DB code: D00015

CATH domain 3.50.50.60 : FAD/NAD(P)-binding domain Catalytic domain
3.30.410.10 : Cholesterol Oxidase; domain 2 Catalytic domain
E.C. 1.1.3.6
CSA 1coy
M-CSA 1coy
MACiE

CATH domain Related DB codes (homologues)
3.50.50.60 : FAD/NAD(P)-binding domain M00163 D00041 D00042 D00045 D00064 D00071 T00004 T00015 T00017 T00025 T00211 T00213 T00233 T00242

Uniprot Enzyme Name
UniprotKB Protein name Synonyms Pfam
P22637 Cholesterol oxidase
CHOD
EC 1.1.3.6
PF05199 (GMC_oxred_C)
PF00732 (GMC_oxred_N)
[Graphical View]
P12676 Cholesterol oxidase
CHOD
EC 1.1.3.6
PF05199 (GMC_oxred_C)
PF00732 (GMC_oxred_N)
[Graphical View]

KEGG enzyme name
cholesterol oxidase
cholesterol- O2 oxidoreductase
3beta-hydroxy steroid oxidoreductase
3beta-hydroxysteroid:oxygen oxidoreductase

UniprotKB: Accession Number Entry name Activity Subunit Subcellular location Cofactor
P22637 CHOD_BREST Cholesterol + O(2) = cholest-4-en-3-one + H(2)O(2). Secreted. FAD.
P12676 CHOD_STRS0 Cholesterol + O(2) = cholest-4-en-3-one + H(2)O(2). Monomer. Secreted. FAD.

KEGG Pathways
Map code Pathways E.C.
MAP00120 Bile acid biosynthesis

Compound table
Cofactors Substrates Products Intermediates
KEGG-id C00016 C00187 C00007 C00599 C00027
E.C.
Compound FAD Cholesterol O2 Cholest-4-en-3-one H2O2 5-cholesten-3-one
Type amide group,amine group,aromatic ring (only carbon atom),aromatic ring (with nitrogen atoms),carbohydrate,nucleotide lipid,steroid others carbohydrate,lipid,steroid others
ChEBI 16238
 16238
 		16113
 16113
 		15379
 26689
 27140
 15379
 26689
 27140
 		16175
 16175
 		16240
 16240
PubChem 643975
 643975
 		5997
 5997
 		977
 977
 		91477
 91477
 		22326046
 784
 22326046
 784
1b4vA01 Bound:FAD Unbound Unbound Unbound Unbound Unbound
1b8sA01 Bound:FAD Unbound Unbound Unbound Unbound Unbound
1cboA01 Bound:FAD Unbound Unbound Unbound Unbound Unbound
1cc2A01 Bound:FAD Unbound Unbound Unbound Unbound Unbound
1coxA01 Unbound Unbound Unbound Unbound Unbound Unbound
1coyA01 Bound:FAD Analogue:AND Unbound Unbound Unbound Unbound
1ijhA01 Bound:FAD Unbound Unbound Unbound Unbound Unbound
1mxtA01 Analogue:FAE Unbound Bound:OXY Unbound Unbound Unbound
1n1pA01 Bound:FAD Unbound Unbound Unbound Unbound Unbound
1n4uA01 Analogue:FAE Unbound Bound:OXY Unbound Unbound Unbound
1n4vA01 Bound:FAD Unbound Unbound Unbound Unbound Unbound
1n4wA01 Bound:FAD Unbound Unbound Unbound Unbound Unbound
3coxA01 Bound:FAD Unbound Unbound Unbound Unbound Unbound
1b4vA02 Unbound Unbound Unbound Unbound Unbound Unbound
1b8sA02 Unbound Unbound Unbound Unbound Unbound Unbound
1cboA02 Unbound Unbound Unbound Unbound Unbound Unbound
1cc2A02 Unbound Unbound Unbound Unbound Unbound Unbound
1coxA02 Unbound Unbound Unbound Unbound Unbound Unbound
1coyA02 Unbound Unbound Unbound Unbound Unbound Unbound
1ijhA02 Unbound Unbound Unbound Unbound Unbound Unbound
1mxtA02 Unbound Unbound Unbound Unbound Unbound Unbound
1n1pA02 Unbound Unbound Unbound Unbound Unbound Unbound
1n4uA02 Unbound Unbound Unbound Unbound Unbound Unbound
1n4vA02 Unbound Unbound Unbound Unbound Unbound Unbound
1n4wA02 Unbound Unbound Unbound Unbound Unbound Unbound
3coxA02 Unbound Unbound Unbound Unbound Unbound Unbound

