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Purine nucleoside phosphorylase from Pseudoalteromonas sp. Bsi590: molecular cloning, gene expression and characterization of the recombinant protein

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Abstract

The gene encoding purine nucleoside phosphorylase (PNP) from the cold-adapted marine bacterium Pseudoalteromonas sp. Bsi590 was identified, cloned and expressed in Escherichia coli. The gene encodes a polypeptide of 233 amino acids with a calculated molecular weight of 25,018 Da. Pseudoalteromonas sp. Bsi590 PNP (PiPNP) shares 60% amino sequence identity and conservation of amino acid residues involved in catalysis with mesophilic Escherichia coli deoD-encoded purine nucleoside phosphorylase (EcPNP). N-terminal his-tagged PiPNP and EcPNP were purified to apparent homogeneity using Ni2+-chelating column. Compared with EcPNP, PiPNP possessed a lower temperature optimum and thermal stability. As for PNP enzymes in general, PiPNP and EcPNP displayed complicated kinetic properties; PiPNP possessed higher K m and catalytic efficiency (k cat/K m ) compared to EcPNP at 37°C. Substrate specificity results showed PiPNP catalyzed the phosphorolytic cleavage of 6-oxopurine and 6-aminopurine nucleosides (or 2-deoxynucleosides), and to a lesser extent purine arabinosides. PiPNP showed a better activity with inosine while no activity toward pyrimidine nucleosides. The protein conformation was analyzed by temperature perturbation difference spectrum. Results showed that PiPNP had lower conformation transition point temperature than EcPNP; phosphate buffer and KCl had significant influence on PiPNP protein conformation stability and thermostability.

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Abbreviations

PiPNP:

Pseudoalteromonas sp. Bsi590 purine nucleoside phosphorylase

EcPNP:

Escherichia coli purine nucleoside phosphorylase

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Acknowledgments

We sincerely thank Dr. Li Huirong of the Polar Reasearch Institute of China for the donation of Pseudoalteromonas spp. We are also grateful to the State Key Lab of Genetic Engineering for HPLC and CD measurements. We thank Ji Chaoneng and Hu Hairong for expert technical assistance. This work was financially supported by National Natural Science Foundation of China under the grant number 40376001.

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Authors and Affiliations

  1. The State Key Laboratory of Genetic Engineering, Institute of Genetics, Fudan University, 200433, Shanghai, China

    Xiaohui Li, Xinyin Jiang & Daming Ren

  2. SOA Key Laboratory for Polar Science, Polar Research Institute of China, 200136, Shanghai, China

    Huirong Li

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  1. Xiaohui Li
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  2. Xinyin Jiang
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  3. Huirong Li
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  4. Daming Ren
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Correspondence to Daming Ren.

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Communicated by K. Horikoshi.

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Li, X., Jiang, X., Li, H. et al. Purine nucleoside phosphorylase from Pseudoalteromonas sp. Bsi590: molecular cloning, gene expression and characterization of the recombinant protein. Extremophiles 12, 325–333 (2008). https://doi.org/10.1007/s00792-007-0131-9

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  • DOI: https://doi.org/10.1007/s00792-007-0131-9

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