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J. Biol. Chem., Vol. 276, Issue 42, 39232-39242, October 19, 2001

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Three-dimensional Structure of a Hyperthermophilic 5'-Deoxy-5'-methylthioadenosine Phosphorylase from Sulfolobus solfataricus^*

Todd C. Appleby, Irimpan I. Mathews, Marina Porcelli^§, Giovanna Cacciapuoti^§, and Steven E. Ealick^¶

From the  Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853 and the ^§ Dipartimento di Biochimica e Biofisica, Seconda Universita di Napoli, Via Costantinopoli, 16, 80138 Naples, Italy

The structure of 5'-deoxy-5'-methylthioadenosine phosphorylase from Sulfolobus solfataricus (SsMTAP) has been determined alone, as ternary complexes with sulfate plus substrates 5'-deoxy-5'-methylthioadenosine, adenosine, or guanosine, or with the noncleavable substrate analog Formycin B and as binary complexes with phosphate or sulfate alone. The structure of unliganded SsMTAP was refined at 2.5-Å resolution and the structures of the complexes were refined at resolutions ranging from 1.6 to 2.0 Å. SsMTAP is unusual both for its broad substrate specificity and for its extreme thermal stability. The hexameric structure of SsMTAP is similar to that of purine-nucleoside phosphorylase (PNP) from Escherichia coli, however, only SsMTAP accepts 5'-deoxy-5'-methylthioadenosine as a substrate. The active site of SsMTAP is similar to that of E. coli PNP with 13 of 18 nearest residues being identical. The main differences are at Thr⁸⁹, which corresponds to serine in E. coli PNP, and Glu¹⁶³, which corresponds to proline in E. coli PNP. In addition, a water molecule is found near the purine N-7 position in the guanosine complex of SsMTAP. Thr⁸⁹ is near the 5'-position of the nucleoside and may account for the ability of SsMTAP to accept either hydrophobic or hydrophilic substituents in that position. Unlike E. coli PNP, the structures of SsMTAP reveal a substrate-induced conformational change involving Glu¹⁶³. This residue is located at the interface between subunits and swings in toward the active site upon nucleoside binding. The high-resolution structures of SsMTAP suggest that the transition state is stabilized in different ways for 6-amino versus 6-oxo substrates. SsMTAP has optimal activity at 120 °C and retains full activity after 2 h at 100 °C. Examination of the three-dimensional structure of SsMTAP suggests that unlike most thermophilic enzymes, disulfide linkages play a key in role in its thermal stability.

The atomic coordinates and structure factors (code 1JDU (native SsMTAP), IJE1 (complex with guanosine and sulfate), 1JDV (complex with adenosine and sulfate), 1JDT (complex with MTA and sulfate), 1JDS (complex with phosphate (space group: P2₁)), 1JP7 (complex with sulfate), 1JDZ (complex with Formycin B and sulfate), and 1JE0 (complex with phosphate (space group C222₁))) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

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