Reference for Active-site residues
resource references E.C.
PDB;1b4v, 1b8s, 1cbo, 1cc2 & Swiss-prot;P12676,P22637 & literature [19]

Active-site residues
PDB Catalytic residues Cofactor-binding residues Modified residues Main-chain involved in catalysis Comment
1b4vA01 HIS 447;ASN 485
1b8sA01 HIS 447;ASN 485
1cboA01 ;ASN 485 mutant H447N
1cc2A01 ;ASN 485 mutant H447Q
1coxA01 HIS 447;ASN 485
1coyA01 HIS 447;ASN 485
1ijhA01 HIS 447; mutant N485L
1mxtA01 HIS 447;ASN 485
1n1pA01 HIS 447;ASN 485
1n4uA01 HIS 447;ASN 485
1n4vA01 HIS 447;ASN 485
1n4wA01 HIS 447;ASN 485
3coxA01 HIS 447;ASN 485
1b4vA02 GLU 361
1b8sA02 mutant E361Q
1cboA02 GLU 361
1cc2A02 GLU 361
1coxA02 GLU 361
1coyA02 GLU 361
1ijhA02 GLU 361
1mxtA02 GLU 361
1n1pA02 GLU 361
1n4uA02 GLU 361
1n4vA02 GLU 361
1n4wA02 GLU 361
3coxA02 GLU 361

References for Catalytic Mechanism
References Sections No. of steps in catalysis
[2]
Fig.1, p.551-552
[3]
Fig.1, Fig.8, Fig.9, p.11512-11514
[8]
p.17995-17999
[11]
Fig.5, Fig.6, p.1079-1080
[12]
Scheme 1, p.622
[13]
p.4282-4285
[14]
p.149-150
[16]
Fig.1
[17]
Fig.1, Fig.4
[18]
FIG. 1, p.841-842
[19]
p.13785-13785
[20]
p.30438-30440
[23]
Fig.1, p.713-715
[25]
Scheme 2, p.1640-1647

