Conformational Dynamics of Human ALKBH2 Dioxygenase in the Course of DNA Repair As Revealed by Stopped-Flow Fluorescence Spectroscopy

Overview

Journal Molecules

Publisher MDPI

Specialty Biology

Date 2022 Aug 12

PMID 35956910

Authors

Lyubov Yu Kanazhevskaya

Denis A Smyshliaev

Nadezhda A Timofeyeva

Alexander A Ishchenko

Murat Saparbaev

Nikita A Kuznetsov

Olga S Fedorova

Affiliations

Soon will be listed here.

Abstract

Elucidation of physicochemical mechanisms of enzymatic processes is one of the main tasks of modern biology. High efficiency and selectivity of enzymatic catalysis are mostly ensured by conformational dynamics of enzymes and substrates. Here, we applied a stopped-flow kinetic analysis based on fluorescent spectroscopy to investigate mechanisms of conformational transformations during the removal of alkylated bases from DNA by ALKBH2, a human homolog of AlkB dioxygenase. This enzyme protects genomic DNA against various alkyl lesions through a sophisticated catalytic mechanism supported by a cofactor (Fe(II)), a cosubstrate (2-oxoglutarate), and O. We present here a comparative study of conformational dynamics in complexes of the ALKBH2 protein with double-stranded DNA substrates containing N1-methyladenine, N3-methylcytosine, or 1,N6-ethenoadenine. By means of fluorescent labels of different types, simultaneous detection of conformational transitions in the protein globule and DNA substrate molecule was performed. Fitting of the kinetic curves by a nonlinear-regression method yielded a molecular mechanism and rate constants of its individual steps. The results shed light on overall conformational dynamics of ALKBH2 and damaged DNA during the catalytic cycle.

Citing Articles

The Role of Key Amino Acids of the Human Fe(II)/2OG-Dependent Dioxygenase ALKBH3 in Structural Dynamics and Repair Activity toward Methylated DNA.

Kanazhevskaya L, Gorbunov A, Lukina M, Smyshliaev D, Zhdanova P, Lomzov A Int J Mol Sci. 2024; 25(2).

PMID: 38256217 PMC: 10816986. DOI: 10.3390/ijms25021145.

Individual Contributions of Amido Acid Residues Tyr122, Ile168, and Asp173 to the Activity and Substrate Specificity of Human DNA Dioxygenase ABH2.

Davletgildeeva A, Tyugashev T, Zhao M, Kuznetsov N, Ishchenko A, Saparbaev M Cells. 2023; 12(14).

PMID: 37508504 PMC: 10377887. DOI: 10.3390/cells12141839.

References

Cetica V, Genitori L, Giunti L, Sanzo M, Bernini G, Massimino M . Pediatric brain tumors: mutations of two dioxygenases (hABH2 and hABH3) that directly repair alkylation damage. J Neurooncol. 2009; 94(2):195-201. DOI: 10.1007/s11060-009-9837-0. View

Bleijlevens B, Shivarattan T, van den Boom K, de Haan A, van der Zwan G, Simpson P . Changes in protein dynamics of the DNA repair dioxygenase AlkB upon binding of Fe(2+) and 2-oxoglutarate. Biochemistry. 2012; 51(16):3334-41. DOI: 10.1021/bi201699e. View

Chen F, Bian K, Tang Q, Fedeles B, Singh V, Humulock Z . Oncometabolites d- and l-2-Hydroxyglutarate Inhibit the AlkB Family DNA Repair Enzymes under Physiological Conditions. Chem Res Toxicol. 2017; 30(4):1102-1110. PMC: 5498157. DOI: 10.1021/acs.chemrestox.7b00009. View

Dunlap C, Tsai M . Use of 2-aminopurine and tryptophan fluorescence as probes in kinetic analyses of DNA polymerase beta. Biochemistry. 2002; 41(37):11226-35. DOI: 10.1021/bi025837g. View

Blouin S, D Craggs T, Lafontaine D, Penedo J . Functional Studies of DNA-Protein Interactions Using FRET Techniques. Methods Mol Biol. 2015; 1334:115-41. DOI: 10.1007/978-1-4939-2877-4_8. View

Wu S, Xu W, Liu S, Chen B, Wang X, Wang Y . Down-regulation of ALKBH2 increases cisplatin sensitivity in H1299 lung cancer cells. Acta Pharmacol Sin. 2011; 32(3):393-8. PMC: 4002771. DOI: 10.1038/aps.2010.216. View

