Cloning, expression and characterisation of a Family B ATP-dependent phosphofructokinase activity from the hyperthermophilic crenarachaeon Aeropyrum pernix.

We have cloned a Family B sugar kinase gene from the aerobic hyperthermophilic crenarchaeon Aeropyrum pernix and have subsequently expressed the protein in Escherichia coli. The enzyme was purified with its associated histidine-tag by affinity chromatography with a nickel-nitrilotriacetic acid column followed by cation exchange chromatography and possesses a high degree of thermostable ATP-dependent phosphofructokinase activity. The enzyme has an estimated apparent K(m) for ATP and fructose-6-phosphate of 0.027 and 1.212 mM, respectively, that were determined in discontinuous assays at 95 degrees C. The Family B ATP-dependent phosphofructokinase has a half-life of approximately 30 min at 95 degrees C and is indicated to be monomeric. The implications of the presence of a Family B phosphofructokinase in the Crenarchaea are discussed with reference to the origins of the Embden-Meyerhof pathway.

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