Lysine-specific [histone H3] demethylase

 

Jumonji Domain protein 2A (JMJD2A or lysine-specific demethylase 4A, KDM4A) is a 2-oxoglutarate dependent oxygenase which catalyses the hydroxylation of methyl groups present on lysine residues within histone protein 3, utilising a Fe(II) dependent redox reaction, with spontaneous deformylation to produce the demethylated lysine and formaldehyde. The enzyme catalyses the removal of methyl groups specifically from lysine residues K9 and K36. JMJD2A has been shown to be over-expressed relative to normal physiological levels in several cancers, and is of interest for its influence over epigenetic factors associated with cancer progression.

The enzyme substrate specificity is determined by amino acids within the JMJD2A active site, and also the flexibility of residues positioned around the reactive lysine in the peptide backbone. The enzyme is most active with the tri-methyl substrate. Each of the three methyl groups binds in a discrete pocket, restricting rotation around the functional group and demethylation proceeds rapidly. In the presence of two methyl groups, the functional groups can flip between a more reactive and less reactive pocket, one which binds methyl groups weakly, but closer to the reactive Fe(II), and vice versa. JMJD2A is not active towards the mono-methyl lysine substrate. The single methyl group stays bound to the less reactive pocket, and cannot be demethylated.

 

Reference Protein and Structure

Sequence
O75164 UniProt (1.14.11.66, 1.14.11.69) IPR003347 (Sequence Homologues) (PDB Homologues)
Biological species
Homo sapiens (Human) Uniprot
PDB
2ybp - JMJD2A COMPLEXED WITH R-2-HYDROXYGLUTARATE AND HISTONE H3K36me3 PEPTIDE (30-41) (2.02 Å) PDBe PDBsum 2ybp
Catalytic CATH Domains
2.60.120.650 CATHdb (see all for 2ybp)
Cofactors
Iron(3+) (1)
Click To Show Structure

Enzyme Reaction (EC:1.14.11.27)

N(6)-methyl-L-lysinium residue
CHEBI:61929ChEBI
+
2-oxoglutarate(2-)
CHEBI:16810ChEBI
+
dioxygen
CHEBI:15379ChEBI
→
carbon dioxide
CHEBI:16526ChEBI
+
formaldehyde
CHEBI:16842ChEBI
+
succinate(2-)
CHEBI:30031ChEBI
+
L-lysinium residue
CHEBI:29969ChEBI
Alternative enzyme names: JHDM1A, JmjC domain-containing histone demethylase 1A, H3-K36-specific demethylase, Histone-lysine (H3-K36) demethylase, Histone demethylase, Protein-6-N,6-N-dimethyl-L-lysine,2-oxoglutarate:oxygen oxidoreductase, [Histone-H3]-lysine-36 demethylase,

Enzyme Mechanism

Introduction

JmjC domain histone demethylases catalyse hydroxylation of the theta methyl groups, forming an unstable hemiaminal intermediate that spontaneously decomposes to product formaldehyde and the demethylated lysine, in addition to the alpha-ketoglutarate cofactor derived products, carbon dioxide and succinate.

Catalytic Residues Roles

UniProt PDB* (2ybp)
Ser288, Gly170 (main-C), Tyr177 Ser288(310)A, Gly170(192)A (main-C), Tyr177(199)A Hold the substrates in the correct conformation for the reaction to occur. hydrogen bond donor, steric role
His276, Glu190, His188 His276(298)A, Glu190(212)A, His188(210)A Forms part of the catalytic iron binding site. metal ligand
*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 homolytic addition, redox reaction, radical formation, cofactor used, overall reactant used, coordination to a metal ion, intermediate formation, bimolecular nucleophilic addition, electron transfer, radical termination, unimolecular elimination by the conjugate base, intermediate collapse, overall product formed, decoordination from a metal ion, decarboxylation, hydrogen transfer, bimolecular homolytic substitution, intermediate terminated, native state of cofactor regenerated, native state of enzyme is not regenerated, reaction occurs outside the enzyme

