Last Updated: December 9, 2023
Introduction to the 2-Oxoglutarate and Fe2+ (FeII)-Dependent Dioxygenases
The 2-oxoglutarate and Fe2+-dependent dioxygenases (often abbreviated 2OG-oxygenases or 2-OGDD) are a large family of enzymes that catalyze a broad range of hydroxylation and demethylation (via hydroxylation) reactions in humans. These enzymes are so-called because of their requirement for the TCA cycle intermediate, 2-oxoglutarate (commonly called α-ketoglutarate). In addition to the requirement for 2-oxoglutarate and Fe2+, several members of the family also require ascorbate as a co-factor. The critical role of ascorbate in the function of 2OG-oxygenases is the reduction of ferric iron (Fe3+) back to the ferrous state (Fe2+).
The 2-OGDD carry out their hydroxylation reactions by incorporating a single oxygen atom from molecular oxygen (O2) into their substrate. The incorporation of the oxygen atom, to generate the hydroxylated substrate, results in the simultaneous oxidation of 2-oxoglutarate to succinate and CO2. The second oxygen atom, of the molecular oxygen utilized by these enzymes, is incorporated into 2-oxoglutarate during the generation of succinate.
The 2-OGDD that catalyze demethylation reactions do so by generating a product that is an unstable hydroxy-methyl group. The hydroxy-methyl group will then spontaneously fragment producing formaldehyde and the demethylated substrate.
Reactions that are catalyzed by the 2-OGDD in humans include hydroxylation and demethylation of DNA, RNA, proteins, lipids, and many other metabolic intermediates. Humans express at least 80 genes that encode members of the 2-OGDD family with around 45 of the enzymes being hydroxylases/demethylases that modify several members of the histone protein family and many other non-histone proteins. The members of the 2-OGDD family that hydroxylate proteins do so at Pro, Lys, Asn, Arg, Asp, and His residues. Given their wide ranging involvement in biological processes it is not surprising that dysregulation of 2-OGDD enzymes has been found to be associated with a variety of different human diseases that includes cancers, cardiac and pulmonary diseases, and neurological disorders.
Some of the earliest studies of the role of 2-OGDD enzymes in hydroxylation reactions in humans were those that identified their involvement in the processing of collagens. These enzymes were found to be required for the hydroxylation of proline and lysine residues in the various collagen proteins. Clinical significance was recognized when it was determined that defective lysine hydroxylation, due to mutations in the gene (PLOD1) encoding procollagen-lysine 2-oxoglutarate 5-dioxygenase 1, were the cause of one of the many forms of the connective tissue disorder collectively called Ehlers–Danlos syndrome (EDS). The specific form of EDS that results from mutations in the PLOD1 gene is called kyphoscoliosis EDS which was formerly identified as type VIA Ehlers-Danlos syndrome.
Structure-Function Relationships of 2-OGDD
The 2-OGDD family enzymes possess a catalytic domain that is made up from eight antiparallel β strands forming a distorted double-stranded β helix (DSBH). The DSBH fold is also referred to as cupin, derived from the Latin word cupa meaning small barrel. The DSBH orients key amino acids allowing for coordinating cofactor(s) and for substrate binding. The critical Fe2+ is coordinated by a conserved motif containing two histidine (H) residues and an aspartic acid (D) or glutamic acid (E) residue found in the consensus: HxD/E…H. The “x” indicates that any amino acid can occupy this position. The spacing between the two His residues can be variable and thus the inclusion of the three “…”. The Fe2+ is required for binding both O2 and 2-oxoglutarate.
