The Escherichia coli DNA repair enzyme AlkB, a Fe(II)/2-oxoglutarate (2OG)-dependent dioxygenase that directly reverses alkylated bases, exists as a dynamic molten-globule-like apoenzyme that folds into the catalytically competent holoenzyme only when Fe(II) and 2OG bind jointly. The source paper (Bleijlevens et al., Biochemistry 2012) posed the linked open questions of what these dynamics are for: whether the apo state participates in lesion detection, whether AlkB searches DNA by one-dimensional scanning to find methyl lesions and, if so, by what mechanism, whether the human homologues ABH1-ABH8 and FTO display similar cofactor-controlled dynamics, whether the iron chaperones PCBP1 and PCBP2 metalate the mammalian enzymes by binding the dynamic apo form, and how sequence variation shapes DSBH-core dynamics and substrate specificity across the family.

Background

Alkylating agents, both environmental and endogenous, add methyl and other alkyl groups to DNA bases; lesions such as 1-methyladenine and 3-methylcytosine are cytotoxic because they disrupt base pairing and block replication. The Escherichia coli enzyme AlkB repairs such damage directly: it is a Fe(II)/2-oxoglutarate (2OG)-dependent dioxygenase, a member of a large enzyme superfamily that couples the oxidative decarboxylation of 2OG to succinate with oxidation of a second substrate. In AlkB that second substrate is the alkyl group of a damaged nucleobase in single- or double-stranded DNA or RNA; hydroxylation of the alkyl group makes it unstable so that it is spontaneously released, regenerating the intact base. Catalysis requires the active site to bind both Fe(II) and 2OG; the fully loaded enzyme is called the holoenzyme and the cofactor-free protein the apoenzyme (apo state). Nine human homologues exist (ABH1-ABH8 and the obesity-associated FTO), several of which repair alkylated nucleic acids or remove epigenetic methylation marks such as N6-methyladenosine from mRNA. Crystal structures of AlkB and its homologues show a conserved double-stranded beta-helix (DSBH, also called jelly-roll) fold - two four-stranded beta-sheets that sandwich the Fe(II)/2OG active site.

Static crystallography does not reveal how these enzymes behave in solution. Using NMR spectroscopy (in particular 1H-15N HSQC correlation spectra, whose peak count and chemical-shift dispersion report on whether backbone amides occupy well-defined environments), fluorescence, and circular dichroism, the source authors showed that apoAlkB is not a folded globular protein but a molten-globule-like ensemble: it retains the secondary-structure content of the holo form (about 37% beta-sheet) yet lacks a stable hydrophobic core and undergoes conformational exchange on the microsecond-to-millisecond timescale. Binding Fe(II) or 2OG alone leaves this ensemble unchanged; only joint occupancy of both cofactors yields the well-folded holo conformation. Their earlier companion study (Bleijlevens et al. 2008) had shown that the co-product (succinate) complex is again more dynamic than the co-substrate complex, and proposed that this dynamic state would promote one-dimensional scanning along DNA - i.e. that the enzyme slides along the polymer while inspecting bases for lesions. Subsequent enzymological and NMR work (Ergel et al. 2014) demonstrated that an open/closed conformational transition of the nucleotide recognition lid (NRL, a beta-hairpin lid covering the active site) gates DNA binding - the apoenzyme binds DNA substrate roughly 100-fold more weakly than the Fe(II)/2OG complex - and controls the order of substrate binding and the sequestration of the reactive iron-oxo (oxyferryl) intermediate, so protein dynamics demonstrably control the catalytic cycle in vitro.

A separate strand concerns iron delivery. In mammalian cells, insertion of iron into some iron proteins is assisted by iron chaperones - proteins that bind labile intracellular iron and transfer it to target apoenzymes. The poly(rC)-binding proteins PCBP1 and PCBP2 are established iron chaperones for ferritin, for the HIF prolyl hydroxylase (a 2OG dioxygenase involved in oxygen sensing), and for deoxyhypusine hydroxylase (the enzyme that hydroxylates the translation factor eIF5A during its maturation). Whether they metalate the mammalian AlkB homologues is unknown. It is against this background that the source paper's discussion posed its open questions about the functional meaning of cofactor-controlled dynamics in the AlkB family.

Problem Statement

Establish the functional roles of the cofactor-controlled conformational dynamics of AlkB-family Fe(II)/2OG-dependent dioxygenases, as posed by the discussion of Bleijlevens et al. (2012). The single scientific objective uniting the problem is a mechanistic account of how protein dynamics control this enzyme family's function from metalation through lesion search to catalysis, with the following linked questions to be answered by direct evidence:

(1) Apo-state function: is apoAlkB (the cofactor-free, molten-globule-like form - a partly folded ensemble that retains its secondary structure but lacks a fixed hydrophobic core - which binds DNA far more weakly than the holo complex) part of the catalytic cycle and/or directly involved in lesion detection - as opposed to being merely an incompletely assembled precursor?

