The AlkB Family of Fe(II)/α-Ketoglutarate-dependent Dioxygenases: Repairing Nucleic Acid Alkylation Damage and Beyond^*

AlkB Is an Escherichia coli Fe(II)/αKG-dependent Dioxygenase That Reverses DNA Alkylation Damage

E. coli cells exposed to a low dose of a methylating agent such as methylnitronitrosoguanidine upregulate a transcriptional program that confers significant resistance to subsequent, higher levels of alkylation insult. This phenomenon, discovered in 1977 by Samson and Cairns, was coined the adaptive response (1). At its core, the adaptive response relies on the activity of four proteins: Ada, AlkA, AlkB, and AidB, whose overexpression affords the observed bacterial resistance tothe deleterious effects of alkylating agents (1). Three of the four proteins (Ada, AlkA, and AlkB) are DNA repair proteins that combat the mutagenic and toxic effects of alkylated bases. Although the biological functions of Ada (a methyltransferase) and AlkA (a glycosylase) were established relatively quickly, the function of AlkB remained mysterious until 2001, when Aravind and Koonin, using sequence homology alignments, predicted that AlkB is an Fe(II)- and α-ketoglutarate (αKG)⁶-dependent dioxygenase (2). Soon after, AlkB was established as a prototypical oxidative dealkylation DNA repair enzyme (3–5) that protects the bacterial genome against alkylation damage (6–10). AlkB uses molecular oxygen to oxidize the alkyl groups on alkylation-damaged nucleic acid bases, such as 1-methyl-adenine (m1A), 3-methylcytosine (m3C) (7, 8), and 1,N⁶-ethenoadenine (ϵA) (9, 11); the oxidized alkyl groups are subsequently released as aldehydes, regenerating the undamaged bases (Fig. 1). The decade of research that followed the 2002 discovery of the enzymatic properties of AlkB (7, 8) greatly expanded our understanding of the biological functions of the AlkB dioxygenases. This minireview summarizes the most salient aspects of this research.

Bacterial Homologs of the E. coli AlkB

The E. coli AlkB protein is by far the best studied enzyme in the family. However, AlkB homologs exist in most bacteria and almost all eukaryotes. Speaking to the remarkable versatility and ubiquity of the AlkB proteins, even certain single-stranded plant-infecting RNA viruses (e.g. Flexiviridae (now classified as Alphaflexiviridae, Betaflexiviridae, and Gammaflexiviridae)) encode an AlkB homolog that can repair alkylated bases in DNA and, preferably, RNA (12).

The majority of aerobic bacterial species express AlkB proteins, which show a great diversity of substrate specificity (4, 13, 14). Given the AlkB requirement for molecular oxygen, obligate anaerobic bacteria (e.g. Clostridium, Bacteroides, Bifidobacterium) do not appear to have AlkB type proteins (4, 14). A careful bioinformatics analysis of the homologous sequences found four phylogenetic groups of bacterial AlkB proteins denoted 1A, 1B, 2A, and 2B (14). Group 1A proteins, which include the E. coli AlkB, are characterized by robust oxidative dealkylation activities and broad substrate specificities. This group includes the Streptomyces AlkB proteins, which share 79% sequence identity with the E. coli AlkB and have been shown to repair both methylated and etheno lesions (14). Group 1B proteins, primarily found in the β and γ subdivisions of Proteobacteria and in cyanobacteria, also show wide substrate preferences. They are most closely related to the eukaryotic ALKBH2 and ALKBH3 homologs. Group 2A proteins, often found in the α Proteobacteria (e.g. Agrobacterium, Rickettsia, and Rhizobium), share strong homology with the ALKBH8 proteins from animals and plants, which have been implicated in tRNA post-transcriptional modification rather than true DNA or RNA repair. Finally, group 2B proteins, often found in soil bacteria (e.g. Actinobacteria) but also in Xanthomonas and Burkholderia, are most likely to show substrate specialization; some enzymes have high activity on monoalkyl lesion, but no activity on bridged adducts (e.g. MT-2B from Mycobacterium tuberculosis), whereas others are exactly the opposite (e.g. XC-2B from Xanthomonas campestris) (14). Such specialization presumably reflects an adaptation to a specific environmental stressor. Additionally, unlike E. coli, many bacterial genomes contain two or even three AlkB family proteins (14). So far, no AlkB homologs have been found in Archaea (14).

Eukaryotic and Mammalian AlkB Homologs

Most eukaryotic cells have several AlkB homologs, with the notable exception of Saccharomyces cerevisiae, which lacks this class of DNA repair enzymes. Mammalian cells have nine homologs (2, 4, 15); the first eight have been denoted ALKBH1–8, whereas the ninth is known as FTO (fat mass and obesity-associated) (16). Evolutionarily speaking, ALKBH5 and FTO are the newest AlkB proteins, being found only in vertebrates (17, 18). The other seven AlkB homologs are conserved across all metazoans, including worms and fruit flies (13).

