Astrobiology Workshop, Macquarie University July 12-13 2001

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Characterisation of a Family B ATP-dependent phosphofructokinase activity from the hyperthermophilic crenarchaeal Aeropyrum pernix: implications for the evolution of metabolism

Ron Ronimus (University of Waikato - Thermophile Research Unit)

Phosphofructokinase (PFK) represents a defining enzymatic activity of the glycolytic or Embden-Meyerhof pathway and is responsible for catalysing the (inter)conversion of fructose-6-phosphate (F-6-P) to fructose-1,6-bisphosphate (F-1,6-P2). The glycolytic pathway is present in each of the domains although there are a number of variations of the classical theme that are found in the archaea and hypethermophilic bacteria. Due to the universal presence of the pathway within the three domains the biochemical properties and phylogenies of enzymes of the pathway can aid in our elucidation of probable scenarios for the development of metabolism in the early archaean time. Three gene sequence families are now recognised that code for PFKs. The PFKA family currently consists of both ATP-PFKs and PPi-PFKs and are found in the domains Bacteria and Eukarya with the only exception known being a PPi-PFK being found in the hyperthermophilic crenarchaeal Thermoproteus tenax. Family B PFKs are ATP-dependent and very few have been characterised. The best example is that from E. coli where it represents a minor PFK, accounting for approximately 10% of the total PFK activity. A third family of PFKs are only found in some members of the Euryarchaea and are ADP-dependent. We have recently finished the cloning, expression and characterisation of a Family B ATP-PFK activity from the aerobic crenarchaeon Aeropyrum pernix which grows optimally at 95°C. The enzyme has a pH optimum of 5.5-6.5, a KCl optimum of 50-100 mM, a Mg2+ optimum of 20-30 mM. The apparent Km values for ATP and F-6-P were 0.027 mM and 1.212 mM, respectively. The implications of the first known Family B PFK activity within the archaea is discussed with respect to the origins of the Embden-Meyerhof pathway. In addition, the distribution of glycolytic/gluconeogenic enzymes in hyperthemophiles is reviewed.