References

  1. Ng SS et al. (2007), Nature, 448, 87-91. Crystal structures of histone demethylase JMJD2A reveal basis for substrate specificity. DOI:10.1038/nature05971. PMID:17589501.
  2. Markolovic S et al. (2016), Curr Opin Struct Biol, 41, 62-72. Structure–function relationships of human JmjC oxygenases—demethylases versus hydroxylases. DOI:10.1016/j.sbi.2016.05.013. PMID:27309310.
  3. Williams ST et al. (2014), Epigenetics, 9, 1596-1603. Studies on the catalytic domains of multiple JmjC oxygenases using peptide substrates. DOI:10.4161/15592294.2014.983381. PMID:25625844.
  4. Chowdhury R et al. (2011), EMBO Rep, 12, 463-469. The oncometabolite 2-hydroxyglutarate inhibits histone lysine demethylases. DOI:10.1038/embor.2011.43. PMID:21460794.
  5. Heightman TD (2011), Curr Chem Genomics, 5, 62-71. Chemical Biology of Lysine Demethylases. DOI:10.2174/1875397301005010062. PMID:21966346.
  6. Zhang QJ et al. (2011), J Clin Invest, 121, 2447-2456. The histone trimethyllysine demethylase JMJD2A promotes cardiac hypertrophy in response to hypertrophic stimuli in mice. DOI:10.1172/jci46277. PMID:21555854.
  7. Couture JF et al. (2007), Nat Struct Mol Biol, 14, 689-695. Specificity and mechanism of JMJD2A, a trimethyllysine-specific histone demethylase. DOI:10.1038/nsmb1273. PMID:17589523.
  8. Tsukada Y et al. (2006), Nature, 439, 811-816. Histone demethylation by a family of JmjC domain-containing proteins. DOI:10.1038/nature04433. PMID:16362057.
  9. Chen Z et al. (2006), Cell, 125, 691-702. Structural Insights into Histone Demethylation by JMJD2 Family Members. DOI:10.1016/j.cell.2006.04.024. PMID:16677698.
  10. Hewitson KS et al. (2005), Philos Trans A Math Phys Eng Sci, 363, 807-828. Oxidation by 2-oxoglutarate oxygenases: non-haem iron systems in catalysis and signalling. DOI:10.1098/rsta.2004.1540. PMID:15901537.

Step 1. Fe(II) donates a single electron to the dioxygen molecule, forming a covalent bond between dioxygen and the resulting Fe(III).

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Catalytic Residues Roles

Residue Roles
Tyr177(199)A hydrogen bond donor, steric role
Gly170(192)A (main-C) hydrogen bond acceptor, steric role
Glu190(212)A hydrogen bond acceptor
Ser288(310)A steric role, hydrogen bond donor
His188(210)A metal ligand
His276(298)A metal ligand
Glu190(212)A metal ligand

Chemical Components

ingold: bimolecular homolytic addition, redox reaction, radical formation, cofactor used, overall reactant used, coordination to a metal ion, intermediate formation

Step 2. Fe(III) donates a single electron to the bound peroxo group. The terminal oxygen of the peroxo group attacks the carbonyl carbon of the 2-oxoglutarate cofactor in a nucleophilic addition.

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Catalytic Residues Roles

Residue Roles
Tyr177(199)A hydrogen bond donor, steric role
Gly170(192)A (main-C) hydrogen bond acceptor, steric role
Glu190(212)A steric role, attractive charge-charge interaction
Ser288(310)A steric role, hydrogen bond donor
His188(210)A metal ligand
His276(298)A metal ligand
Glu190(212)A metal ligand

Chemical Components

ingold: bimolecular nucleophilic addition, electron transfer, radical termination, intermediate formation, cofactor used

