TY - JOUR

T1 - Conformational Dynamics of Human ALKBH2 Dioxygenase in the Course of DNA Repair as Revealed by Stopped-Flow Fluorescence Spectroscopy

AU - Kanazhevskaya, Lyubov Yu

AU - Smyshliaev, Denis A.

AU - Timofeyeva, Nadezhda A.

AU - Ishchenko, Alexander A.

AU - Saparbaev, Murat

AU - Kuznetsov, Nikita A.

AU - Fedorova, Olga S.

N1 - Funding Information: This work was supported partially by the Russian Federal Ministry of Science and Higher Education (project No. 121031300041-4) to O.S.F. and N.A.K., by Electricité de France (RB 2020-02 and RB 2021-05, to M.S.), and Fondation ARC (PJA-2021060003796) to A.A.I. The part of the work involving the analysis of ALKBH2 conformational dynamics and activity was specifically funded by Russian Science Foundation grant No. 21-14-00018. Publisher Copyright: © 2022 by the authors.

PY - 2022/8

Y1 - 2022/8

N2 - 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 Escherichia coli 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 O2. 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.

AB - 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 Escherichia coli 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 O2. 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.

KW - aminopurine

KW - conformational dynamics

KW - dioxygenase ALKBH2

KW - DNA methylation

KW - DNA repair

KW - fluorescent spectroscopy

KW - FRET analysis

KW - pre-steady-state kinetics

KW - stopped-flow

KW - Dioxygenases/genetics

KW - Humans

KW - Spectrometry, Fluorescence

KW - Escherichia coli/metabolism

KW - DNA Repair/physiology

KW - DNA/chemistry

KW - AlkB Homolog 2, Alpha-Ketoglutarate-Dependent Dioxygenase/genetics

KW - Escherichia coli Proteins/metabolism

KW - Protein Conformation

KW - Kinetics

UR - http://www.scopus.com/inward/record.url?scp=85137038630&partnerID=8YFLogxK

U2 - 10.3390/molecules27154960

DO - 10.3390/molecules27154960

M3 - Article

C2 - 35956910

AN - SCOPUS:85137038630

VL - 27

JO - Molecules

JF - Molecules

SN - 1420-3049

IS - 15

M1 - 4960

ER -