Pilzys T, Marcinkowski M, Kukwa W, Garbicz D, Dylewska M, Ferenc K . ALKBH overexpression in head and neck cancer: potential target for novel anticancer therapy. Sci Rep. 2019; 9(1):13249. PMC: 6744417. DOI: 10.1038/s41598-019-49550-x. View

Lenz S, Li D, Wetmore S . Insights into the Direct Oxidative Repair of Etheno Lesions: MD and QM/MM Study on the Substrate Scope of ALKBH2 and AlkB. DNA Repair (Amst). 2020; 96:102944. PMC: 7669730. DOI: 10.1016/j.dnarep.2020.102944. View

Ringvoll J, Moen M, Nordstrand L, Meira L, Pang B, Bekkelund A . AlkB homologue 2-mediated repair of ethenoadenine lesions in mammalian DNA. Cancer Res. 2008; 68(11):4142-9. PMC: 4725713. DOI: 10.1158/0008-5472.CAN-08-0796. View

10.

Kuzmic P . Program DYNAFIT for the analysis of enzyme kinetic data: application to HIV proteinase. Anal Biochem. 1996; 237(2):260-73. DOI: 10.1006/abio.1996.0238. View

11.

Krebs C, Fujimori D, Walsh C, Bollinger Jr J . Non-heme Fe(IV)-oxo intermediates. Acc Chem Res. 2007; 40(7):484-92. PMC: 3870002. DOI: 10.1021/ar700066p. View

12.

Chen B, Liu H, Sun X, Yang C . Mechanistic insight into the recognition of single-stranded and double-stranded DNA substrates by ABH2 and ABH3. Mol Biosyst. 2010; 6(11):2143-9. DOI: 10.1039/c005148a. View

13.

Monsen V, Sundheim O, Aas P, P Westbye M, Sousa M, Slupphaug G . Divergent ß-hairpins determine double-strand versus single-strand substrate recognition of human AlkB-homologues 2 and 3. Nucleic Acids Res. 2010; 38(19):6447-55. PMC: 2965238. DOI: 10.1093/nar/gkq518. View

14.

Kanazhevskaya L, Alekseeva I, Fedorova O . A Single-Turnover Kinetic Study of DNA Demethylation Catalyzed by Fe(II)/α-Ketoglutarate-Dependent Dioxygenase AlkB. Molecules. 2019; 24(24). PMC: 6943663. DOI: 10.3390/molecules24244576. View

15.

Bleijlevens B, Shivarattan T, Sedgwick B, Rigby S, Matthews S . Replacement of non-heme Fe(II) with Cu(II) in the alpha-ketoglutarate dependent DNA repair enzyme AlkB: spectroscopic characterization of the active site. J Inorg Biochem. 2007; 101(7):1043-8. DOI: 10.1016/j.jinorgbio.2007.03.018. View

16.

Bian K, Chen F, Humulock Z, Tang Q, Li D . Copper Inhibits the AlkB Family DNA Repair Enzymes under Wilson's Disease Condition. Chem Res Toxicol. 2017; 30(10):1794-1796. PMC: 6885016. DOI: 10.1021/acs.chemrestox.7b00230. View

17.

GUTFREUND H . Rapid-flow techniques and their contributions to enzymology. Trends Biochem Sci. 1999; 24(11):457-60. DOI: 10.1016/s0968-0004(99)01468-1. View

18.

Ke B, Ye K, Cheng S . ALKBH2 inhibition alleviates malignancy in colorectal cancer by regulating BMI1-mediated activation of NF-κB pathway. World J Surg Oncol. 2020; 18(1):328. PMC: 7731553. DOI: 10.1186/s12957-020-02106-0. View

19.

Giri N, Sun H, Chen H, Costa M, Maroney M . X-ray absorption spectroscopy structural investigation of early intermediates in the mechanism of DNA repair by human ABH2. Biochemistry. 2011; 50(22):5067-76. PMC: 3124014. DOI: 10.1021/bi101668x. View

20.

Ergel B, Gill M, Brown L, Yu B, Palmer 3rd A, Hunt J . Protein dynamics control the progression and efficiency of the catalytic reaction cycle of the Escherichia coli DNA-repair enzyme AlkB. J Biol Chem. 2014; 289(43):29584-601. PMC: 4207975. DOI: 10.1074/jbc.M114.575647. View