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

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 fluor...

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Detalles Bibliográficos
Autores principales: Kanazhevskaya, Lyubov Yu., Smyshliaev, Denis A., Timofeyeva, Nadezhda A., Ishchenko, Alexander A., Saparbaev, Murat, Kuznetsov, Nikita A., Fedorova, Olga S.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9370705/
https://www.ncbi.nlm.nih.gov/pubmed/35956910
http://dx.doi.org/10.3390/molecules27154960
Descripción
Sumario: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 O(2). 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.