Citation: Kanazhevskaya, L.Y.; Gorbunov, A.A.; Zhdanova, P.V.; Koval, V.V.Dataset for Spectroscopic, Structural and Dynamic Analysis of Human Fe(II)/2OG-Dependent Dioxygenase ALKBH3. Data 2023, 8, 57. https://doi.org/ 10.3390/data8030057 Academic Editor: Pufeng Du Received: 7 December 2022 Revised: 26 February 2023 Accepted: 1 March 2023 Published: 3 March 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). data Data Descriptor Dataset for Spectroscopic, Structural and Dynamic Analysis of Human Fe(II)/2OG-Dependent Dioxygenase ALKBH3 Lyubov Yu. Kanazhevskaya 1, * , Alexey A. Gorbunov 2 , Polina V. Zhdanova 1 and Vladimir V. Koval 1,2, * 1 Institute of Chemical Biology and Fundamental Medicine (ICBFM), 8 Lavrentiev Ave., 630090 Novosibirsk, Russia 2 Department of Natural Sciences, Novosibirsk State University, 1 Pirogova St., 630090 Novosibirsk, Russia * Correspondence: lyubov.kanazhevskaya@niboch.nsc.ru (L.Y.K.); koval@niboch.nsc.ru (V.V.K.) Abstract: Fe(II)/2OG-dependent dioxygenases of the AlkB family catalyze a direct removal of alky- lated damages in the course of DNA and RNA repair. A human homolog of the E. coli AlkB ALKBH3 protein is able to hydroxylate N1-methyladenine, N3-methylcytosine, and N1-methylguanine in single-stranded DNA and RNA. Due to its contribution to an antitumor drug resistance, this enzyme is considered a promising therapeutic target. The elucidation of ALKBH3’s structural peculiarities is important to establish a detailed mechanism of damaged DNA recognition and processing, as well as to the development of specific inhibitors. This work presents new data on the wild type ALKBH3 protein and its four mutant forms (Y143F, Y143A, L177A, and H191A) obtained by circular dichroism (CD) spectroscopy. The dataset includes the CD spectra of proteins measured at different temperatures and a 3D visualization of the ALKBH3–DNA complex where the mutated amino acid residues are marked. These results show how substitution of the key amino acids influences a secondary structure content of the protein. Dataset: https://doi.org/10.17632/7bfsjtkgtb.1 Dataset License: CC BY 4.0 Keywords: CD spectroscopy; fluorescent spectroscopy; dioxygenase; AlkB-like proteins; ALKBH3; DNA methylation 1. Summary The ALKBH3 protein belongs to a large family of non-heme dioxygenases involved in the dealkylation of nucleic and amino acids [1]. This enzyme has a broad substrate specificity to methyl, ethyl, and etheno modifications of single-stranded DNA and RNA [2]. Increased level of expression of ALKBH3 in some human carcinomas stimulated attempts to control its expression by creating effective inhibitors [3–5]. This requires a clear under- standing of the mechanism of a lesion coordination within the enzyme’s active site. To date, there is a single crystal structure of wild type (WT) ALKBH3 bound to the Fe(II) ion and 2-oxoglutarate co-substrate but lack of DNA substrate [6]. Therefore, further research involving not only X-ray crystallography, but also various spectroscopic techniques is required to clarify the ALKBH3 active site structure and selectivity. Here, we present a comprehensive set of spectroscopic data which evaluate the secondary structure content and a thermal denaturation profile for WT and mutant variants of ALKBH3 dioxygenase. A 3D-structure of the catalytic complex of ALKBH3 with Fe(II), 2OG, and methylated DNA was visualized using the UCSF Chimera software (University of California, San Francisco, USA) [7]. Based on the structure, the mutant forms Y143F, Y143A, L177A, and H191A ALKBH3 have been selected for analysis by circular dichroism (CD) spectroscopy. The optimal conditions for the ALKBH3 samples and the buffer preparation as well as the Data 2023, 8, 57. https://doi.org/10.3390/data8030057 https://www.mdpi.com/journal/data