Several members of the 2-OGDD family possess a DSBH domain that has homology to a portion of a protein originally identified as Jumonji. Mutations in the Jumonji gene resulted in an abnormal morphology of the neural plates such that they looked like a cross and “jumonji” mean cruciform in Japanese. The jumonji protein was shown to have a domain at the N-terminus and another at the C-terminus that were similar to domains in numerous other proteins, e.g. several transcription factors. These domains were thus called the JmjN and JmjC domains. The JmjC domain is found in 2-OGDD that catalyze lysine demthylation reactions. The largest subfamily of JmjC hydroxylases catalyze lysyl demethylation of histones. Histone demethylation plays a significant role in the process of epigenetic control of gene expression
In addition to the DSBH domain found in all 2-OGDD, most of the enzymes in this large family also contain additional non-catalytic domains involved in substrate recognition as well as subcellular localization. For example, the DNA demethylases contain DNA-binding domains of the zinc finger family. The JmjC domain-containing histone demethylases contain methylated histone binding domains, Tudor domains, and ARID domains. The Tudor domain was originally identified in the Tudor protein which is a Drosophila protein involved in early embryonic development. Mutations in the gene encoding Tudor are lethal and the gene was therefore, given the name for the Tudor King Henry VIII and the several miscarriages experienced by his wives. The ARID domain (A–T Rich Interaction Domain) is a DNA-binding domain similar to the helix–turn–helix motif.
Processes Involving Human 2-OGDD
Collagen Proline and Lysine Hydroxylation
Collagens are the most abundant proteins in humans. The various collagens constitute the major proteins comprising the extracellular matrix (ECM). Collagens are synthesized as preproproteins and undergo extensive co- and post-translational processing that includes amino acid hydroxylation and carbohydrate additions. Specific proline residues are hydroxylated by prolyl 3-hydroxylases and prolyl 4-hydroxylases. Specific lysine residues also are hydroxylated at the C-5 position by lysyl hydroxylases. Both prolyl and lysyl hydroxylases are absolutely dependent upon ascorbic acid (vitamin C) as co-factor. The prolyl 3- and prolyl 4-hydroxylases, and the lysyl 5-hydroxylases all belong to the large family of 2-OGDD.
Humans express three distinct prolyl 3-hydroxylase genes (P3H1, P3H2, and P3H3). The original nomenclature for the prolyl 3-hydroxylases was leucine- and proline-enriched proteoglycan 1 (leprecan, LEPRE1) such that P3H1 was LEPRE1, P3H2 was LEPREL1 (leprecan-like 1), and P3H3 was LEPREL2 (leprecan-like 2). Humans contain a fourth gene, P3H4, in the prolyl 3-hydroxylase gene family but the encoded protein is not active as a prolyl 3-hydroxylase.
The P3H1 encoded protein functions in a trimeric complex with the CRTAP (cartilage-associated protein; also known as leprecan-like 3) encoded protein and the protein encoded by the peptidylprolyl isomerase B (PPIB) gene. Mutations in any one of these three genes results in various forms of osteogenesis imperfecta.
Human prolyl 4-hydroxylases are functional as heterotetrameric enzymes composed of two α-subunits (the catalytic subunits) and two β-subunits. Humans express four distinct prolyl 4-hydroxylase α-subunit genes (P4HA1, P4HA2, P4HA3, and P4HTM) and one β-subunit gene (P4HB).
Humans express three distinct lysyl hydroxylase genes identified as PLOD1, PLOD2, and PLOD3. PLOD stands for procollagen-lysine, 2-oxoglutarate 5-dioxygenase.
Carnitine Synthesis
Lysine is the precursor for the synthesis of carnitine (γ-trimethyl-hydroxybutyrobetaine). Carnitine is required for the transport of long-chain fatty acids into the mitochondria for β-oxidation.
Free lysine does not serve as the precursor for the synthesis of carnitine, rather the modified lysine (6-N-trimethyllysine), found in certain proteins, is the precursor. The conversion of trimethyllysine to carnitine occurs in a series of four reactions where the first and final reactions are catalyzed by enzymes that are members of the 2-OGDD family. The first enzyme in this pathway is trimethyllysine hydroxylase, epsilon (also called trimethyllysine dioxygenase) which is encoded by the TMLEH gene. The final reaction of the pathway is catalyzed by γ-butyrobetaine dioxygenase (also called γ-butyrobetaine 2-oxoglutarate dioxygenase and also called γ-butyrobetaine hydroxylase) which is encoded by the BBOX1 gene.