Step 3. Carbon dioxide is eliminated from the intermediate to form succinate and an iron-bound oxo anion. Succinate is thought to decoordinate from the active site prior to hydroxylation [PMID:21555854]. Stopped-flow absorption and freeze-quench Mossbauer experiments have suggested the Fe centre to hold a highly oxidising (IV) intermediate state.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Tyr177(199)A hydrogen bond donor, steric role
Gly170(192)A (main-C) hydrogen bond acceptor, steric role
Glu190(212)A attractive charge-charge interaction, steric role
Ser288(310)A hydrogen bond donor, steric role
His188(210)A metal ligand
His276(298)A metal ligand
Glu190(212)A metal ligand

Chemical Components

ingold: unimolecular elimination by the conjugate base, intermediate collapse, intermediate formation, overall product formed, decoordination from a metal ion, decarboxylation

Step 4. The iron-bound oxygen donates an electron to the Fe(IV) centre and abstracts a proton from the lysine-methyl group, forming a carbon radical centre. The active site residues are important for determining the substrate selectivity, both in terms of peptide backbone recognition and methylation state. JMJD2A is more active with tri- rather than di-methylated substrate, and inactive in the presence of mono-methylated lysine peptides [PMID:21966346, PMID:17589523].

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Tyr177(199)A hydrogen bond donor, steric role
Gly170(192)A (main-C) hydrogen bond acceptor, steric role
Glu190(212)A attractive charge-charge interaction, steric role
Ser288(310)A hydrogen bond donor, steric role
His188(210)A metal ligand
His276(298)A metal ligand
Glu190(212)A metal ligand

Chemical Components

redox reaction, hydrogen transfer, radical formation, overall reactant used, intermediate formation

Step 5. The carbon radical initiates a homolytic substitution which forms the 1-hydroxy-2-amino-lysine product and results in a single electron transfer to the Fe(III) centre.

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Catalytic Residues Roles

Residue Roles
Tyr177(199)A hydrogen bond donor, steric role
Gly170(192)A (main-C) hydrogen bond acceptor, steric role
Glu190(212)A attractive charge-charge interaction, steric role
Ser288(310)A hydrogen bond donor, steric role
His188(210)A metal ligand
His276(298)A metal ligand
Glu190(212)A metal ligand

Chemical Components

radical termination, ingold: bimolecular homolytic substitution, decoordination from a metal ion, intermediate terminated, overall product formed, native state of cofactor regenerated, native state of enzyme is not regenerated

Step 6. The hydroxy-methyl lysine decomposes outside of the active site to produce formaldehyde and the demethylated histone lysine residue.

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Catalytic Residues Roles

Residue Roles

Chemical Components

reaction occurs outside the enzyme
Download: Image, Marvin File

Step 1. Fe(II) donates a single electron to the dioxygen molecule, forming a covalent bond between dioxygen and the resulting Fe(III).

Step 2. Fe(III) donates a single electron to the bound peroxo group. The terminal oxygen of the peroxo group attacks the carbonyl carbon of the 2-oxoglutarate cofactor in a nucleophilic addition.

Step 3. Carbon dioxide is eliminated from the intermediate to form succinate and an iron-bound oxo anion. Succinate is thought to decoordinate from the active site prior to hydroxylation [PMID:21555854]. Stopped-flow absorption and freeze-quench Mossbauer experiments have suggested the Fe centre to hold a highly oxidising (IV) intermediate state.

Step 4. The iron-bound oxygen donates an electron to the Fe(IV) centre and abstracts a proton from the lysine-methyl group, forming a carbon radical centre. The active site residues are important for determining the substrate selectivity, both in terms of peptide backbone recognition and methylation state. JMJD2A is more active with tri- rather than di-methylated substrate, and inactive in the presence of mono-methylated lysine peptides [PMID:21966346, PMID:17589523].

Step 5. The carbon radical initiates a homolytic substitution which forms the 1-hydroxy-2-amino-lysine product and results in a single electron transfer to the Fe(III) centre.

Step 6. The hydroxy-methyl lysine decomposes outside of the active site to produce formaldehyde and the demethylated histone lysine residue.

Products.

Contributors

Sophie T. Williams, Gemma L. Holliday