(2) Scanning mechanism: does AlkB search DNA by a one-dimensional scanning (sliding) mechanism to efficiently identify and repair methyl lesions, and if so, by what mechanism and do the cofactor-controlled dynamics characterized for AlkB facilitate it?

(3) Family conservation: do the human AlkB homologues ABH1-ABH8 and FTO display cofactor-controlled dynamic characteristics similar to E. coli AlkB? A systematic comparison across the family members is required, not only isolated characterizations.

(4) Metalation route: do the iron chaperones PCBP1 and PCBP2 (proteins that bind intracellular iron and deliver it to target enzymes) metalate mammalian AlkB homologues - in particular, do they bind preferentially to the dynamic apo form and are they released upon formation of the folded holo complex?

(5) Sequence determinants: how do sequence variations modulate the dynamics of the DSBH core (the conserved double-stranded beta-helix fold that forms the catalytic domain and scaffolds the Fe(II)/2OG active site) across the family, and how do those dynamics contribute to substrate specificity?

The five components jointly serve the stated common objective and are independently answerable.

References

  1. Bleijlevens B, Shivarattan T, van den Boom KS, de Haan A, van der Zwan G, Simpson PJ, Matthews SJ. Changes in Protein Dynamics of the DNA Repair Dioxygenase AlkB upon Binding of Fe2+ and 2-Oxoglutarate. Biochemistry 51(16):3334-3341 (2012). DOI 10.1021/bi201699e, https://doi.org/10.1021/bi201699e
  2. Bleijlevens B, Shivarattan T, Flashman E, Yang Y, Simpson PJ, Koivisto P, Sedgwick B, Schofield CJ, Matthews SJ. Dynamic states of the DNA repair enzyme AlkB regulate product release. EMBO Reports 9(9):872-877 (2008). DOI 10.1038/embor.2008.120, https://doi.org/10.1038/embor.2008.120
  3. Yu B, Edstrom WC, Benach J, Hamuro Y, Weber PC, Gibney BR, Hunt JF. Crystal structures of catalytic complexes of the oxidative DNA/RNA repair enzyme AlkB. Nature 439(7078):879-884 (2006). DOI 10.1038/nature04561, https://doi.org/10.1038/nature04561
  4. Ergel B, Gill ML, Brown L, Yu B, Palmer AG, Hunt JF. Protein dynamics control the progression and efficiency of the catalytic reaction cycle of the Escherichia coli DNA-repair enzyme AlkB. Journal of Biological Chemistry 289(43):29584-29601 (2014). DOI 10.1074/jbc.M114.575647, https://doi.org/10.1074/jbc.M114.575647
  5. Kanazhevskaya LY, Alekseeva IV, Fedorova OS. A Single-Turnover Kinetic Study of DNA Demethylation Catalyzed by Fe(II)/alpha-Ketoglutarate-Dependent Dioxygenase AlkB. Molecules 24(24):4576 (2019). DOI 10.3390/molecules24244576, https://doi.org/10.3390/molecules24244576
  6. Kanazhevskaya LY, Smyshliaev DA, Alekseeva IV, Fedorova OS. Conformational Dynamics of Dioxygenase AlkB and DNA in the Course of Catalytically Active Enzyme-Substrate Complex Formation. Russian Journal of Bioorganic Chemistry 45(6):630-640 (2019). DOI 10.1134/S1068162019060190, https://doi.org/10.1134/S1068162019060190
  7. Purslow JA, Nguyen TT, Egner TK, Dotas RR, Khatiwada B, Venditti V. Active Site Breathing of Human Alkbh5 Revealed by Solution NMR and Accelerated Molecular Dynamics. Biophysical Journal 115(10):1895-1905 (2018). DOI 10.1016/j.bpj.2018.10.004, https://doi.org/10.1016/j.bpj.2018.10.004
  8. Purslow JA, Nguyen TT, Khatiwada B, Singh A, Venditti V. N6-methyladenosine binding induces a metal-centered rearrangement that activates the human RNA demethylase Alkbh5. Science Advances 7(34):eabi8215 (2021). DOI 10.1126/sciadv.abi8215, https://doi.org/10.1126/sciadv.abi8215
  9. Khatiwada B, Nguyen TT, Purslow JA, Venditti V. Solution structure ensemble of human obesity-associated protein FTO reveals druggable surface pockets at the interface between the N- and C-terminal domain. Journal of Biological Chemistry 298(5):101907 (2022). DOI 10.1016/j.jbc.2022.101907, https://doi.org/10.1016/j.jbc.2022.101907
