A journey down to hell: new thermostable protein-tags for biotechnology at high temperatures

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

“…However, the experiments were performed at 65 • C instead of the standard procedure at 50 • C, as described for SsOGT [39,59,60], due to the strong thermophilicity of this enzyme. This behaviour was effectively confirmed by differential scan fluorimetry analysis where the temperature melting (T m ) of PfuOGT was found to be 80 • C, much higher than that of SsOGT (68 • C) [89]. It is worth noting that, in order to obtain a the sigmoidal melting curve for PfuOGT, a slower heating rate (10 min/ • C × cycle) was set up, whereas the T m value measurement is usually performed at 1 min/ • C × cycle [93].…”

“…The PF1878 ORF is relative to a protein of 20.1 kDa. From its primary structure, the relative polypeptide seems to be closely related to the MGMT from Pyrococcus kodakarensis KOD1 (Pk-MGMT) [48,89,92]. The extreme thermostability was confirmed by in vitro biochemical studies on the heterologous expressed and characterized OGT protein from P. furiosus PfuOGT.…”

“…As shown in Figure 3, the IC 50 value was similar to that obtained for SsOGT. Surprisingly, the enzyme from T. neapolitana exhibited a very high activity at low temperatures [89], similar to that possessed by the mutant SsOGT-H 5 (Table 3) [52,59]. Superimposition analysis between a TnOGT 3D model and the free form of SsOGT (ID PDB: 4ZYE) revealed in both structures the presence of a serine residue in the active site loop (S132 in SsOGT, see Figure 2a), which was replaced in SsOGT-H 5 by a glutamic acid to improve its activity at lower temperatures.…”

“…SDS-PAGE gel imaging analysis on lyophilized T. neapolitana cells incubated with the AGT fluorescent substrate showed a strong fluorescent signal with a molecular weight close to that of SsOGT. The observed molecular weight and, above all, the sensitivity to the O 6 -BG derivative, led to the cloning and heterologous expression of the Thermotoga neapolitana OGT protein (TnOGT) in E. coli [89]. This protein, like most AGTs, has a role in DNA repair, as confirmed by competitive fluorescent assay in the presence of methylated dsDNA.…”

“…To extend the SNAP-tag technology to hyperthermophilic microorganisms for in vivo studies, an O 6 -alklylguanine-DNA alkyltransferase has been recently characterized from the archaeon Pyrococcus furiosus [89]. This extremophilic microorganism was originally isolated from hot marine sediments in Vulcano Island (Italy) [90], with an optimum growth temperature around 100 • C, thus thriving under extremely harsh conditions.…”