Tina A. Müller

FeII/α-ketoglutarate hydroxylases involved in nucleobase, nucleoside, nucleotide, and chromatin metabolism

Fe(II)/alpha-ketoglutarate-dependent hydroxylases uniformly possess a double-stranded beta-helix fold with two conserved histidines and one carboxylate coordinating their mononuclear ferrous ions. Oxidative decomposition of the alpha-keto acid is proposed to generate a ferryl-oxo intermediate capable of hydroxylating unactivated carbon atoms in a myriad of substrates. This Perspective focuses on a subgroup of these enzymes that are involved in pyrimidine salvage, purine decomposition, nucleoside and nucleotide hydroxylation, DNA/RNA repair, and chromatin modification. The varied reaction schemes are presented, and selected structural and kinetic information is summarized.

Human AlkB homologue 1 (ABH1) exhibits DNA lyase activity at abasic sites

Bacterial AlkB and three human AlkB homologues (ABH1, ABH2, and ABH3) are Fe(2+)/2-oxoglutarate-dependent oxygenases that directly repair alkylation-damaged DNA. Here, we show that ABH1 unexpectedly has a second activity, cleaving DNA at abasic (AP) sites such as those arising spontaneously from alkylation-dependent depurination reactions. The DNA cleavage activity of ABH1 does not require added Fe(2+) or 2-oxoglutarate, is not inhibited by EDTA, and is unaffected by mutation of the putative metal-binding residues, indicating that this activity arises from an active site distinct from that used for demethylation. AP-specific DNA cleavage was shown to occur by a lyase mechanism, rather than by hydrolysis, with the enzyme remaining associated with the DNA product. ABH1 can cleave at closely spaced AP-sites on opposite DNA strands yielding double-strand breaks in vitro and this reaction may relate to the physiological role of this unexpected AP lyase activity.

ALKBH1 is dispensable for abasic site cleavage during base excision repair and class switch recombination.

Potential roles of the abasic site lyase activity associated with AlkB homolog 1 (ALKBH1) were assessed by studies focusing on the two cellular processes that create abasic sites as intermediates: base excision repair and class switch recombination. Alkbh1−/− pups (lacking exon 3) were born at a lower than expected frequency from heterozygous parents, suggesting a reduced survival rate and non-Mendelian inheritance, and they exhibited a gender bias in favor of males (70% males and 30% females). To study ALKBH1’s potential involvement in DNA repair, fibroblasts were isolated from Alkbh1−/− mice, spontaneously immortalized and tested for resistance to DNA damaging agents. Alkbh1−/− and isogenic cells expressing hALKBH1 showed no difference in survival to the DNA damaging agents methyl-methionine sulfate or H2O2. This result indicates that ALKBH1 does not play a major role in the base excision repair pathway. To assess ALKBH1’s role in class switch recombination, splenic B cells were isolated from Alkbh1−/− and Alkbh1+/+ mice and subjected to switching from IgM to IgG1. No differences were found in IgG1 switching, suggesting that Alkbh1 is not involved in class switch recombination of the immunoglobulin heavy chain during B lymphocyte activation.

Structural basis for the enantiospecificities of R‐ and S‐specific phenoxypropionate/α‐ketoglutarate dioxygenases

(R)‐ and (S)‐dichlorprop/α‐ketoglutarate dioxygenases (RdpA and SdpA) catalyze the oxidative cleavage of 2‐(2,4‐dichlorophenoxy)propanoic acid (dichlorprop) and 2‐(4‐chloro‐2‐methyl‐phenoxy)propanoic acid (mecoprop) to form pyruvate plus the corresponding phenol concurrent with the conversion of α‐ketoglutarate (αKG) to succinate plus CO2. RdpA and SdpA are strictly enantiospecific, converting only the (R) or the (S) enantiomer, respectively. Homology models were generated for both enzymes on the basis of the structure of the related enzyme TauD (PDB code 1OS7). Docking was used to predict the orientation of the appropriate mecoprop enantiomer in each protein, and the predictions were tested by characterizing the activities of site‐directed variants of the enzymes. Mutant proteins that changed at residues predicted to interact with (R)‐ or (S)‐mecoprop exhibited significantly reduced activity, often accompanied by increased Km values, consistent with roles for these residues in substrate binding. Four of the designed SdpA variants were (slightly) active with (R)‐mecoprop. The results of the kinetic investigations are consistent with the identification of key interactions in the structural models and demonstrate that enantiospecificity is coordinated by the interactions of a number of residues in RdpA and SdpA. Most significantly, residues Phe171 in RdpA and Glu69 in SdpA apparently act by hindering the binding of the wrong enantiomer more than the correct one, as judged by the observed decreases in Km when these side chains are replaced by Ala.