References
[1]
Resource
Comments
Medline ID
PubMed ID 6528973
Journal Anal Biochem
Year 1984
Volume 142
Pages 347-50
Authors Omodeo Sale F, Marchesini S, Fishman PH, Berra B
Title A sensitive enzymatic assay for determination of cholesterol in lipid extracts.
Related PDB
Related UniProtKB
[2]
Resource
Comments X-RAY CRYSTALLOGRAPHY (1.8 ANGSTROMS)
Medline ID 91269328
PubMed ID 2051487
Journal J Mol Biol
Year 1991
Volume 219
Pages 533-54
Authors Vrielink A, Lloyd LF, Blow DM
Title Crystal structure of cholesterol oxidase from Brevibacterium sterolicum refined at 1.8 A resolution.
Related PDB 1cox
Related UniProtKB P22637
[3]
Resource
Comments X-ray crystallography
Medline ID
PubMed ID 8218217
Journal Biochemistry
Year 1993
Volume 32
Pages 11507-15
Authors Li J, Vrielink A, Brick P, Blow DM
Title Crystal structure of cholesterol oxidase complexed with a steroid substrate: implications for flavin adenine dinucleotide dependent alcohol oxidases.
Related PDB 3cox 1coy
Related UniProtKB
[4]
Resource
Comments
Medline ID
PubMed ID 8459768
Journal Mol Microbiol
Year 1993
Volume 7
Pages 419-28
Authors Molnar I, Hayashi N, Choi KP, Yamamoto H, Yamashita M, Murooka Y
Title Bacterial cholesterol oxidases are able to act as flavoprotein-linked ketosteroid monooxygenases that catalyse the hydroxylation of cholesterol to 4-cholesten-6-ol-3-one.
Related PDB
Related UniProtKB
[5]
Resource
Comments
Medline ID
PubMed ID 8812988
Journal J Struct Biol
Year 1996
Volume 116
Pages 317-9
Authors Croteau N, Vrielink A
Title Crystallization and preliminary X-ray analysis of cholesterol oxidase from Brevibacterium sterolicum containing covalently bound FAD.
Related PDB
Related UniProtKB
[6]
Resource
Comments
Medline ID
PubMed ID 9001407
Journal FEBS Lett
Year 1997
Volume 400
Pages 247-51
Authors Medina M, Vrielink A, Cammack R
Title Electron spin echo envelope modulation studies of the semiquinone anion radical of cholesterol oxidase from Brevibacterium sterolicum.
Related PDB
Related UniProtKB
[7]
Resource
Comments
Medline ID
PubMed ID 9153088
Journal Protein Eng
Year 1997
Volume 10
Pages 231-5
Authors Nishiya Y, Harada N, Teshima SI, Yamashita M, Fujii I, Hirayama N, Murooka Y
Title Improvement of thermal stability of Streptomyces cholesterol oxidase by random mutagenesis and a structural interpretation.
Related PDB
Related UniProtKB
[8]
Resource
Comments
Medline ID
PubMed ID 9922167
Journal Biochemistry
Year 1998
Volume 37
Pages 17990-8000
Authors Kass IJ, Sampson NS
Title Evaluation of the role of His447 in the reaction catalyzed by cholesterol oxidase.
Related PDB
Related UniProtKB
[9]
Resource
Comments
Medline ID
PubMed ID 9548964
Journal Biochemistry
Year 1998
Volume 37
Pages 5770-8
Authors Sampson NS, Kass IJ, Ghoshroy KB
Title Assessment of the role of an omega loop of cholesterol oxidase: a truncated loop mutant has altered substrate specificity.
Related PDB
Related UniProtKB
[10]
Resource
Comments
Medline ID
PubMed ID 9749912
Journal Protein Eng
Year 1998
Volume 11
Pages 609-11
Authors Nishiya Y, Yamashita M, Murooka Y, Fujii I, Hirayama N
Title Effect of non-ionic detergents on apparent enzyme mechanism: V121A mutant of Streptomyces cholesterol oxidase endowed with enhanced sensitivity towards detergents.
Related PDB
Related UniProtKB
[11]
Resource
Comments
Medline ID
PubMed ID 9876929
Journal Protein Eng
Year 1998
Volume 11
Pages 1075-81
Authors Yamashita M, Toyama M, Ono H, Fujii I, Hirayama N, Murooka Y
Title Separation of the two reactions, oxidation and isomerization, catalyzed by Streptomyces cholesterol oxidase.
Related PDB
Related UniProtKB
[12]
Resource
Comments
Medline ID
PubMed ID 10417325
Journal Biochem J
Year 1999
Volume 341
Pages 621-7
Authors Doukyu N, Aono R
Title Two moles of O2 consumption and one mole of H2O2 formation during cholesterol peroxidation with cholesterol oxidase from Pseudomonas sp. strain ST-200.
Related PDB
Related UniProtKB
[13]
Resource
Comments X-RAY CRYSTALLOGRAPHY (1.5 ANGSTROMS), AND MUTAGENESIS
Medline ID 99211873
PubMed ID 10194345
Journal Biochemistry
Year 1999
Volume 38
Pages 4277-86
Authors Yue QK, Kass IJ, Sampson NS, Vrielink A
