1cc2: Difference between revisions

Revision as of 13:04, 21 February 2008

CHOLESTEROL OXIDASE FROM STREPTOMYCES HIS447GLN MUTANT

OverviewOverview

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.

About this StructureAbout this Structure

1CC2 is a Single protein structure of sequence from Streptomyces sp. with as ligand. Active as Cholesterol oxidase, with EC number 1.1.3.6 Full crystallographic information is available from OCA.

ReferenceReference

Crystal structure determination of cholesterol oxidase from Streptomyces and structural characterization of key active site mutants., Yue QK, Kass IJ, Sampson NS, Vrielink A, Biochemistry. 1999 Apr 6;38(14):4277-86. PMID:10194345

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-[[Image:1cc2.gif|left|200px]]<br /><applet load="1cc2" size="450" color="white" frame="true" align="right" spinBox="true"
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 caption="1cc2, resolution 2.2&Aring;" />
 '''CHOLESTEROL OXIDASE FROM STREPTOMYCES HIS447GLN MUTANT'''<br />
 ==Overview==
-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.
+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.
 ==About this Structure==
-CC2 is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Streptomyces_sp. Streptomyces sp.] with FAD as [http://en.wikipedia.org/wiki/ligand ligand]. Active as [http://en.wikipedia.org/wiki/Cholesterol_oxidase Cholesterol oxidase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.1.3.6 1.1.3.6] Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=1CC2 OCA].
+CC2 is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Streptomyces_sp. Streptomyces sp.] with <scene name='pdbligand=FAD:'>FAD</scene> as [http://en.wikipedia.org/wiki/ligand ligand]. Active as [http://en.wikipedia.org/wiki/Cholesterol_oxidase Cholesterol oxidase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.1.3.6 1.1.3.6] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1CC2 OCA].
 ==Reference==
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 [[Category: Streptomyces sp.]]
 [[Category: Vrielink, A.]]
-[[Category: Yue, Q.K.]]
+[[Category: Yue, Q K.]]
 [[Category: FAD]]
 [[Category: flavoenzyme]]
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 [[Category: steroid metabolism]]
-''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Tue Nov 20 12:19:58 2007''
+''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 12:04:30 2008''