Calorimetric assessment of Fe2+ binding to α-ketoglutarate/taurine dioxygenase: Ironing out the energetics of metal coordination by the 2-his-1-carboxylate facial triad

The thermodynamic properties of Fe(2+) binding to the 2-His-1-carboxylate facial triad in α-ketoglutarate/taurine dioxygenase (TauD) were explored using isothermal titration calorimetry. Direct titrations of Fe(2+) into TauD and chelation experiments involving the titration of ethylenediaminetetraacetic acid into Fe(2+)-TauD were performed under an anaerobic environment to yield a binding equilibrium of 2.4 (±0.1) × 10(7) (Kd = 43 nM) and a ΔG° value of -10.1 (±0.03) kcal/mol. Further analysis of the enthalpy/entropy contributions indicates a highly enthalpic binding event, where ΔH = -11.6 (±0.3) kcal/mol. Investigations into the unfavorable entropy term led to the observation of water molecules becoming organized within the Fe(2+)-TauD structure.

CHAPTER 8:AlkB and Its Homologues – DNA Repair and Beyond

AlkB is an Fe(ii)/2-oxoglutarate-dependent dioxygenase that is part of the adaptive response to alkylating agents in Escherichia coli. AlkB hydroxylates a wide variety of alkylated DNA bases producing unstable intermediates which decompose to restore the non-alkylated bases. Homologues exist in other bacteria, metazoa (e.g. nine in humans), plants and viruses, but not in archaea, with many catalysing the same oxidative demethylation reactions as for AlkB. The mammalian enzymes Alkbh2 and Alkbh3 catalyse direct DNA repair, Alkbh5 and FTO (Alkbh9) are RNA demethylases, and Alkbh8 is used to synthesize a tRNA, while the remaining mammalian homologues have alternative functions. Alkbh1 is an apurinic/apyrimidinic lyase in addition to exhibiting demethylase activities, but no clear role for the Alkbh1 protein has emerged. Alkbh4 is involved in cell division and potentially demethylates actin, whereas the mitochondrial homologue Alkbh7 has a role in obesity; however, no enzymatic activity has been linked to Alkbh4 or Alkbh7. Here, we discuss AlkB as the ‘archetype’ of this class of hydroxylases, compare it to Alkbh2 and Alkbh3, and then briefly review the diverse (and largely unknown) functions of Alkbh1, Alkbh4, Alkbh6 and Alkbh7. Alkbh5, Alkbh8 and Alkbh9 (FTO) are described separately.

Homology modeling, molecular dynamics, and site-directed mutagenesis study of AlkB human homolog 1 (ALKBH1)

The ability to repair DNA is important for the conservation of genetic information of living organisms. Cells have a number of ways to restore damaged DNA, such as direct DNA repair, base excision repair, and nucleotide excision repair. One of the proteins that can perform direct repair of DNA bases is Escherichia coli AlkB. In humans, there are 9 identified AlkB homologs, including AlkB homolog 1 (ALKBH1). Many of these proteins catalyze the direct oxidative dealkylation of DNA and RNA bases and, as such, have an important role in repairing DNA from damage induced by alkylating agents. In addition to the dealkylase activity, ALKBH1 can also function as an apyrimidinic/apurinic lyase and was proposed to have a distinct lyase active site. To our knowledge, no crystal structure or complete homology model of ALKBH1 protein is available. In this study, we have used homology modeling to predict the structure of ALKBH1 based on AlkB and Duffy-binding-like domain crystal structures as templates. Molecular dynamics simulations were subsequently performed on the predicted structure of ALKBH1. The positions of two disulfide bonds or a zinc-finger motif and a disulfide bond were predicted and the importance of these features was tested by mutagenesis. Possible locations for the lyase active site are proposed based on the analysis of our predicted structures and previous experimental results.

ALKBH7 Variant Related to Prostate Cancer Exhibits Altered Substrate Binding

The search for prostate cancer biomarkers has received increased attention and several DNA repair related enzymes have been linked to this dysfunction. Here we report a targeted search for single nucleotide polymorphisms (SNPs) and functional impact characterization of human ALKBH family dioxygenases related to prostate cancer. Our results uncovered a SNP of ALKBH7, rs7540, which is associated with prostate cancer disease in a statistically significantly manner in two separate cohorts, and maintained in African American men. Comparisons of molecular dynamics (MD) simulations on the wild-type and variant protein structures indicate that the resulting alteration in the enzyme induces a significant structural change that reduces ALKBH7’s ability to bind its cosubstrate. Experimental spectroscopy studies with purified proteins validate our MD predictions and corroborate the conclusion that this cancer-associated mutation affects productive cosubstrate binding in ALKBH7.