DNA-alkyl-transferases (called AGT, OGT or MGMT) are ubiquitous proteins involved in the repair and protection of mutagenic and carcinogenic DNA adducts induced by exposure to alkylating agents [1; 2]. On the other hand, they also play a role in the onset of resistance to chloroethylanting and methylanting agents used for the treatment of several cancer types [3]. Dealkylation reaction catalysed by DNA-alkyl-transferases occurs by transfer of the alkylic group from O6-alkyl-guanine or O4-alkyl-thymine to a cysteine residue in the active site of the protein. The stoichiometry of the reaction is 1:1 and the alkylated form of the protein is irreversibly inactivated. Therefore, DNA-alkyl-transferases are not properly enzymes, rather they act as suicide or "kamikaze" proteins. Human AGT (hAGT) is strongly affected by O6-benzyl-guanine (O6-BG), a non-toxic powerful inhibitor that prevents the activity of hAGT by covalent transfer of the benzylic group to the active site cysteine [2]. The unusual covalent bond between the cysteine in the hAGT active site and the benzylic group of this substrate/inhibitor can be exploited to label AGT, provided that the O6-BG is conjugated with appropriate chemical groups. hAGT and its variants have been successfully used as protein tags fused to target proteins, for many applications in biochemistry and cell biology [4-6]. Over the last decades, proteins from (hyper)thermophilic organisms have proven useful in several biotechnological applications, due to their intrinsic stability. Likewise, the use of a thermostable AGT as a protein tag could outperform the hAGT for its inherent stability and activity in a broad range of reaction conditions. We have cloned, purified and characterized the DNA-alkyl-transferase from the hyperthermophilic archaeon Sulfolobus solfataricus (SsOGT). This protein shows interesting biochemical features and its potentiality, as a flexible biotechnological tool for several applications, will be discussed. [1]Fang Q., Kanugula S., and Pegg A.E. (2005). Function of domains of human O6-alkylguanine DNA alkyltransferase. Biochemistry, 44, 46: 15396-405. [2]Yang C.G., Garcia K., and He C. (2009). Damage detection and base flipping in direct DNA alkylation repair. Chembiochem, 10, 3: 417-23. [3]Rabik C.A., Njoku M.C., and Dolan M.E. (2006). Inactivation of O6-alkylguanine DNA alkyltransferase as a means to enhance chemotherapy. Cancer Treat. Rev., 32, 4: 261-276. [4]Keppler A., Gendreizig S., Gronemeyer T., Pick H., Vogel H., and Johnsson K. (2003). A general method for the covalent labeling of fusion proteins with small molecules in vivo. Nat. Biotechnol., 21, 86-89. [5]Keppler A, Pick H, Arrivoli C, Vogel H, Johnsson K. (2004). Labeling of fusion proteins with synthetic fluorophores in live cells. Proc. Natl. Acad. Sci. USA, 10: 9955-9959. [6]Gautier A., Juillerat A., Heinis C., Corrêa I.R. Jr, Kindermann M., Beaufils F., and Johnsson K. (2008). An engineered protein tag for multiprotein labeling in living cells. Chem. Biol., 15: 128-136.
The Sulfolobus solfataricus DNA alkyltransferase and its application as a protein tag
Perugino Giuseppe
2010
Abstract
DNA-alkyl-transferases (called AGT, OGT or MGMT) are ubiquitous proteins involved in the repair and protection of mutagenic and carcinogenic DNA adducts induced by exposure to alkylating agents [1; 2]. On the other hand, they also play a role in the onset of resistance to chloroethylanting and methylanting agents used for the treatment of several cancer types [3]. Dealkylation reaction catalysed by DNA-alkyl-transferases occurs by transfer of the alkylic group from O6-alkyl-guanine or O4-alkyl-thymine to a cysteine residue in the active site of the protein. The stoichiometry of the reaction is 1:1 and the alkylated form of the protein is irreversibly inactivated. Therefore, DNA-alkyl-transferases are not properly enzymes, rather they act as suicide or "kamikaze" proteins. Human AGT (hAGT) is strongly affected by O6-benzyl-guanine (O6-BG), a non-toxic powerful inhibitor that prevents the activity of hAGT by covalent transfer of the benzylic group to the active site cysteine [2]. The unusual covalent bond between the cysteine in the hAGT active site and the benzylic group of this substrate/inhibitor can be exploited to label AGT, provided that the O6-BG is conjugated with appropriate chemical groups. hAGT and its variants have been successfully used as protein tags fused to target proteins, for many applications in biochemistry and cell biology [4-6]. Over the last decades, proteins from (hyper)thermophilic organisms have proven useful in several biotechnological applications, due to their intrinsic stability. Likewise, the use of a thermostable AGT as a protein tag could outperform the hAGT for its inherent stability and activity in a broad range of reaction conditions. We have cloned, purified and characterized the DNA-alkyl-transferase from the hyperthermophilic archaeon Sulfolobus solfataricus (SsOGT). This protein shows interesting biochemical features and its potentiality, as a flexible biotechnological tool for several applications, will be discussed. [1]Fang Q., Kanugula S., and Pegg A.E. (2005). Function of domains of human O6-alkylguanine DNA alkyltransferase. Biochemistry, 44, 46: 15396-405. [2]Yang C.G., Garcia K., and He C. (2009). Damage detection and base flipping in direct DNA alkylation repair. Chembiochem, 10, 3: 417-23. [3]Rabik C.A., Njoku M.C., and Dolan M.E. (2006). Inactivation of O6-alkylguanine DNA alkyltransferase as a means to enhance chemotherapy. Cancer Treat. Rev., 32, 4: 261-276. [4]Keppler A., Gendreizig S., Gronemeyer T., Pick H., Vogel H., and Johnsson K. (2003). A general method for the covalent labeling of fusion proteins with small molecules in vivo. Nat. Biotechnol., 21, 86-89. [5]Keppler A, Pick H, Arrivoli C, Vogel H, Johnsson K. (2004). Labeling of fusion proteins with synthetic fluorophores in live cells. Proc. Natl. Acad. Sci. USA, 10: 9955-9959. [6]Gautier A., Juillerat A., Heinis C., Corrêa I.R. Jr, Kindermann M., Beaufils F., and Johnsson K. (2008). An engineered protein tag for multiprotein labeling in living cells. Chem. Biol., 15: 128-136.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
