Purification and characterization of 5'-methylthioadenosine phosphorylase from the hyperthermophilic archaeon Pyrococcus furiosus: substrate specificity and primary structure analysis

Extremophiles : Life Under Extreme Conditions
G CacciapuotiM Porcelli


5'-Methylthioadenosine phosphorylase (MTAP) was purified to homogeneity from the hyperthermophilic archaeon Pyrococcus furiosus. The protein is a homoexamer of 180 kDa. The enzyme is highly thermoactive, with an optimum temperature of 125 degrees C, and extremely thermostable, retaining 98% residual activity after 5 h at 100 degrees C and showing a half-life of 43 min at 130 degrees C. In the presence of 100 mM phosphate, the apparent T(m) (137 degrees C) increases to 139 degrees C. The enzyme is extremely stable to proteolytic cleavage and after incubation with protein denaturants, detergents, organic solvents, and salts even at high temperature. Thiol groups are not involved in the catalytic process, whereas disulfide bond(s) are present, since incubation with 0.8 M dithiothreitol significantly reduces the thermostability of the enzyme. N-Terminal sequence analysis of the purified enzyme is 100% identical to the predicted amino acid sequence of the gene PF0016 from the partially sequenced P. furiosus genome. The deduced amino acid sequence of the gene revealed a high degree of identity (52%) with human MTAP. Nevertheless, unlike human MTAP, MTAP from P. furiosus is not specific for 5'-methylthioadenosine, since it phosphoroly...Continue Reading


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Related Concepts

5'-Methylthioadenosine phosphorylase
Enzyme Stability
Nicotinamide Riboside Phosphorylase
Substrate Specificity
Homologous Sequences, Amino Acid
Reducing Agents
Genes, Archaeal
Pyrococcus furiosus

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