Open AccessCloning, expression and characterisation of a Family B ATP‐dependent phosphofructokinase activity from the hyperthermophilic crenarachaeon Aeropyrum pernix
Author(s) -
Ronimus Ron S.,
Kawarabayasi Yutaka,
Kikuchi Hisasi,
Morgan Hugh W.
Publication year - 2001
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.2001.tb10784.x
Subject(s) - phosphofructokinase , biochemistry , biology , affinity chromatography , histidine , fructose , enzyme , thermostability , escherichia coli , microbiology and biotechnology , glycolysis , gene
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°C. The Family B ATP‐dependent phosphofructokinase has a half‐life of approximately 30 min at 95°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.