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. 2001 Dec;177(1):62-9. doi: 10.1007/s00203-001-0359-1. Epub 2001 Oct 25.

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 degrees 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; Mg(2+), which was most effective, could partially be replaced by Co(2+), Ni(2+), or Mn(2+). The enzyme had a temperature optimum of 90 degrees C and showed a significant thermostability up to 100 degrees 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphate / metabolism*
  • Allosteric Regulation
  • Amino Acid Sequence
  • Cloning, Molecular
  • Desulfurococcaceae / enzymology*
  • Desulfurococcaceae / genetics
  • Hot Temperature
  • Kinetics
  • Molecular Sequence Data
  • Phosphofructokinases / genetics
  • Phosphofructokinases / metabolism*
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Sequence Alignment
  • Sequence Analysis, DNA*

Substances

  • Recombinant Proteins
  • Adenosine Triphosphate
  • Phosphofructokinases