Among the nine human AlkB homologs, ALKBH2 (19–25) and ALKBH3 (25–29) are bona fide nuclear DNA repair enzymes, being the only homologs that can complement the function of E. coli AlkB in vivo (25, 30) and protect cells against alkylation damage both in cell culture and in animal models (22, 28, 30). ALKBH1, featuring the highest sequence homology to E. coli AlkB, can function as a mitochondrial nucleic acid demethylase (31) but also exhibits apyrimidinic/apurinic lyase activity (32–34). The lyase activity has also been observed in Abh1, the Schizosaccharomyces pombe homolog, which, however, has no demethylase activity (35). Additionally, ALKBH1 can also demethylate H2A histone (36), an activity more consistent with the impaired development phenotype of the ALKBH1 KO mice (37, 38).

The remaining homologs have no known activity on DNA substrates; instead, they demethylate RNA or proteins. Both FTO (39–41), whose overexpression is closely linked to obesity and diabetes (16, 42, 43), and ALKBH5 (44–48) repair primarily N⁶-methyladenine (m6A) in RNA (Fig. 1). FTO also repairs 3-methylthymine (m3T) and 3-methyluracil (m3U) in ssDNA (49). ALKBH8 (50–54) is involved in the maturation of tRNA featuring both an S-adenosylmethionine-dependent methyl-transferase domain that methylates 5-carboxymethyluridine (cm5U) to 5-methoxycarbonylmethyluridine (mcm5U), and a dioxygenase domain that hydroxylates mcm5U to generate (S)-5-methoxycarbonylhydroxymethyluridine (mchm5U), a common functional modification at the wobble position of tRNA (51, 52). The main function of ALKBH4 seems to be actin demethylation (55, 56). ALKBH7, which plays a role in alkylation-induced necrosis (57–59), is believed to act on protein substrates, but their identity is not currently known. Finally, the function and substrates of ALKBH6 remain to be established. Although they are also members of the Fe(II)/αKG dioxygenase superfamily, the mammalian TET (ten-eleven translocation) proteins, which play an important role in the epigenetic reprogramming of the cell (60), are only distantly related to the AlkB proteins, and they will be covered in other minireviews.

Structural Features of the AlkB Dioxygenases

Crystal structures of the E. coli AlkB (61–64) and the human homologs ALKBH2 (62), ALKBH3 (29), ALKBH5 (48), ALKBH7 (59), ALKBH8 (53), and FTO (66) have provided significant insight into the molecular mechanism of the oxidative dealkylation catalyzed by AlkB dioxygenases. The active site of these enzymes is contained on a characteristic double-stranded β-helix domain, also known as a “jelly-roll” fold, consisting of eight β-strands arranged in pairs in a helical conformation to form the core that binds the Fe(II) and αKG cofactors using conserved residues (61, 62) (Fig. 2A). The E. coli AlkB also contains a unique 90-residue N-terminal subdomain that interacts with the oligonucleotide substrate containing the damaged base and covers the active site by forming a “nucleotide recognition lid.” Being conformationally flexible, this lid can accommodate substrates of variable sizes, which can explain the diverse substrate specificity of the enzyme (see below). However, recent data on bacterial AlkB homologs suggest that this domain is not the only factor governing the substrate specificity (67). The active site of AlkB contains a 3 Å-wide oxygen diffusion tunnel from the protein surface to the oxygen binding site (61). His-131, Asp-133, and His-187 of E. coli AlkB, together with molecular oxygen (or water under anaerobic conditions) and αKG bound in a bidentate fashion, form the octahedral primary coordination sphere around the non-heme iron (Fig. 2, B–D). The αKG cofactor is held in place by two salt bridge interactions between the Arg-204, Arg-210, and the carboxylates of αKG (61, 63). The octahedral coordination and the global geometry are conserved in AlkB complexes in which Fe(II) is replaced with Co(II) or Mn(II), but such replacement results in closure of the oxygen diffusion tunnel, producing an oxygen-resistant phenotype (63).

The Mechanism of AlkB-catalyzed Dealkylation

Similar to other non-heme iron dioxygenases, the AlkB-catalyzed oxidative dealkylation reaction is initiated by the binding of molecular oxygen to Fe(II) in the active site by replacing the bound water molecule (68–75). Cleavage of molecular oxygen generates an Fe(IV)-oxo reactive moiety, whereas the αKG ligand is oxidatively decarboxylated to succinate. Once CO₂ is released from the active site, the reactive Fe(IV)-oxo ligand migrates adjacent to the target alkyl group on the nucleobase substrate and hydroxylates the alkyl moiety (72–75). The resulting carbinoliminium is typically unstable and dissociates (in a spontaneous or catalyzed fashion) into an aldehyde product and the unmodified (repaired) base.

