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 hyperthermophi lic crenarchaeon Aeropyrum pernix and have subsequently expressed the prote in in Escherichia coli. The enzyme was purified with its associated histidi ne-tag by affinity chromatography with a nitrilotriacetic acid column follo wed by cation exchange chromatography and possesses a high degree of thermo stable ATP-dependent phosphofructokinase activity. The enzyme has an estima ted apparent K-m for ATP and fructose-6-phosphate of 0.027 and 1.212 mM, re spectively, that were determined in discontinuous assays at 95 degreesC. Th e Family B ATP-dependent phosphofructokinase has a half-life of approximate ly 30 min at 95 degreesC and is indicated to be monomeric. The implications of the presence of a Family B phosphofructokinase in the Crenarchaea are d iscussed with reference to the origins of the Embden-Meyerhof pathway. (C) 2001 Federation of European Microbiological Societies. Published by Elsevie r Science B.V. All rights reserved.