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2.39Å
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R-factor:
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0.216
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R-free:
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0.279
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Authors:
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A.C.Wohlkonig
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Key ref:
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A.N.Volkov
et al.
(2013).
Expression, purification, characterization, and solution nuclear magnetic resonance study of highly deuterated yeast cytochrome C peroxidase with enhanced solubility.
Biochemistry,
52,
2165-2175.
PubMed id:
DOI:
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Date:
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19-Feb-13
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Release date:
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10-Apr-13
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PROCHECK
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Headers
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References
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P00431
(CCPR_YEAST) -
Cytochrome c peroxidase, mitochondrial from Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
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Seq:
Struc:
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361 a.a.
291 a.a.
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Key: |
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PfamA domain |
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Secondary structure |
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CATH domain |
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Enzyme class:
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E.C.1.11.1.5
- cytochrome-c peroxidase.
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Reaction:
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2 Fe(II)-[cytochrome c] + H2O2 + 2 H+ = 2 Fe(III)-[cytochrome c] + 2 H2O
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2
×
Fe(II)-[cytochrome c]
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+
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H2O2
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+
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2
×
H(+)
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=
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2
×
Fe(III)-[cytochrome c]
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+
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2
×
H2O
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Cofactor:
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Heme
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Heme
Bound ligand (Het Group name =
HEM)
matches with 95.45% similarity
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Molecule diagrams generated from .mol files obtained from the
KEGG ftp site
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DOI no:
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Biochemistry
52:2165-2175
(2013)
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PubMed id:
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Expression, purification, characterization, and solution nuclear magnetic resonance study of highly deuterated yeast cytochrome C peroxidase with enhanced solubility.
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A.N.Volkov,
A.Wohlkonig,
S.H.Soror,
N.A.van Nuland.
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ABSTRACT
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Here we present the preparation, biophysical characterization, and nuclear
magnetic resonance (NMR) spectroscopy study of yeast cytochrome c peroxidase
(CcP) constructs with enhanced solubility. Using a high-yield Escherichia coli
expression system, we routinely produced uniformly labeled [(2)H,(13)C,(15)N]CcP
samples with high levels of deuterium incorporation (96-99%) and good yields
(30-60 mg of pure protein from 1 L of bacterial culture). In addition to
simplifying the purification procedure, introduction of a His tag at either
protein terminus dramatically increases its solubility, allowing preparation of
concentrated, stable CcP samples required for multidimensional NMR spectroscopy.
Using a range of biophysical techniques and X-ray crystallography, we
demonstrate that the engineered His tags neither perturb the structure of the
enzyme nor alter the heme environment or its reactivity toward known ligands.
The His-tagged CcP constructs remain catalytically active yet exhibit
differences in the interaction with cytochrome c, the physiological binding
partner, most likely because of steric occlusion of the high-affinity binding
site by the C-terminal His tag. We show that protein perdeuteration greatly
increases the quality of the double- and triple-resonance NMR spectra, allowing
nearly complete backbone resonance assignments and subsequent study of the CcP
by heteronuclear NMR spectroscopy.
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Links
