A novel hyperthermostable 5'-deoxy-5'-methylthioadenosine phosphorylase from the archaeon Sulfolobus solfataricus

The FEBS Journal
Giovanna CacciapuotiMarina Porcelli

Abstract

We report herein the first molecular characterization of 5'-deoxy-5'-methylthio-adenosine phosphorylase II from Sulfolobus solfataricus (SsMTAPII). The isolated gene of SsMTAPII was overexpressed in Escherichia coli BL21. Purified recombinant SsMTAPII is a homohexamer of 180 kDa with an extremely low Km (0.7 microm) for 5'-deoxy-5'-methylthioadenosine. The enzyme is highly thermophilic with an optimum temperature of 120 degrees C and extremely thermostable with an apparent Tm of 112 degrees C that increases in the presence of substrates. The enzyme is characterized by high kinetic stability and remarkable SDS resistance and is also resistant to guanidinium chloride-induced unfolding with a transition midpoint of 3.3 m after 22-h incubation. Limited proteolysis experiments indicated that the only one proteolytic cleavage site is localized in the C-terminal region and that the C-terminal peptide is necessary for the integrity of the active site. Moreover, the binding of 5'-deoxy-5'-methylthioadenosine induces a conformational transition that protected the enzyme against protease inactivation. By site-directed mutagenesis we demonstrated that Cys259, Cys261 and Cys262 play an important role in the enzyme stability since the mutant...Continue Reading

References

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Citations

Nov 20, 2012·Applied Microbiology and Biotechnology·Shaozhou ZhuGuojun Zheng
Sep 24, 2011·Archaea : an International Microbiological Journal·Julien Jorda, Todd O Yeates
Oct 20, 2015·Journal of Cellular Biochemistry·Giovanna CacciapuotiMarina Porcelli
Mar 8, 2006·The FEBS Journal·Maria G TozziPiero L Ipata
Mar 31, 2015·Nature Chemical Biology·Riku AonoHaruyuki Atomi
Feb 2, 2008·The Biochemical Journal·Joaquín Fernández-IrigoyenFernando J Corrales

Related Concepts

5'-Methylthioadenosine phosphorylase
Enzyme Stability
Protein Conformation
Nicotinamide Riboside Phosphorylase
Thermodynamics
Determination, Sequence Homology
Sulfolobus solfataricus

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