Aspartate Hydroxylation
Aspartate beta hydroxylase (ASPH) is a type II transmembrane protein that hydroxylates Asp and Asn residues in epidermal growth factor (EGF)-like domains of various proteins, including Notch receptors and ligands, and several proteins of the coagulation cascades including factors VII, IX, and X, and protein C.
Expression of the ASPH gene occurs predominately during embryogenesis to promote cell migration for organ development. In adult tissues ASPH expression is very low. Expression of the ASPH gene is controlled by two promoters and following transcription there is extensive alternative splicing that results in the encoding of four distinct protein isoforms. These proteins are ASPH, junctin (a structural protein of the sarcoplasmic reticulum), humbug (ASPH-type junctate), and junctin-type junctate. Only ASPH contains the C-terminal catalytic domain that carries out the Asp and Asn hydroxylations.
Overexpression of ASPH has been observed in a number of malignancies, including hepatocellular carcinoma, prostate cancer, cholangiocarcinoma, lung, breast, and colon cancer, and neoplasms of the nervous system. In hepatocellular carcinoma patients there is a significant association between ASPH overexpression and higher recurrence and lower survival rate following surgery.
Hypoxia-Induced Factor Proline Hydroxylation
The hypoxia-induced factor (HIF) pathway, which is activated by conditions of hypoxia (low oxygen tension), is a major homeostatic mechanism for cellular responses to changes in the level of oxygen within cells. The HIF complexes are heterodimeric complexes composed of an α-subunit and a β-subunit. The β-subunit is constitutively expressed while the α-subunit expression and activity are increased in response to changes in cellular oxygen content. There are three related HIF complexes identified as HIF-1, HIF-2, and HIF-3 that are defined by the particular α-subunit of the complex.
Humans express three α-subunit genes, HIF1α (HIF1A gene), HIF2α (EPAS1 gene, for endothelial PAS domain protein 1), and HIF3α (HIF3A gene). The PAS domain is so-called because of the three proteins in which the domain was originally identified: Per (period circadian protein), ARNT (aryl hydrocarbon receptor nuclear translocator), and Sim (simple-minded protein).
The HIF α-subunits possess an oxygen-dependent degradation (ODD) domain. The ODD domain is hydroxylated by a member of the prolyl hydroxylase domain (PHD) family of proline hydroxylating enzymes. The PHD family includes the prolyl hydroxylases described above that incorporate hydroxyl groups into proline residues in collagens. The prolyl hydroxylases that hydroxylate HIF α-subunits are all 2-OGDD. There are three genes that encode the PHD enzymes that hydroxylate the HIF α-subunit proteins. These genes are identified as EGLN2 (encoding PHD1), EGLN1 (encoding PHD2), and EGLN3 (encoding PHD3). The designation of EGLN refers to the fact that these three genes are homologs of the Caenorhabditis elegans egg laying-9 (Egl-9) gene.
In addition to the hydroxylation of HIF α-subunits, the PHD3 enzyme also hydroxylates the cardiac isoform of acetyl-CoA carboxylase 2 (ACC2).
Hypoxia-Induced Factor Asparagine Hydroxylation
The activity of HIF1α is also regulated via the hydroxylation of a specific asparagine residue (N803) found in the C-terminal transactivation domain. The N803 hydroxylation is catalyzed by the 2-OGDD originally identified as factor-inhibiting HIF-1 (FIH1; also identified as FIH). FIH1 is encoded by the HIF1AN (hypoxia inducible factor 1 alpha subunit inhibitor) gene. The consequences of the β-hydroxylation of N803 are that HIF1α can no longer interact with the transcriptional co-activators CBP [cAMP-response element-binding protein (CREB)-binding protein] and p300 resulting in inhibition of HIF-1 activity. CBP and p300 are closely related proteins that constitute the p300-CBP family of histone acetyltransferases.
