1. Biochemical characterization and mutational studies of the 8-oxoguanine DNA glycosylase from the hyperthermophilic and radioresistant archaeon Thermococcus gammatolerans
- Author
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Zhihui Yang, Yuting Li, Haoqiang Shi, Likui Zhang, Jianting Zheng, Dai Zhang, Philippe Oger, Yangzhou University, Peking University [Beijing], Agricultural University of Hebei, Microbiologie, adaptation et pathogénie (MAP), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Institut National des Sciences Appliquées (INSA)-Université de Lyon-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS), Adaptation aux milieux extrêmes (AME), Institut National des Sciences Appliquées (INSA)-Université de Lyon-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Institut National des Sciences Appliquées (INSA)-Université de Lyon-Institut National des Sciences Appliquées (INSA)-Université de Lyon-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon, and Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées de Lyon (INSA Lyon)
- Subjects
Guanine ,DNA Mutational Analysis ,Mutant ,Enzyme Activators ,Cleavage (embryo) ,Applied Microbiology and Biotechnology ,DNA Glycosylases ,03 medical and health sciences ,chemistry.chemical_compound ,Enzyme Stability ,Enzyme Inhibitors ,ComputingMilieux_MISCELLANEOUS ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,biology ,030306 microbiology ,Chemistry ,Temperature ,Thermococcus gammatolerans ,[SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology ,General Medicine ,Base excision repair ,Hydrogen-Ion Concentration ,biology.organism_classification ,Thermococcus ,Enzyme ,Biochemistry ,DNA glycosylase ,Mutant Proteins ,DNA ,Biotechnology - Abstract
8-oxoguanine (GO) is a major lesion found in DNA that arises from guanine oxidation. The hyperthermophilic and radioresistant euryarchaeon Thermococcus gammatolerans encodes an archaeal GO DNA glycosylase (Tg-AGOG). Here, we characterized biochemically Tg-AGOG and probed its GO removal mechanism by mutational studies. Tg-AGOG can remove GO from DNA at high temperature through a β-elimination reaction. The enzyme displays an optimal temperature, ca.85–95 °C, and an optimal pH, ca.7.0–8.5. In addition, Tg-AGOG activity is independent on a divalent metal ion. However, both Co2+ and Cu2+ inhibit its activity. The enzyme activity is also inhibited by NaCl. Furthermore, Tg-AGOG specifically cleaves GO-containing dsDNA in the order: GO:C, GO:T, GO:A, and GO:G. Moreover, the temperature dependence of cleavage rates of the enzyme was determined, and from this, the activation energy for GO removal from DNA was first estimated to be 16.9 ± 0.9 kcal/mol. In comparison with the wild-type Tg-AGOG, the R197A mutant has a reduced cleavage activity for GO-containing DNA, whereas both the P193A and F167A mutants exhibit similar cleavage activities for GO-containing DNA. While the mutations of P193 and F167 to Ala lead to increased binding, the mutation of R197 to Ala had no significant effect on binding. These observations suggest that residue R197 is involved in catalysis, and residues P193 and F167 are flexible for conformational change.
- Published
- 2019
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