Cholesterol oxidase (GMC type)

 

Cholesterol oxidase, a monomeric flavoenzyme unique to bacteria, catalyses the oxidation and isomerisation of cholesterol to cholest-4-en-3-one. This is the first step in cholesterol catabolism used by a wide range of soil bacteria which can use cholesterol as sole carbon source. Two forms of the cholesterol oxidase are known which share no sequence homology. One contains a FAD cofactor covalently linked through a histidine residue (PDB:1i19) while the other, represented in this entry, contains a non-covalently bound FAD cofactor. This enzyme is part of a wider family the Cholesterol-Methane-Glucose oxidoreductases or GMC oxidoreductases.

The enzyme may be used as a potent insecticidal agent, active against boll weevil larvae and other insects. The enzyme acts by lysing the cells of the mid-gut epithelium resulting in larval death.

 

Reference Protein and Structure

Sequence
P22637 UniProt (1.1.3.6, 5.3.3.1) IPR000172 (Sequence Homologues) (PDB Homologues)
Biological species
Brevibacterium sterolicum (Bacteria) Uniprot
PDB
1coy - CRYSTAL STRUCTURE OF CHOLESTEROL OXIDASE COMPLEXED WITH A STEROID SUBSTRATE. IMPLICATIONS FOR FAD DEPENDENT ALCOHOL OXIDASES (1.8 Å) PDBe PDBsum 1coy
Catalytic CATH Domains
3.50.50.60 CATHdb 3.30.410.10 CATHdb (see all for 1coy)
Cofactors
Fadh2(2-) (1)
Click To Show Structure

Enzyme Reaction (EC:1.1.3.6)

cholesterol
CHEBI:16113ChEBI
+
dioxygen
CHEBI:15379ChEBI
→
cholest-5-en-3-one
CHEBI:63906ChEBI
+
hydrogen peroxide
CHEBI:16240ChEBI
Alternative enzyme names: 3-beta-hydroxysteroid:oxygen oxidoreductase, 3-beta-hydroxy steroid oxidoreductase, Cholesterol-O(2) oxidoreductase,

Enzyme Mechanism

Introduction

The reductive-half reaction is initiated by the abstraction of the C3-OH proton by Glu361, and concomitant hydride transfer to the N5 of the non-covalently bound FAD cofactor. The oxidative-half reaction is thought to occur before the isomersation step, regenerating the FAD cofactor and deprotonating Glu361 by the reduction of molecular oxygen to hydrogen peroxide. The same glutamate residue acts as the general base in the isomerisation step, abstracting an alpha-keto proton which initiates double bond rearrangement between C4, C5 and C6.

Catalytic Residues Roles

UniProt PDB* (1coy)
His492 His447A Acts as a general acid/base. Also thought to contribute towards hydride transfer by stabilising the partial anion character of the oxygen atom. proton acceptor, hydrogen bond donor, electrostatic stabiliser, proton donor
Glu406 Glu361A Acts as a general acid/base. Responsible for the initial abstraction of a proton from the hydroxyl group of cholesterol. proton acceptor, proton donor
Asn530 Asn485A Helps activate both the Glu and His general acid/bases. increase basicity, activator, electrostatic stabiliser, increase acidity
*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, intermediate formation, cofactor used, overall reactant used, inferred reaction step, assisted keto-enol tautomerisation, native state of cofactor regenerated, overall product formed

References

  1. Vrielink A (2010), Subcell Biochem, 51, 137-158. Cholesterol Oxidase: Structure and Function. DOI:10.1007/978-90-481-8622-8_5. PMID:20213543.
  2. Harb LH et al. (2017), Proteins, 85, 1645-1655. Computational site-directed mutagenesis studies of the role of the hydrophobic triad on substrate binding in cholesterol oxidase. DOI:10.1002/prot.25319. PMID:28508424.
  3. 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.
  4. Yue QK et al. (1999), Biochemistry, 38, 4277-4286. Crystal Structure Determination of Cholesterol Oxidase fromStreptomycesand Structural Characterization of Key Active Site Mutants†,‡. DOI:10.1021/bi982497j. PMID:10194345.
  5. Kass IJ et al. (1998), Biochemistry, 37, 17990-18000. Evaluation of the role of His447 in the reaction catalyzed by cholesterol oxidase. DOI:10.1021/bi982115+. PMID:9922167.
  6. 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.
  7. Cavener DR (1992), J Mol Biol, 223, 811-814. GMC oxidoreductases. A newly defined family of homologous proteins with diverse catalytic activities. PMID:1542121.
  8. Vrielink A et al. (1991), J Mol Biol, 219, 533-554. Crystal structure of cholesterol oxidase from Brevibacterium sterolicum refined at 1.8 Å resolution. DOI:10.1016/0022-2836(91)90192-9. PMID:2051487.

Step 1. Glu361 abstracts the C3-OH proton, initiating hydride transfer from the cholesterol to N5 of the flavin cofactor, forming cholester-6-en-5-one.

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Catalytic Residues Roles

Residue Roles
His447A hydrogen bond donor, electrostatic stabiliser
Asn485A electrostatic stabiliser, increase basicity
Glu361A proton acceptor

Chemical Components

proton transfer, hydride transfer, intermediate formation, cofactor used, overall reactant used

Step 2. Glu361acts as a general acid towards the reduced FAD cofactor.

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Catalytic Residues Roles

Residue Roles
His447A hydrogen bond donor
His447A electrostatic stabiliser
Asn485A electrostatic stabiliser, increase acidity
Glu361A proton donor

Chemical Components

proton transfer, cofactor used, inferred reaction step

Step 3. The anionic Glu361 acts as a general base, abstracting the alpha-keto proton to form an enolate, stabilised through hydrogen bonding with His447.

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Catalytic Residues Roles

Residue Roles
His447A hydrogen bond donor, electrostatic stabiliser
Asn485A activator, electrostatic stabiliser
His447A proton donor
Glu361A proton acceptor

Chemical Components

proton transfer, assisted keto-enol tautomerisation

Step 4. The enolate intermediate collapses, initiating a conjugate double bond rearrangement, and formation of the isomeric product.

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Catalytic Residues Roles

Residue Roles
His447A hydrogen bond donor, electrostatic stabiliser
Asn485A activator, electrostatic stabiliser
Glu361A proton donor
His447A proton acceptor

Chemical Components

proton transfer, assisted keto-enol tautomerisation

Step 5. The oxidative half reaction reoxidizes the enzyme-bound flavin cofactor by the reduction of molecular oxygen to hydrogen peroxide.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His447A hydrogen bond donor, electrostatic stabiliser
Asn485A electrostatic stabiliser

Chemical Components

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

Step 1. Glu361 abstracts the C3-OH proton, initiating hydride transfer from the cholesterol to N5 of the flavin cofactor, forming cholester-6-en-5-one.

Step 2. Glu361acts as a general acid towards the reduced FAD cofactor.

Step 3. The anionic Glu361 acts as a general base, abstracting the alpha-keto proton to form an enolate, stabilised through hydrogen bonding with His447.

Step 4. The enolate intermediate collapses, initiating a conjugate double bond rearrangement, and formation of the isomeric product.

Step 5. The oxidative half reaction reoxidizes the enzyme-bound flavin cofactor by the reduction of molecular oxygen to hydrogen peroxide.

Products.

Contributors

Sophie T. Williams, Nozomi Nagano, Craig Porter, Gemma L. Holliday