  10. Burns D, Khatiwada B, Singh A, Purslow JA, Potoyan DA, Venditti V. An alpha-ketoglutarate conformational switch controls iron accessibility, activation, and substrate selection of the human FTO protein. Proceedings of the National Academy of Sciences USA 121(25):e2404457121 (2024). DOI 10.1073/pnas.2404457121, https://doi.org/10.1073/pnas.2404457121
  11. Faal B, Purslow JA, Venditti V. 1H, 15N, 13C backbone resonance assignment of human Alkbh7. Biomolecular NMR Assignments 19(1):65-69 (2025). DOI 10.1007/s12104-025-10219-4, https://doi.org/10.1007/s12104-025-10219-4
  12. Das S, Purslow JA, Malcom K, Knott J, Venditti V. 1H, 15N, 13C backbone resonance assignment of Escherichia coli AlkB in complex with Zn(II) and alpha-ketoglutarate. Biomolecular NMR Assignments 20(1):23 (2026). DOI 10.1007/s12104-026-10273-6, https://doi.org/10.1007/s12104-026-10273-6
  13. Kanazhevskaya LY, Smyshliaev DA, Timofeyeva NA, Ishchenko AA, Saparbaev M, Kuznetsov NA, Fedorova OS. Conformational Dynamics of Human ALKBH2 Dioxygenase in the Course of DNA Repair as Revealed by Stopped-Flow Fluorescence Spectroscopy. Molecules 27(15):4960 (2022). DOI 10.3390/molecules27154960, https://doi.org/10.3390/molecules27154960
  14. Kanazhevskaya LY, Gorbunov AA, Lukina MV, Smyshliaev DA, Zhdanova PV, Lomzov AA, Koval VV. The Role of Key Amino Acids of the Human Fe(II)/2OG-Dependent Dioxygenase ALKBH3 in Structural Dynamics and Repair Activity toward Methylated DNA. International Journal of Molecular Sciences 25(2):1145 (2024). DOI 10.3390/ijms25021145, https://doi.org/10.3390/ijms25021145
  15. Nandal A, Ruiz JC, Subramanian P, Ghimire-Rijal S, Sinnamon RA, Stemmler TL, Bruick RK, Philpott CC. Activation of the HIF Prolyl Hydroxylase by the Iron Chaperones PCBP1 and PCBP2. Cell Metabolism 14(5):647-657 (2011). DOI 10.1016/j.cmet.2011.08.015, https://doi.org/10.1016/j.cmet.2011.08.015
  16. Frey AG, Nandal A, Park JH, Smith PM, Yabe T, Ryu MS, Ghosh MC, Lee J, Rouault TA, Park MH, Philpott CC. Iron chaperones PCBP1 and PCBP2 mediate the metallation of the dinuclear iron enzyme deoxyhypusine hydroxylase. Proceedings of the National Academy of Sciences USA 111(22):8031-8036 (2014). DOI 10.1073/pnas.1402732111, https://doi.org/10.1073/pnas.1402732111
  17. Philpott CC. Coming into View: Eukaryotic Iron Chaperones and Intracellular Iron Delivery. Journal of Biological Chemistry 287(17):13518-13523 (2012). DOI 10.1074/jbc.r111.326876, https://doi.org/10.1074/jbc.r111.326876
  18. Philpott CC, Patel SJ, Protchenko O. Management versus miscues in the cytosolic labile iron pool: The varied functions of iron chaperones. Biochimica et Biophysica Acta Molecular Cell Research 1867(11):118830 (2020). DOI 10.1016/j.bbamcr.2020.118830, https://doi.org/10.1016/j.bbamcr.2020.118830
  19. Aik W, McDonough MA, Thalhammer A, Chowdhury R, Schofield CJ. Role of the jelly-roll fold in substrate binding by 2-oxoglutarate oxygenases. Current Opinion in Structural Biology 22(6):691-700 (2012). DOI 10.1016/j.sbi.2012.10.001, https://doi.org/10.1016/j.sbi.2012.10.001
  20. Xu B, Liu D, Wang Z, Tian R, Zuo Y. Multi-substrate selectivity based on key loops and non-homologous domains: new insight into ALKBH family. Cellular and Molecular Life Sciences 78(1):129-141 (2021). DOI 10.1007/s00018-020-03594-9, https://doi.org/10.1007/s00018-020-03594-9
  21. Yang S, Xing J, Bao L, Cui J, Liu D, Wen S, Xu S, Zuo Y. Functional emergence driven by structural evolution of the DSBH domain in Fe2+ and alpha-ketoglutarate-dependent family. The FEBS Journal (2026). DOI 10.1111/febs.70565, https://doi.org/10.1111/febs.70565