Dataset Information

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

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 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.

SUBMITTER: Kanazhevskaya LY

PROVIDER: S-EPMC9370705 | biostudies-literature | 2022 Aug

REPOSITORIES: biostudies-literature

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

Kanazhevskaya Lyubov Yu LY Smyshliaev Denis A DA Timofeyeva Nadezhda A NA Ishchenko Alexander A AA Saparbaev Murat M Kuznetsov Nikita A NA Fedorova Olga S OS

Molecules (Basel, Switzerland) 20220804 15

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 <i>Escherichia coli</i> AlkB dioxygenase. T ...[more]

PMID: 35956910

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Project description:IR spectroscopy has been widely applied in the study of photo-activated biological processes such as photosynthesis, but has not been applied to the study of reacting systems which require rapid mixing of aqueous solutions. This has been due in part to the high pressure needed to make an aqueous solution flow rapidly through the 50 microns optical pathlength between the plates in an IR cuvette suitable for use with 2H2O and the high viscosity of the concentrated protein solutions required to generate measurable IR signals. An apparatus, based largely on conventional stopped-flow technology, is described which achieves mixing well within the time-resolved performance (approximately 40 ms) of modern Fourier-transform IR (FTIR) spectrometers, since the dead time of the mixing device is approximately 15 ms. It has proved possible to achieve efficient mixing by using a simple six-jet mixing device. This is probably at least in part because of the high back pressure which develops when aqueous fluid is passed rapidly through the short pathlength of the cuvette and which promotes turbulent flow. Several examples of measurements of the deacylation of acylchymotrypsins are provided which demonstrate the operation of the apparatus in conjunction with a spectrometer capable of scanning at four scans/s. For cinnamoyl-chymotrypsin, isotope-edited spectra have been obtained which show somewhat lower resolution than is achieved by conventional scanning methods, since some smoothing has to be applied to the spectra. Difference spectra of the acylation of chymotrypsin by glycylglycine p-nitrophenyl ester have been obtained by averaging ten stopped-flow shots and show good signal-to-noise ratio without smoothing. It is predicted that this apparatus is likely to find a variety of applications in the study of enzyme-catalysed reactions, since the spectra are relatively rich in structural information, and isotope editing greatly enhances the interpretability of the spectra.

Dataset Information

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

Publications

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

Similar Datasets

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