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Enhancement of gene expression in Escherichia coli and characterization of highly stable ATP-dependent glucokinase from Pyrobaculum calidifontis

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Abstract

The genome of the hyperthermophilic archaeon Pyrobaculum calidifontis contains an open reading frame, Pcal_1032, annotated as glucokinase. Amino acid sequence analysis showed that Pcal_1032 belonged to ROK (repressor, open reading frame, and kinase) family of sugar kinases. To examine the properties of Pcal_1032, the coding gene was cloned and expressed in Escherichia coli. However, expression of the gene was low resulting in a poor yield of the recombinant protein. A single site directed mutation in Pcal_1032 gene, without altering the amino acid sequence, resulted in approximately tenfold higher expression. Purified recombinant Pcal_1032 efficiently phosphorylated various hexoses with a marked preference for glucose. ATP was the most preferred phosphoryl group donor. Optimum temperature and pH for the glucokinase activity of Pcal_1032 were 95 °C and 8.5, respectively. Catalytic efficiency (k cat/K m) towards glucose was 437 mM−1 s−1. The recombinant enzyme was highly stable against temperature with a half-life of 25 min at 100 °C. In addition, Pcal_1032 was highly stable in the presence of denaturants. There was no significant change in the CD spectra and enzyme activity of Pcal_1032 even after overnight incubation in the presence of 8 M urea. To the best of our knowledge, Pcal_1032 is the most active and highly stable glucokinase characterized to date from archaea, and this is the first description of the characterization of a glucokinase from genus Pyrobaculum.

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Acknowledgements

This work was partly supported by an NRPU Grant No. 20-2024 to NR from Higher Education Commission of Pakistan.

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Authors and Affiliations

  1. School of Biological Sciences, University of the Punjab, Lahore, 54590, Pakistan

    Tahira Bibi, Musadiq Ali, Naeem Rashid, Majida Atta Muhammad & Muhammad Akhtar

  2. School of Biological Sciences, University of Southampton, Southampton, SO16 7PX, UK

    Muhammad Akhtar

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  1. Tahira Bibi
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  2. Musadiq Ali
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  3. Naeem Rashid
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  4. Majida Atta Muhammad
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  5. Muhammad Akhtar
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Correspondence to Naeem Rashid.

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Communicated by L. Huang.

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Bibi, T., Ali, M., Rashid, N. et al. Enhancement of gene expression in Escherichia coli and characterization of highly stable ATP-dependent glucokinase from Pyrobaculum calidifontis . Extremophiles 22, 247–257 (2018). https://doi.org/10.1007/s00792-017-0993-4

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  • DOI: https://doi.org/10.1007/s00792-017-0993-4

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