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PDBsum entry 1b8s
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Contents |
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* Residue conservation analysis
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References listed in PDB file
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Key reference
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Title
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Crystal structure determination of cholesterol oxidase from streptomyces and structural characterization of key active site mutants.
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Authors
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Q.K.Yue,
I.J.Kass,
N.S.Sampson,
A.Vrielink.
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Ref.
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Biochemistry, 1999,
38,
4277-4286.
[DOI no: ]
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PubMed id
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Abstract
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Cholesterol oxidase is a monomeric flavoenzyme which catalyzes the oxidation and
isomerization of cholesterol to cholest-4-en-3-one. The enzyme interacts with
lipid bilayers in order to bind its steroid substrate. The X-ray structure of
the enzyme from Brevibacterium sterolicum revealed two loops, comprising
residues 78-87 and residues 433-436, which act as a lid over the active site and
facilitate binding of the substrate [Vrielink et al. (1991) J. Mol. Biol. 219,
533-554; Li et al. (1993) Biochemistry 32, 11507-11515]. It was postulated that
these loops must open, forming a hydrophobic channel between the membrane and
the active site of the protein and thus sequestering the cholesterol substrate
from the aqueous environment. Here we describe the three-dimensional structure
of the homologous enzyme from Streptomyces refined to 1.5 A resolution.
Structural comparisons to the enzyme from B. sterolicum reveal significant
conformational differences in these loop regions; in particular, a region of the
loop comprising residues 78-87 adopts a small amphipathic helical turn with
hydrophobic residues directed toward the active site cavity and hydrophilic
residues directed toward the external surface of the molecule. It seems
reasonable that this increased rigidity reduces the entropy loss that occurs
upon binding substrate. Consequently, the Streptomyces enzyme is a more
efficient catalyst. In addition, we have determined the structures of three
active site mutants which have significantly reduced activity for either the
oxidation (His447Asn and His447Gln) or the isomerization (Glu361Gln). Our
structural and kinetic data indicate that His447 and Glu361 act as general base
catalysts in association with conserved water H2O541 and Asn485. The His447,
Glu361, H2O541, and Asn485 hydrogen bond network is conserved among other
oxidoreductases. This catalytic tetrad appears to be a structural motif that
occurs in flavoenzymes that catalyze the oxidation of unactivated alcohols.
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