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.
Ronimus
R S
RS
Thermophile Research Unit, University of Waikato, Hamilton, New Zealand. ronimus@waikato.ac.nz
Kawarabayasi
Y
Y
Kikuchi
H
H
Morgan
H W
HW
D000255:Adenosine Triphosphate / Q000378:metabolism*; D003001:Cloning, Molecular; D019615:Crenarchaeota / Q000201:enzymology* / Q000235:genetics*; D019143:Evolution, Molecular; D019766:Genes, Archaeal / Q000235:genetics; D006019:Glycolysis / Q000502:physiology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D025221:Phosphofructokinases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D013379:Substrate Specificity; D013696:Temperature
D000255:Adenosine Triphosphate; D025221:Phosphofructokinases