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Oxidoreductase
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PDB id
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1ijh
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Contents |
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* Residue conservation analysis
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Enzyme class:
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E.C.1.1.3.6
- Cholesterol oxidase.
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Reaction:
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Cholesterol + O2 = cholest-4-en-3-one + H2O2
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Cholesterol
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+
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O(2)
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=
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cholest-4-en-3-one
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+
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H(2)O(2)
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Cofactor:
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FAD
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FAD
Bound ligand (Het Group name =
FAD)
corresponds exactly
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Molecule diagrams generated from .mol files obtained from the
KEGG ftp site
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Gene Ontology (GO) functional annotation
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Cellular component
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extracellular region
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1 term
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Biological process
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oxidation reduction
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4 terms
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Biochemical function
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oxidoreductase activity
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4 terms
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DOI no:
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Biochemistry
40:13779-13787
(2001)
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PubMed id:
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The presence of a hydrogen bond between asparagine 485 and the pi system of FAD modulates the redox potential in the reaction catalyzed by cholesterol oxidase.
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Y.Yin,
N.S.Sampson,
A.Vrielink,
P.I.Lario.
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ABSTRACT
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Cholesterol oxidase catalyzes the oxidation and isomerization of cholesterol to
cholest-4-en-3-one. An asparagine residue (Asn485) at the active site is
believed to play an important role in catalysis. To test the precise role of
Asn485, we mutated it to a leucine and carried out kinetic and crystallographic
studies. Steady-state kinetic analysis revealed a 1300-fold decrease in the
oxidation k(cat)/K(m) for the mutant enzyme whereas the k(cat)/K(m) for
isomerization is only 60-fold slower. The primary kinetic isotope effect in the
mutant-catalyzed reaction indicates that 3alpha-H transfer remains the
rate-determining step. Measurement of the reduction potentials for the wild-type
and N485L enzymes reveals a 76 mV decrease in the reduction potential of the FAD
for the mutant enzyme relative to wild type. The crystal structure of the
mutant, determined to 1.5 A resolution, reveals a repositioning of the side
chain of Met122 near Leu485 to form a hydrophobic pocket. Furthermore, the
movement of Met122 facilitates the binding of an additional water molecule,
possibly mimicking the position of the equatorial hydroxyl group of the steroid
substrate. The wild-type enzyme shows a novel N-H...pi interaction between the
side chain of Asn485 and the pyrimidine ring of the cofactor. The loss of this
interaction in the N485L mutant destabilizes the reduced flavin and accounts for
the decreased reduction potential and rate of oxidation. Thus, the observed
structural rearrangement of residues at the active site, as well as the kinetic
data and thermodynamic data for the mutant, suggests that Asn485 is important
for creating an electrostatic potential around the FAD cofactor enhancing the
oxidation reaction.
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Literature references that cite this PDB file's key reference
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PubMed id
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Reference
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A.Y.Lyubimov,
L.Chen,
N.S.Sampson,
and
A.Vrielink
(2009).
A hydrogen-bonding network is important for oxidation and isomerization in the reaction catalyzed by cholesterol oxidase.
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Acta Crystallogr D Biol Crystallogr, 65,
1222-1231.
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PDB codes:
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I.Dreveny,
A.S.Andryushkova,
A.Glieder,
K.Gruber,
and
C.Kratky
(2009).
Substrate binding in the FAD-dependent hydroxynitrile lyase from almond provides insight into the mechanism of cyanohydrin formation and explains the absence of dehydrogenation activity.
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Biochemistry, 48,
3370-3377.
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PDB codes:
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H.Takahashi,
H.Ohno,
R.Kishi,
M.Nakano,
and
N.Matubayasi
(2008).
Computation of the free energy change associated with one-electron reduction of coenzyme immersed in water: a novel approach within the framework of the quantum mechanical/molecular mechanical method combined with the theory of energy representation.
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J Chem Phys, 129,
205103.
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J.E.Lee,
E.Bae,
C.A.Bingman,
G.N.Phillips,
and
R.T.Raines
(2008).
Structural basis for catalysis by onconase.
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J Mol Biol, 375,
165-177.
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PDB codes:
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L.Chen,
A.Y.Lyubimov,
L.Brammer,
A.Vrielink,
and
N.S.Sampson
(2008).
The binding and release of oxygen and hydrogen peroxide are directed by a hydrophobic tunnel in cholesterol oxidase.
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Biochemistry, 47,
5368-5377.
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PDB code:
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A.Y.Lyubimov,
K.Heard,
H.Tang,
N.S.Sampson,
and
A.Vrielink
(2007).
Distortion of flavin geometry is linked to ligand binding in cholesterol oxidase.
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Protein Sci, 16,
2647-2656.
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PDB codes:
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N.M.Nesbitt,
and
N.S.Sampson
(2007).
Antifungal tradecraft by cholesterol oxidase.
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Chem Biol, 14,
238-241.
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J.Zhang,
F.E.Frerman,
and
J.J.Kim
(2006).
Structure of electron transfer flavoprotein-ubiquinone oxidoreductase and electron transfer to the mitochondrial ubiquinone pool.
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Proc Natl Acad Sci U S A, 103,
16212-16217.
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PDB codes:
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V.Anantharaman,
L.Aravind,
and
E.V.Koonin
(2003).
Emergence of diverse biochemical activities in evolutionarily conserved structural scaffolds of proteins.
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Curr Opin Chem Biol, 7,
12-20.
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The most recent references are shown first.
Citation data come partly from CiteXplore and partly
from an automated harvesting procedure. Note that this is likely to be
only a partial list as not all journals are covered by
either method. However, we are continually building up the citation data
so more and more references will be included with time.
Where a reference describes a PDB structure, the PDB
codes are
shown on the right.
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Links