Ribosomal Protein Proline Hydroxylation
Several members of the 2-OGDD family catalyze proline hydroxylation of ribosomal proteins. The first enzyme characterized to carry out ribosomal protein hydroxylation is encoded by the OGFOD1 (2-oxoglutarate and Fe2+-dependent oxygenase domain-containing protein 1) gene. The OGFOD1 encoded enzyme hydroxylates Pro-62 in the small ribosomal subunit protein, RPS23. OGFOD1 plays a critical role in the regulation of protein translation and cellular growth as evidenced by the fact that reduced, or absent, expression of the OGFOD1 gene is associated with a marked reduction in growth and the onset of a phenotype associated with translational stress. There are two additional genes related to OGFOD1 identified as OGFOD2 and OGFOD3 whose functions have yet to be fully characterized.
Ribosomal Protein Histidine Hydroxylation
Two well characterized 2-OGDD that catalyze ribosomal protein histidine hydroxylation are encoded by the RIOX1 and RIOX2 genes. RIOX1 was originally identified as NO66 for nucleolar protein 66. RIOX2 was originally identified as MINA53 for myc-induced nuclear antigen 53. Both of these enzymes belong to the JmjC domain-containing subfamily of 2-OGDD. However, unlike most other JmjC domain-containing 2OGDD, the RIOX1 and RIOX2 encoded enzymes do not possess any other obvious functional domains. Both RIOX1 and RIOX2 hydroxylate the C-3 carbon of His residues in ribosomal proteins. RIOX1 hydroxylates His-216 of the large ribosomal subunit protein, RPL8. RIOX2 hydroxylates His-39 of the large ribosomal protein, RPL27A.
Protein Lysine Demethylases
Lysine demethylases (KDM) represent a large family of enzymes that demethylate (via hydroxylation) the various states of lysine methylation (mono-, di-, and trimethylation). Many of the KDM enzymes play a role in the regulation of epigenetics via their demethylation of histone proteins. A large subfamily of KDM enzymes belong to the 2-OGDD family of enzymes, specifically the JmjC domain-containing demethylases. The JmjC domain-containing demethylases can demethylate all states of lysine methylation. There are at least 33 human genes that encode JmjC domain-containing proteins and these 33 proteins can be subdivided into 8 subfamilies. Of these 33 genes encoding JmjC domain-containing proteins, 24 are 2-OGDD enzymes.
The 24 JmjC domain-containing 2-OGDD family members are KDM1A, KDM1B, KDM2A, KDM2B, KDM3A, KDM3B, KDM4A, KDM4B, KDM4C, KDM4D, KDM4E, KDM4F, KDM5A, KDM5B, KDM5C, KDM5D, KDM6A, KDK6B, KDM7A, KDM8, JMJD1C, UTY, PHF2, and PHF8. Many KDM enzymes are also identified by the JMJD designation. For example, the KDM3A gene has also been identified as JMJD1A, KDM3B is also known as JMJD1B, KDM4A is also known as JMJD2A, KDM4B is also known as JMJD2B, KDM4C is also known as JMJD2C, KDM4D is also known as JMJD2D, KDM6B is also known as JMJD3, and KDM8 is also known as JMJD5
Some members of the JmjC domain-containing demethylases are identified as JmjC domain-containing histone demethylases (JHDM) and are also identified as the JMJD family. Several JMJD enzymes can also reverse all three known states of lysine methylation in histones as well as many other proteins. Examples include JMJD4, JMJD6, JMJD7.
The JMJD4 enzyme demethylates eukaryotic peptide chain release factor subunit 1 (ETF1: eukaryotic translation termination factor 1). ETF1 directs the termination of nascent protein synthesis.
The JMJD6 enzyme catalyzes the demethylation of not only histones but proteins of the mRNA splicing machinery, p53, Hsp70, and estrogen receptor alpha (ERα). The JMJD6 enzyme is , thus far, the only demethylase involved arginine demethylation
The JMJD7 enzyme demethylates developmentally regulated GTP binding protein 1 (DRG1) a protein involved in the regulation of microtubule polymerization and stability. The actions of DRG1 are required for mitotic spindle assembly during the prophase to anaphase transition of the cell cycle.
