Abstract.
The gene (ORF APF0012) encoding the ATP-dependent 6-phosphofructokinase (ATP-PFK) of the hyperthermophilic archaeon Aeropyrum pernix was identified, cloned, and functionally expressed in Escherichia coli. The deduced amino acid sequence showed similarity (25–40%) to members of PFK-B sugar kinases. The purified recombinant enzyme is a heterotetramer of 115 kDa, composed of 34-kDa subunits. Rate dependence (at 85 °C) on both fructose 6-phosphate (F-6-P) and ATP followed Michaelis-Menten kinetics with apparent K m values of 0.25 mM and 0.68 mM, respectively; apparent V max values were about 5 U/mg. The enzyme was specific for ATP as phosphoryl donor, but showed a broader spectrum of phosphoryl acceptors: in addition to F-6-P, glucose 6-phosphate, adenosine, fructose, ribose 5-phosphate, and ribose were accepted. Enzyme activity required divalent cations; Mg2+, which was most effective, could partially be replaced by Co2+, Ni2+, or Mn2+. The enzyme had a temperature optimum of 90 °C and showed a significant thermostability up to 100 °C. ATP-PFK activity was not allosterically regulated by classical effectors of ATP-PFKs of eukarya and bacteria, such as ADP and phosphoenolpyruvate. In accordance, this archaeal ATP-PFK did not contain the typical conserved binding sites for these effectors. This is the first report of a sequence of an archaeal ATP-PFK related to the PFK-B sugar kinase family.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
Author information
Authors and Affiliations
Additional information
Electronic Publication
Rights and permissions
About this article
Cite this article
Hansen, T., Schönheit, P. Sequence, expression, and characterization of the first archaeal ATP-dependent 6-phosphofructokinase, a non-allosteric enzyme related to the phosphofructokinase-B sugar kinase family, from the hyperthermophilic crenarchaeote Aeropyrum pernix. Arch Microbiol 177, 62–69 (2001). https://doi.org/10.1007/s00203-001-0359-1
Received:
Revised:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/s00203-001-0359-1