Cloning, Expression, and Characterization of the First Archaeal ATR-Dependent Glucokinase from Aerobic Hyperthermophilic Archaeon <i>Aeropyrum pernix</i>

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  • Sakuraba Haruhiko
    Department of Biological Science and Technology, Faculty of Engineering, The University of Tokushima
  • Mitani Yuri
    Department of Biological Science and Technology, Faculty of Engineering, The University of Tokushima
  • Goda Shuichiro
    Department of Biological Science and Technology, Faculty of Engineering, The University of Tokushima
  • Kawarabayasi Yutaka
    The National Institute of Advanced Industrial Science and Technology
  • Ohshima Toshihisa
    Department of Biological Science and Technology, Faculty of Engineering, The University of Tokushima

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  • Cloning, Expression, and Characterization of the First Archaeal ATP-Dependent Glucokinase from Aerobic Hyperthermophilic Archaeon, Aeropyrum pernix

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The gene encoding the ATP-dependent glucokinase of hyperthermophilic archaeon Aeropyrum pernix was identified, cloned, and functionally expressed in Escherichia coli. The deduced amino acid sequence showed 40% identity to that of the putative glucokinase from hyperthermophilic archaeon Pyrobacurum aerophilum. The purified recombinant enzyme was a monomer with a molecular mass of 35 kDa. The enzyme retained its full activity on heating at 70°C for 10 min and retained 65% of the activity after 10-min incubation at 100°C. The enzyme exclusively catalyzed the phosphorylation of D-glucose using ATP as a phosphoryl donor. ITP was accepted in addition to ATP. The rate dependence with both glucose and ATP followed Michaelis-Menten kinetics, with apparent Km values of 0.054 and 0.50mM, respectively. The enzyme activity required divalent cations; Mg2+, which was most effective, could partially be replaced by Mn2+ or Ca2+. Phylogenetic analysis revealed that the glucokinase from A. pernix does not belong to the clusters of enzymes found in bacteria and eukarya. This is the first description of the characteristics of an ATP-dependent glucokinase from an archaeon.

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