Cholesterol oxidase

 

Cholesterol oxidase catalyses the oxidation and isomerisation of cholesterol to form cholest-4-en-3-one using FAD as a cofactor. The two forms of the enzyme found in Brevibacterium BCO1 and BCO2 do not show any structural or sequence homology, despite carrying out the same reaction. As a result their mechanisms are believed to be different. Study of the enzymes that degrade cholesterol is clearly of great relevance to medicine due to the role of the steroid in cardiovascular disease.

 

Reference Protein and Structure

Sequence
Q7SID9 UniProt IPR015213 (Sequence Homologues) (PDB Homologues)
Biological species
Brevibacterium sterolicum (Bacteria) Uniprot
PDB
1i19 - CRYSTAL STRUCTURE OF CHOLESTEROL OXIDASE FROM B.STEROLICUM (1.7 Å) PDBe PDBsum 1i19
Catalytic CATH Domains
3.30.43.10 CATHdb 3.40.462.10 CATHdb (see all for 1i19)
Cofactors
Fadh2(2-) (1)
Click To Show Structure

Enzyme Reaction (EC:1.1.3.-)

cholesterol
CHEBI:16113ChEBI
+
dioxygen
CHEBI:15379ChEBI
→
hydrogen peroxide
CHEBI:16240ChEBI
+
cholest-4-en-3-one
CHEBI:16175ChEBI

Enzyme Mechanism

Introduction

The enzyme is able to carry out three interrelated reactions. First, in the oxidative half reaction, hydride transfer from the C3 of the steroid to N5 of FAD, with deprotonation of the 3C OH by Glu 475 oxidises cholesterol to form cholest-3-one. The proton is transferred to Glu 311, to allow Glu 475 to abstract a proton from the 4C and add it to the 6C to isomerise the cholest-3-one to cholest-4-en-3-one. Meanwhile, in the reductive half reaction, the reduced FAD, stabilised by contacts with Arg 477, transfers a hydride to a dioxygen molecule, which is protonated by Glu 311, forming H2O2.

Catalytic Residues Roles

UniProt PDB* (1i19)
His69 His121(69)B Forms a covalent attachment to the FAD cofactor and modifies its redox potential. covalently attached, alter redox potential
Glu423 Glu475(423)B Acts as a general acid/base in the oxidative half reaction where it deprotonates the 3C OH group, and in the isomerisation step where it transfers a proton between the 4C and the 6C of the steroid ring system. proton acceptor, proton donor
Arg425 Arg477(425)B Stabilises the negative charge that develops on the flavin ring system during its reduction. electrostatic stabiliser
Glu259 Glu311(259)B Accepts a proton from Glu 475 in order to allow Glu 475 to function as a base in both the oxidation and isomerisation of cholesterol. Then transfers this proton to the oxygen molecule to allow it to be reduced to H2O2. proton acceptor, proton donor
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

proton transfer, hydride transfer, overall reactant used, overall product formed, native state of enzyme regenerated, native state of cofactor regenerated

References

  1. Coulombe R et al. (2001), J Biol Chem, 276, 30435-30441. Oxygen Access to the Active Site of Cholesterol Oxidase through a Narrow Channel Is Gated by an Arg-Glu Pair. DOI:10.1074/jbc.m104103200. PMID:11397813.
  2. Vrielink A et al. (2009), FEBS J, 276, 6826-6843. Cholesterol oxidase: biochemistry and structural features. DOI:10.1111/j.1742-4658.2009.07377.x. PMID:19843169.
  3. Lim L et al. (2006), Biochem J, 400, 13-22. Structural and kinetic analyses of the H121A mutant of cholesterol oxidase. DOI:10.1042/BJ20060664. PMID:16856877.
  4. Li J et al. (1993), Biochemistry, 32, 11507-11515. Crystal structure of cholesterol oxidase complexed with a steroid substrate: Implications for flavin adenine dinucleotide dependent alcohol oxidases. DOI:10.1021/bi00094a006. PMID:8218217.

Step 1. In the oxidative half reaction the hydroxyl group of the substrate is deprotonated by Glu475 and a hydride is transferred from C3 of the steroid to N5 of FAD. Cholest-3-one is formed.

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Catalytic Residues Roles

Residue Roles
His121(69)B covalently attached
His121(69)B alter redox potential
Arg477(425)B electrostatic stabiliser
Glu475(423)B proton acceptor

Chemical Components

proton transfer, hydride transfer, overall reactant used

Step 2. Glu311 abstracts a proton from Glu475.

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Catalytic Residues Roles

Residue Roles
Glu475(423)B proton donor
Glu311(259)B proton acceptor

Chemical Components

proton transfer

Step 3. Glu475 abstracts a proton from C4 of the oxidised intermediate.

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Catalytic Residues Roles

Residue Roles
Glu475(423)B proton acceptor

Chemical Components

proton transfer

Step 4. The proton abstracted by Glu475 is transferred to C6 to isomerise cholest-3-one to the product, cholest-4-en-3-one.

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Catalytic Residues Roles

Residue Roles
Glu475(423)B proton donor

Chemical Components

proton transfer, overall product formed

Step 5. In the reductive half reaction FADH- transfers a hydride to a dioxygen molecule which is simultaneously protonated by Glu311, forming H2O2 and restoring the active site.

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Catalytic Residues Roles

Residue Roles
His121(69)B alter redox potential
Arg477(425)B electrostatic stabiliser
His121(69)B covalently attached
Glu311(259)B proton donor

Chemical Components

hydride transfer, proton transfer, overall product formed, native state of enzyme regenerated, native state of cofactor regenerated
Download: Image, Marvin File

Step 1. In the oxidative half reaction the hydroxyl group of the substrate is deprotonated by Glu475 and a hydride is transferred from C3 of the steroid to N5 of FAD. Cholest-3-one is formed.

Step 2. Glu311 abstracts a proton from Glu475.

Step 3. Glu475 abstracts a proton from C4 of the oxidised intermediate.

Step 4. The proton abstracted by Glu475 is transferred to C6 to isomerise cholest-3-one to the product, cholest-4-en-3-one.

Step 5. In the reductive half reaction FADH- transfers a hydride to a dioxygen molecule which is simultaneously protonated by Glu311, forming H2O2 and restoring the active site.

Products.

Contributors

Peter Sarkies, Gemma L. Holliday, Amelia Brasnett