Interdomain interactions rearrangements control the reaction steps of a thermostable DNA alkyltransferase

O6-alkylguanine-DNA Alkyltransferases in Microbes Living on the Edge: From Stability to Applicability

“…This non-fluorescent substrate lowers the final fluorescent signal on gel imaging analysis, depending on its concentration. In this way, it is possible to measure the activity of AGTs for their natural substrate, giving an indirect measure of methylation repair efficiency (Figure 3) [38,39,46,52,59,60]. By using this methodology, it was even possible to discriminate the SsOGT activity regarding the position of the O 6 -MG on DNA (see below; [39]), in line with previous data on hMGMT [64].…”

“…Because AGT covalently binds a benzyl-fluorescein moiety of its substrate after reaction, it is possible to immediately load the protein product on a SDS-PAGE-the gel-imaging analysis of the fluorescence intensity gives a direct measure of the protein activity because of the 1:1 stoichiometry of protein/substrate (Figure 3). Signals of fluorescent protein (corrected by the amount of loaded protein by Coomassie staining analysis) obtained at different times are plotted, and a second order reaction rate is determined [38,39,46,52,59,60]. This method can be applied to all AGTs that bind O 6 -BG, with the exception of the E. coli Ada-C [61,62].…”

“…The chimeric proteins were correctly folded, and the tag did not interfere with the enzymatic activity of the tetrameric S. solfataricus β-glycosidase (Ssβgly) [59], nor with the hyperthermophile-specific DNA topoisomerase reverse gyrase [68][69][70][71][72]. Furthermore, the stability of H 5 made possible a heat treatment of the cell-free extract to remove most of the E. coli proteins and performing the β-glycosidase assay at high temperatures without the need of removing the tag [60].…”

Understanding of the activity of a protein involved in DNA repair by biochemical, structural and in silico approaches

“…With this protein, we were able to highlight conformational changes and perturbations of intramolecular interaction occurring during lesion recognition and catalysis, confirming our previous hypothesis that coordination between the N-and C-terminal domains of SsOGT is important for protein activity and stability [2]. All the data allowed us to propose a general model of structural rearrangements occurring during the reaction cycle of AGTs [3], and proposing it as a starting point to design strategies to modulate AGT activity in therapeutic settings.…”

Targeting Genome Integrity in Mycobacterium Tuberculosis: From Nucleotide Synthesis to DNA Replication and Repair

“…MTB OGT, as the orthologous proteins of other organisms, invariably performs the removal of alkyl adducts on modified guanines through a suicidal mechanism, by catalyzing the stoichiometric transfer of the O6-alkyl group to the strictly conserved cysteine residue in the protein active site, which is hosted in the C-terminal domain [93][94][95]. The protein's overall structure and mechanistic aspects of the suicidal reaction are highly conserved among prokaryotic OGTs, as well as in the human equivalent enzyme [96][97][98][99][100], which is a validated target for cancer chemotherapy [101]. Small-molecule inhibitors of the human enzyme, which are currently used as adjuvants in antineoplastic therapeutic regimens, have not been tested on MTB; indeed, their potential exploitability as anti-tubercular drugs is limited by their cross-reactivity with the human protein, possibly resulting in a genotoxic effect.…”