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Structure of dimeric, recombinant Sulfolobus solfataricus phosphoribosyl diphosphate synthase: a bent dimer defining the adenine specificity of the substrate ATP

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Abstract

The enzyme 5-phosphoribosyl-1-α-diphosphate (PRPP) synthase (EC 2.7.6.1) catalyses the Mg2+-dependent transfer of a diphosphoryl group from ATP to the C1 hydroxyl group of ribose 5-phosphate resulting in the production of PRPP and AMP. A nucleotide sequence specifying Sulfolobus solfataricus PRPP synthase was synthesised in vitro with optimised codon usage for expression in Escherichia coli. Following expression of the gene in E. coli PRPP synthase was purified by heat treatment and ammonium sulphate precipitation and the structure of S. solfataricus PRPP synthase was determined at 2.8 Å resolution. A bent dimer oligomerisation was revealed, which seems to be an abundant feature among PRPP synthases for defining the adenine specificity of the substrate ATP. Molecular replacement was used to determine the S. solfataricus PRPP synthase structure with a monomer subunit of Methanocaldococcus jannaschii PRPP synthase as a search model. The two amino acid sequences share 35 % identity. The resulting asymmetric unit consists of three separated dimers. The protein was co-crystallised in the presence of AMP and ribose 5-phosphate, but in the electron density map of the active site only AMP and a sulphate ion were observed. Sulphate ion, reminiscent of the ammonium sulphate precipitation step of the purification, seems to bind tightly and, therefore, presumably occupies and blocks the ribose 5-phosphate binding site. The activity of S. solfataricus PRPP synthase is independent of phosphate ion.

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Abbreviations

PRPP:

5-Phospho-d-ribosyl 1-α-diphosphate

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Acknowledgments

We thank Qunxin She (University of Copenhagen) for kindly providing DNA of S. solfataricus, Tonny D. Hansen, Jens-Christian N. Poulsen and Dorthe Boelskifte for pertinent technical assistance. The MAXLAB and DANSCATT are gratefully acknowledged for providing the infrastructure facilitating collection of synchrotron data.

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical standards

The experiments performed in this study comply with the current laws of Denmark.

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Authors and Affiliations

  1. Department of Chemistry, University of Copenhagen, 5 Universitetsparken, 2100, Copenhagen Ø, Denmark

    Rune W. Andersen, Leila Lo Leggio, Bjarne Hove-Jensen & Anders Kadziola

Authors
  1. Rune W. Andersen
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  2. Leila Lo Leggio
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  3. Bjarne Hove-Jensen
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  4. Anders Kadziola
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Corresponding authors

Correspondence to Bjarne Hove-Jensen or Anders Kadziola.

Additional information

Communicated by S. Albers.

The atomic coordinates and structure factors have been deposited in the Protein Data Bank (PDB code 4TWB), Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org).

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Andersen, R.W., Leggio, L.L., Hove-Jensen, B. et al. Structure of dimeric, recombinant Sulfolobus solfataricus phosphoribosyl diphosphate synthase: a bent dimer defining the adenine specificity of the substrate ATP. Extremophiles 19, 407–415 (2015). https://doi.org/10.1007/s00792-014-0726-x

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  • DOI: https://doi.org/10.1007/s00792-014-0726-x

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