Title Crystal structure determination of cholesterol oxidase from Streptomyces and structural characterization of key active site mutants.
Related PDB 1b4v 1b8s 1cbo 1cc2
Related UniProtKB P12676
[14]
Resource
Comments
Medline ID
PubMed ID 10447682
Journal Eur J Biochem
Year 1999
Volume 264
Pages 140-51
Authors Pollegioni L, Wels G, Pilone MS, Ghisla S
Title Kinetic mechanisms of cholesterol oxidase from Streptomyces hygroscopicus and Brevibacterium sterolicum.
Related PDB
Related UniProtKB
[15]
Resource
Comments
Medline ID
PubMed ID 10949695
Journal Appl Biochem Biotechnol
Year 2000
Volume 87
Pages 141-51
Authors Yotova LK, Ivanov IP
Title Kinetic studies and analytical application of cholesterol oxidase and peroxidase immobilized to synthetic polymer.
Related PDB
Related UniProtKB
[16]
Resource
Comments
Medline ID
PubMed ID 10822008
Journal J Steroid Biochem Mol Biol
Year 2000
Volume 72
Pages 169-95
Authors MacLachlan J, Wotherspoon AT, Ansell RO, Brooks CJ
Title Cholesterol oxidase: sources, physical properties and analytical applications.
Related PDB
Related UniProtKB
[17]
Resource
Comments
Medline ID
PubMed ID 10694883
Journal Trends Biochem Sci
Year 2000
Volume 25
Pages 126-32
Authors Fraaije MW, Mattevi A
Title Flavoenzymes: diverse catalysts with recurrent features.
Related PDB
Related UniProtKB
[18]
Resource
Comments
Medline ID
PubMed ID 11761331
Journal Antioxid Redox Signal
Year 2001
Volume 3
Pages 839-46
Authors Sampson NS
Title Dissection of a flavoenzyme active site: the reaction catalyzed by cholesterol oxidase.
Related PDB
Related UniProtKB
[19]
Resource
Comments X-ray crystallography
Medline ID
PubMed ID 11705367
Journal Biochemistry
Year 2001
Volume 40
Pages 13779-87
Authors Yin Y, Sampson NS, Vrielink A, Lario PI
Title 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.
Related PDB 1ijh
Related UniProtKB
[20]
Resource
Comments
Medline ID
PubMed ID 11397813
Journal J Biol Chem
Year 2001
Volume 276
Pages 30435-41
Authors Coulombe R, Yue KQ, Ghisla S, Vrielink A
Title Oxygen access to the active site of cholesterol oxidase through a narrow channel is gated by an Arg-Glu pair.
Related PDB
Related UniProtKB
[21]
Resource
Comments
Medline ID
PubMed ID 11359791
Journal J Biol Chem
Year 2001
Volume 276
Pages 18024-30
Authors Motteran L, Pilone MS, Molla G, Ghisla S, Pollegioni L
Title Cholesterol oxidase from Brevibacterium sterolicum. The relationship between covalent flavinylation and redox properties.
Related PDB
Related UniProtKB
[22]
Resource
Comments
Medline ID
PubMed ID 12051668
Journal Arch Biochem Biophys
Year 2002
Volume 402
Pages 235-42
Authors Yin Y, Liu P, Anderson RG, Sampson NS
Title Construction of a catalytically inactive cholesterol oxidase mutant: investigation of the interplay between active site-residues glutamate 361 and histidine 447.
Related PDB
Related UniProtKB
[23]
Resource
Comments
Medline ID
PubMed ID 12974654
Journal Acc Chem Res
Year 2003
Volume 36
Pages 713-22
Authors Sampson NS, Vrielink A
Title Cholesterol oxidases: a study of nature's approach to protein design.
Related PDB
Related UniProtKB
[24]
Resource
Comments X-ray crystallography
Medline ID
PubMed ID 14558826
Journal J Am Chem Soc
Year 2003
Volume 125
Pages 12787-94
Authors Lario PI, Vrielink A
Title Atomic resolution density maps reveal secondary structure dependent differences in electronic distribution.
Related PDB 1n1p
Related UniProtKB
[25]
Resource
Comments X-ray crystallography
Medline ID
PubMed ID 12595270
Journal J Mol Biol
Year 2003
Volume 326
Pages 1635-50
Authors Lario PI, Sampson N, Vrielink A
Title Sub-atomic resolution crystal structure of cholesterol oxidase: what atomic resolution crystallography reveals about enzyme mechanism and the role of the FAD cofactor in redox activity.
Related PDB 1mxt
Related UniProtKB

Comments
This enzyme catalyzes three subsequent reactions;
(A) Hydride transfer from FADH2 to O2, giving FAD and H2O2.
(B) Hydride transfer from cholesterol (or 5-cholestan-3-ol) to FAD, giving 5-chelesten-3-one and FADH2.
(C) Isomerization (or shift of double-bonde) of 5-cholesten-3-one, producing 4-cholesten-3-one (shift from C4-C5=C6 to C4=C5-C6).

Created Updated
2004-03-24 2009-03-16