For simple alkyl substrates, AlkB oxidizes the carbon attached to a nucleobase nitrogen atom (i.e. ring nitrogen or exocyclic amine). The resulting carbinoliminium species (e.g. 3-hydroxymethylcytosine from m3C (Fig. 1), or 1-hydroxy-methyladenine from m1A) have been experimentally observed in AlkB oxidation reactions performed in crystallo (64). Specifically, the AlkB-substrate complex, together with Fe(II) and αKG, is first crystalized under anaerobic conditions. The reaction is then initiated by exposing the crystals to air (64).

In the case of etheno lesions (e.g. ϵA), the AlkB dealkylation was proposed to involve the oxidation of the etheno bridge to an epoxide, followed by hydrolysis of the epoxide to a glycol and subsequent release of the dialdehyde glyoxal (Fig. 1) (9, 11). The glycol corresponding to ϵA has been experimentally observed in crystallo (64). The epoxide formation, however, has been recently challenged in a computational study, which suggested instead that the AlkB reaction with ϵA proceeds via a zwitterion intermediate (76). Nevertheless, further experimental work is needed to establish the identity of the reaction intermediate.

The binding affinity of E. coli AlkB to damaged DNA is influenced by both the damaged base and the backbone phosphates of the substrate (61). Residues Thr-51, Tyr-76, and Arg-161, conserved across all eubacterial AlkB homologs, form extensive hydrogen-bonding interactions with the backbone phosphates (29, 61, 62). In the AlkB repair complex, the central alkylated base is splayed out of the DNA helix; the stacking interactions between the normal flanking bases stabilize the resulting backbone conformation. The flipped-out alkylated base is sandwiched between AlkB conserved residues Trp-69 and His-131, whereas the DNA backbone is distorted such that the bases flanking the flipped damaged base stack with each other (62).

Unlike E. coli AlkB, ALKBH2 promotes the flipping of the damaged base by using an aromatic finger residue, Phe-102, which intercalates into the duplex DNA and fills the gap left by the flipped base (62). Moreover, although AlkB only interacts with the lesion strand, ALKBH2 interacts with both DNA strands, which may explain the observed differences in substrate preference: ALKBH2 prefers dsDNA, whereas E. coli AlkB prefers ssDNA or RNA (61, 62).

Substrate Specificity of AlkB Dioxygenases

In addition to the originally reported AlkB substrates, m1A and m3C, a wide variety of modified bases are substrates for the AlkB family proteins, including monoalkylated bases with alkyl moieties of various sizes (Table 1) and bases featuring exocyclic bridged adducts (Table 2). Additionally, AlkB proteins can process both single-stranded and double-stranded substrates, and in both DNA and RNA.

Studying DNA Repair by AlkB Family Dioxygenases

Future Directions and Perspective

As the body of knowledge regarding the specific cellular functions of the AlkB family dioxygenases expands, and the complete list of substrates for each enzyme, particularly for the human homologs, becomes known, the field will be poised to explore in more depth the regulation of AlkB proteins. From the point of view of DNA repair, two directions merit attention. First, as gatekeepers of genomic integrity, certain AlkB homologs may function as tumor suppressor genes (92). When their activity is impaired, excessive DNA alkylation damage may accumulate, which can lead to mutations and malignant transformation or cell death (93, 94). Understanding the biochemical mechanisms by which AlkB enzymes are rendered inoperative may help connect environmental or endogenous factors to mutagenesis and cancer. Molecules that compete for binding with αKG (e.g. the oncometabolite 2-hydroxyglutarate) or metal ions that compete with the required Fe(II) (e.g. Ni(II)) have already been shown to inhibit the activity of certain Fe(II)/αKG-dependent dioxygenases (95, 96). However, the relevance of these mechanisms of inhibition to the AlkB family of enzymes has not been fully evaluated.

Second, from an opposite perspective, AlkB family proteins may also be key factors that help tumor cells withstand chemotherapy and promote tumor cell growth (24, 26, 27). Here, the development of potent and specific inhibitors of Fe(II)/αKG-dependent dioxygenases becomes an important challenge (65, 97). By using the structural and mechanistic information available, the development of anti-cancer agents or adjuvants that target AlkB homologs is certainly within reach.

The last decade of research on the AlkB family dioxygenases has been filled with unexpected findings that have propelled the field in leaps and bounds. One can only wonder about the still-to-be-discovered surprises this family of enzymes has to offer for the decades to come.