Homogentisate 1,2-dioxygenase

 

Homogentistate dioxygenase (HGO) catalyses the metabolic degredation of Phe and Tyr amino acids. The ring opening reaction requires non-heme Fe(2+) to incorporate both atoms of molecular oxygen into homogentisate. The accumulation of this substrate, when insufficient levels of HGO are present, results in the deposition of of insoluble ochromotic pigments in conective tissues, leading to degenerative arthritis.

 

Reference Protein and Structure

Sequence
Q93099 UniProt (1.13.11.5) IPR005708 (Sequence Homologues) (PDB Homologues)
Biological species
Homo sapiens (Human) Uniprot
PDB
1ey2 - HUMAN HOMOGENTISATE DIOXYGENASE WITH FE(II) (2.3 Å) PDBe PDBsum 1ey2
Catalytic CATH Domains
2.60.120.10 CATHdb (see all for 1ey2)
Cofactors
Iron(2+) (1)
Click To Show Structure

Enzyme Reaction (EC:1.13.11.5)

dioxygen
CHEBI:15379ChEBI
+
homogentisate
CHEBI:16169ChEBI
→
hydron
CHEBI:15378ChEBI
+
4-maleylacetoacetate
CHEBI:17105ChEBI
Alternative enzyme names: Homogentisate dioxygenase, Homogentisate oxidase, Homogentisate oxygenase, Homogentisic acid oxidase, Homogentisic acid oxygenase, Homogentisic oxygenase, Homogentisicase, Homogentisate:oxygen 1,2-oxidoreductase (decyclizing),

Enzyme Mechanism

Introduction

Direct bidentate coordination occurs between Fe(2+) and the homogentisate 1-acetate and 2-hydroxylate groups, while His 292 hydrogen bonds to to the 5-hydroxyl group. The essential metal ion is coordinated to His 371, His 335 and Glu 341. Molecular dioxygen binds to iron and reacts with homogentisate, forming a peroxo-bridged intermediate. The second step is a homolytic cleave of the O-O bond in this intermediate, which generates an arene oxide radical. Lastly, nucleophilic attack of the iron bound OH group at the carbonyl of the arene oxide radical, opening the epoxide ring. In the mechanism proposed above, His 292 is expected to remain neutral, limiting its role to substrate binding. However, a second mechanism, higher in energy, proposed by Borowski et al. implicates His 292 as a general acid/base in a proton assisted Criegee Rearrangement for HGO.

Catalytic Residues Roles

UniProt PDB* (1ey2)
His292 His292(318)A Helps activate and stabilise the reactive intermediates and transition states formed during the course of the reaction. enhance reactivity, electrostatic stabiliser
His335, Glu341, His371 His335(361)A, Glu341(367)A, His371(397)A Forms part of the catalytic iron binding site. metal ligand
His365 His365(391)A Acts as a general acid/base during the course of the reaction. 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

bimolecular nucleophilic addition, overall reactant used, proton transfer, electron transfer, homolysis, intramolecular homolytic addition, intramolecular homolytic elimination, native state of cofactor regenerated, overall product formed, inferred reaction step, native state of enzyme regenerated

References

  1. Borowski T et al. (2005), J Am Chem Soc, 127, 17303-17314. Catalytic Reaction Mechanism of Homogentisate Dioxygenase:  A Hybrid DFT Study. DOI:10.1021/ja054433j. PMID:16332080.
  2. Buongiorno D et al. (2013), Coord Chem Rev, 257, 541-563. Structure and function of atypically coordinated enzymatic mononuclear non-heme-Fe(II) centers. DOI:10.1016/j.ccr.2012.04.028. PMID:24850951.
  3. Titus GP et al. (2000), Nat Struct Biol, 7, 542-546. Crystal structure of human homogentisate dioxygenase. DOI:10.1038/76756. PMID:10876237.

Step 1. The iron-bound dioxygen attacks the aromatic ring at the C2 carbon

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Catalytic Residues Roles

Residue Roles
Glu341(367)A metal ligand
His371(397)A metal ligand
His335(361)A metal ligand
His292(318)A enhance reactivity

Chemical Components

ingold: bimolecular nucleophilic addition, overall reactant used

Step 2. The peroxo group abstracts a proton from His365.

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Catalytic Residues Roles

Residue Roles
Glu341(367)A metal ligand
His371(397)A metal ligand
His335(361)A metal ligand
His292(318)A enhance reactivity
His365(391)A proton donor

Chemical Components

proton transfer

Step 3. A single electron from the iron(II) ion is donated to the peroxo group, which initiates the homolytic cleavage of the peroxo bond.

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Catalytic Residues Roles

Residue Roles
Glu341(367)A metal ligand
His371(397)A metal ligand
His335(361)A metal ligand
His292(318)A enhance reactivity

Chemical Components

electron transfer, homolysis

Step 4. An epoxide ring is formed via homolytic rearrangement.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His292(318)A enhance reactivity
Glu341(367)A metal ligand
His371(397)A metal ligand
His335(361)A metal ligand

Chemical Components

ingold: intramolecular homolytic addition

Step 5. The hydroxide group attacks the carbonyl group bound to the iron(III) ion to form the new carboxylic acid group.

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Catalytic Residues Roles

Residue Roles
Glu341(367)A metal ligand
His371(397)A metal ligand
His335(361)A metal ligand
His292(318)A enhance reactivity

Chemical Components

ingold: bimolecular nucleophilic addition

Step 6. The epoxide group collapses.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu341(367)A metal ligand
His371(397)A metal ligand
His335(361)A metal ligand
His292(318)A enhance reactivity

Chemical Components

ingold: intramolecular homolytic elimination

Step 7. The hydroxyl radical initiates a homolytic rearrangement that results in the formation of the final product and a single electron transfer to the Fe(III) centre to regenerate the cofactor.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu341(367)A metal ligand
His371(397)A metal ligand
His335(361)A metal ligand
His292(318)A enhance reactivity

Chemical Components

ingold: intramolecular homolytic elimination, native state of cofactor regenerated, overall product formed

Step 8. Inferred step in which His365 abstracts a proton from the product to regenerate the active site.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu341(367)A metal ligand
His371(397)A metal ligand
His335(361)A metal ligand
His292(318)A electrostatic stabiliser
His365(391)A proton acceptor

Chemical Components

inferred reaction step, proton transfer, native state of enzyme regenerated
Download: Image, Marvin File

Step 1. The iron-bound dioxygen attacks the aromatic ring at the C2 carbon

Step 2. The peroxo group abstracts a proton from His365.

Step 3. A single electron from the iron(II) ion is donated to the peroxo group, which initiates the homolytic cleavage of the peroxo bond.

Step 4. An epoxide ring is formed via homolytic rearrangement.

Step 5. The hydroxide group attacks the carbonyl group bound to the iron(III) ion to form the new carboxylic acid group.

Step 6. The epoxide group collapses.

Step 7. The hydroxyl radical initiates a homolytic rearrangement that results in the formation of the final product and a single electron transfer to the Fe(III) centre to regenerate the cofactor.

Step 8. Inferred step in which His365 abstracts a proton from the product to regenerate the active site.

Products.

Contributors

James W. Murray, Craig Porter, Gemma L. Holliday