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.
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.
Hansen
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Institut für Allgemeine Mikrobiologie, Christian-Albrechts-Universität Kiel, Am Botanischen Garten 1-9, 24118 Kiel, Germany.
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D000255:Adenosine Triphosphate / Q000378:metabolism*; D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D019620:Desulfurococcaceae / Q000201:enzymology* / Q000235:genetics; D006358:Hot Temperature; D007700:Kinetics; D008969:Molecular Sequence Data; D025221:Phosphofructokinases / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D025221:Phosphofructokinases