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Purification and characterization of 5′-methylthioadenosine phosphorylase from the hyperthermophilic archaeon Pyrococcus furiosus

Substrate specificity and primary structure analysis

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Abstract

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 °C, and extremely thermostable, retaining 98% residual activity after 5 h at 100 °C and showing a half-life of 43 min at 130 °C. In the presence of 100 mM phosphate, the apparent T m (137 °C) increases to 139 °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 phosphorolytically cleaves adenosine, inosine, and guanosine. The calculated k cat/K m values for 5′-methylthioadenosine and adenosine, about 20-fold higher than for inosine and guanosine, indicate that 6-amino purine nucleosides are preferred substrates of MTAP from P. furiosus. The structural features and the substrate specificity of MTAP from P. furiosus document that it represents a 5′-methylthioadenosine-metabolizing enzyme different from those previously characterized among Archaea, Bacteria, and Eukarya. The functional and structural relationships among MTAP from P. furiosus, human MTAP, and two putative MTAPs from P. furiosus and Sulfolobus solfataricus are discussed here for the first time.

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Acknowledgements

We sincerely thank Dr. V. Carratore, CNR, Naples, Italy, for performing NH2-terminal analysis.

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Authors and Affiliations

  1. Dipartimento di Biochimica e Biofisica "F. Cedrangolo", Seconda Università di Napoli, Via Costantinopoli 16, 80138, Naples, Italy

    Giovanna Cacciapuoti, Assunta Brio, Vincenzo Zappia & Marina Porcelli

  2. Technical Microbiology, Technical University Hamburg-Harburg, Kasernenstrasse 12, 21073 , Hamburg, Germany

    Costanzo Bertoldo

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  1. Giovanna Cacciapuoti
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  2. Costanzo Bertoldo
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  3. Assunta Brio
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  4. Vincenzo Zappia
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  5. Marina Porcelli
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Correspondence to Giovanna Cacciapuoti.

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Communicated by G. Antranikian

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Cacciapuoti, G., Bertoldo, C., Brio, A. et al. Purification and characterization of 5′-methylthioadenosine phosphorylase from the hyperthermophilic archaeon Pyrococcus furiosus . Extremophiles 7, 159–168 (2003). https://doi.org/10.1007/s00792-002-0307-2

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