Nucleic Acid Demethylation via Hydroxylation
The E. coli AlkB protein was identified as a 2-OGDD responsible for the repair of alkylated DNA. Several AlkB homologues (ALKBH) have been identified in the human genome and identified as ALKBH1-9 (where ALKBH9 is identified as FTO: fat mass and obesity-associated). Like the E. coli AlkB enzyme, the human ALKBH genes encode enzymes that are members of 2-OGDD family.
Several of the ALKBH encoded enzymes function in the demethylation of DNA, whereas the FTO and ALKBH5 enzymes have specificity for 6-methyladenosine (6mA) residues in RNA. ALKBH2 and ALKBH3 function to demethylate N1-methyladenosine (1mA) and N3-methylcytosine (3mC) residues in DNA. The ALKBH2 protein is the primary enzyme responsible for demethylation repair of alkylated DNA. The ALKBH3 enzyme is also involved the dealkylation of RNA.
The ALKBH4 gene encodes a lysine demethylase. The function of ALKBH4 is to demethylate monomethylated Lys-84 (K84me1) in actin. This effect results in the regulation of actomyosin-processes.
The ALKBH8 gene encodes a tRNA methyltransferase as outlined in the next section.
tRNA Oxidation
Two members of the 2-OGDD family are involved in the methylation or oxidation of tRNAs.
The ALKBH8 encoded enzyme catalyzes a complex series of reactions involving methylation and hydroxylation to generate 5S-methoxycarbonylhydroxymethyluridine (mchm5U) in the anticodon loop of tRNAs. The enzyme first converts 5-carboxymethyluridine (cm5U) to 5-methoxycarbonylmethyluridine (mcm5U) via methylation and then the hydroxylase domain converts mcm5U to mchm5U. The preferred substrates for ALKBH8 are Arg and Glu tRNAs.
The protein encoded by the TYW5 [tRNA-wybutosine(yW) synthesizing enzyme 5] gene is a member of the large JMJD family of 2-OGDD enzymes. Wybutosine and hydroxywybutosine are hypermodified guanosines found at position 37 of the phenylalanine tRNA (tRNAPhe) in yeast and mammals, respectively. The precise role of this modification of the tRNAPhe is uncertain but may promote translation fidelity via the prevention of ribosome slippage and frameshifting.
DNA Cytosine Demethylation
Given that chromatin structure, and consequently transcriptional activity, can be modified by the addition of a methyl group to cytidine residues, it is not surprising that there are activities in the cell that are responsible for the removal of the methyl group.
Genes encoding human cytosine demethylases were found to be related to a gene that was originally identified in rare cases of acute myeloid and lymphocytic leukemia (MLL). This form of leukemia results from a translocation between chromosome 10 and chromosome 11. The translocation results in the fusion of the mixed-lineage leukemia 1 (MLL1) gene on chromosome 10 and a gene on chromosome 11 that was subsequently given the name TET (ten eleven translocation).
The MLL1 protein is a lysine methyltransferase (KMT) family enzyme encoded by the KMT2A gene. Three TET genes are expressed in humans identified as TET1, TET2, and TET3. The official name for these genes is tet methylcytosine dioxygenase 1, 2, and 3. The TET gene encoded enzymes are distantly related to the human ALKBH genes. All three TET genes encode proteins that are zinc-finger domain-containing 2-OGDD. Each of the three TET enzymes successively oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) within DNA, ultimately resulting in umethylated cytosine.
Lipid Metabolizing 2-OGDD
The peroxisomal metabolism of phytanic acid, derived from the chlorophyll component, phytol, involves the pathway referred to as α-oxidation. One of the enzymes in this pathway, phytanoyl-CoA hydroxylase (PhyH) is a member of the 2-OGDD family. Mutations in the PHYH gene are the causes of Refsum disease.