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


Ontology type: schema:ScholarlyArticle     


Article Info

DATE

2018-03

AUTHORS

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

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. More... »

PAGES

247-257

Identifiers

URI

http://scigraph.springernature.com/pub.10.1007/s00792-017-0993-4

DOI

http://dx.doi.org/10.1007/s00792-017-0993-4

DIMENSIONS

https://app.dimensions.ai/details/publication/pub.1099927584

PUBMED

https://www.ncbi.nlm.nih.gov/pubmed/29275440


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48 schema:description 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 <sub>cat</sub>/K <sub>m</sub>) towards glucose was 437 mM<sup>-1</sup> s<sup>-1</sup>. 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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