Alpha-ketoglutarate-dependent dioxygenase alkB homolog 2
By: Michael Plietz
Alpha-ketoglutarate-dependent dioxygenase alkB homolog 2 (ALKBH2) is an oxidoreductase that operates on DNA. Oxidoreductases are a class of enzyme that catalyzes the transfer of electrons from a reductant to an oxidant. It has a molecular weight of 32,462.29 amu. ALKBH2’s structure (PDB ID: 3RZG) was initially found in homo sapiens. It performs direct DNA repair on N(1)-methyladenine, N(3)-methylcytosine and 1,N(6)-ethenoadenine damage present in DNA bases. 1
ALKBH2 contains 209 amino acids, in one chain. Its secondary structure is made up of 18% alpha helices. There are five helices present from 39 residues. It also contains 38% beta sheets, which make up 18 separate strands from 81 residues.
ALKBH2 has multiple important functional domains. Two Histidine residues and one Aspartic Acid residue perform its primary function (His171, His236 and Asp173). 5 All three bind an Iron (II) molecule, which will already has two bonds formed with alpha-ketoglutarate. This allows the Iron molecule to be in the correct state to bind oxygen and oxidize the unwanted methyl groups on DNA bases. This process is demonstrated in Figure 2.
ALKBH2 also contains a functional domain that is vital to identifying the instability of DNA bases. Its unique structure allows it to probe base pair stability without needing a damage-checking site. Its mechanism “ensures that only cognate lesions are oxidized and reversed to normal bases.” 5 The end result is ALKBH2 is capable of performing the reaction seen in Figure 1 to accomplish the needed base repair. The methyl group on the DNA base is oxidized then removed once it contacts water.
A BLAST comparison of ALKBH2 shows that there is high conservation across the entire protein between species. Query coverage is above 95% for most sequences with significant alignments. This includes all beta pleated sheets and alpha helices within the protein. Analysis of conserved domains demonstrates a high degree of conservation across the 2OG-Fe(II) oxygenase superfamily from residue 8 to 202 in a protein with 209 residues total. Comparative proteins also perform the function of DNA repair in cells of other species. In the Arabidopsis thiana, ALKBH2 has shown to protect against methylation damage, like is seen in human beings 2. ALKBH2 also provides significant protection against a methylating agent in mice 3.
ALKBH2 finds DNA base pairs with reduced stability and oxidizes cognate bases to revert them back to their original form. ALKBH2 is critical to survival after acute inflammation and mutant organisms lacking the gene for ALKBH2 struggle to fight off infection. ALKBH2 is critical to cancer prevention 4. Chemicals can cause alkylation of DNA that modifies DNA bases and is often mutagenic. ALKBH2 reverses this damage through demethylation of the modified DNA bases. It is important for this protein to be studied because understanding its function may be pivotal to curing many diseases, including cancer. The reversal of DNA damage holds the potential to remove cancerous mutations that can result from undesired methylation of DNA bases.
ALKBH2 requires iron, α-ketoglutarate (aKG), and oxygen in order to perform its key demethylation function. Iron is bound to three amino acids in the protein and loses two of its aqua ligands to aKG. The presence of both the substrate and cofactor causes the iron to lose its final aqua ligand and open a site to bind oxygen. Oxidative decarboxylation of aKG takes place leading to formation of an iron atom with a +4 charge double bonded to oxygen. This species hydroxylates the methyl group on the N-atom of the DNA base. The DNA base then undergoes another spontaneous reaction that forms formaldehyde and the demethylated base 5.
The structure of ALKBH2 is highly conserved across different species. This is important because most experiments performed on this protein have been done on species other than human beings. The structure of ALKBH2 allows for it wrap around DNA and reach the bases inside. Since DNA is the source of genetic code for all life forms, every organism requires very similar DNA repair mechanisms for methylation damage.
Point mutations are not currently known for this protein, but studies into this protein are beginning to surface due to its cancer-fighting abilities. Removal of this protein in other species has shown an elevated risk of base-pair mutation and as a result, the chance of acquiring cancer. Other related proteins, such as ALKBH3, have very similar structures and perform the same demethylating function, but have an affinity for DNA and RNA in different scenarios. For example, ALKBH3 prefers to repair only single-stranded DNA and RNA, as opposed to ALKBH2, which focuses on double-stranded DNA.