Characterization of a Family IV uracil DNA glycosylase from the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5

Abstract

The hyperthermophilic euryarchaeon Thermococcus barophilus Ch5 encodes two uracil DNA glycosylases (UDGs): Tba UDG247 and Tba UDG194. In our previous publication, we revealed biochemical characterization of Tba UDG247. Herein, we characterized biochemically Tba UDG194, which is a member of Family IV UDG, demonstrating that this enzyme has similar efficiencies for cleaving uracil-containing ssDNA and dsDNA. Compared with Tba UDG247, Tba UDG194 exhibits different biochemical characteristics. At >85 o C, >90 cleavage percentage was observed, suggesting that Tba UDG194 can remove uracil from DNA at the close physiological temperature of its host. Thus, the enzyme has been currently the most thermophilic glycosylase among all the reported UDGs. Furthermore, the optimal pH of the enzyme activity was estimated to be 10, which is higher than that of Tba UDG247. Similar to Tba UDG247, Tba UDG194 activity is independent on a divalent metal ion. Mn 2+ , Zn 2+ and Cu 2+ display inhibitory effect on the enzyme activity at varied degreed whereas Mg 2+ and Ca 2+ have no detectable effect on the enzyme activity. In addition, Tba UDG194 is a salt-tolerant enzyme that retains compromised activity at 600 mM NaCl. Furthermore, Tba UDG 194 displays the following substrate preference: U≈U/ G>U/T≈U/C>U/A. The Arrhenius activation energy was etsimated to be 20.1 ± 3.4 kcal/mol, theoretically representing the energy barrier for uracil removal from DNA by Tba UDG194. Overall, our observations suggest that Tba UDG194 might be involved in removal of uracil in DNA in Thermococcus cells.

Identification of a novel bifunctional uracil DNA glycosylase from Thermococcus barophilus Ch5

“…Ch5 is both hyperthermophilic (an optimal temperature of 85 o C) and piezophilic (an optimal pressure of 40 MPa) (Marteinsson et al 1999). Our results have shown that both enzymes can remove uracil from DNA at high temperature (Shi et al 2019;Gan et al 2020). Tba UDG194 is a classical family IV UDG member, possessing the conserved motifs present in other family IV members (Gan et al 2020).…”

“…Our results have shown that both enzymes can remove uracil from DNA at high temperature (Shi et al 2019;Gan et al 2020). Tba UDG194 is a classical family IV UDG member, possessing the conserved motifs present in other family IV members (Gan et al 2020). In contrast, Tba UDG247 lacks the conserved motifs found in family IV and family V UDG members, although being most closely related to family V UDGs (Shi et al 2019).…”

Characterization of a novel type III alcohol dehydrogenase from Thermococcus barophilus Ch5

“…The genome of T. barophilus Ch5 encodes 2,679 proteins, two thirds of which have no associated functions [24]. Furthermore, several thermostable enzymes from T. barophilus Ch5 have been characterized [25][26][27][28][29] Three putative alcohol dehydrogenases (Tba ADHs) are encoded in the genome of T. barophilus Ch5, including one short-chain ADH (GenBank: ALM74123) and two putative type III ADHs (GenBank: ALM74514 and ALM74601). Interestingly, these two putative Fe-ADHs from T. barophilus Ch5 have only 26% similarity in amino acid compositions, suggesting that they might harbor distinct characteristics.…”