Sequencing, expression, characterisation and phylogeny of the ADP-dependent phosphofructokinase from the hyperthermophilic, euryarchaeal Thermococcuszilligii

The full-length gene encoding the ADP-dependent phosphofructokinase (PFK) f rom the euryarchaeal Thermococcus zilligii was cloned, using degenerate pri mer polymerase chain reaction (PCR) combined with inverse-PCR techniques, a nd ultimately expressed in Escherichia coli. The expressed enzyme was bioch emically characterised and found to be similar to the native enzyme for mos t properties examined. Sequence database searches suggest that this unique ADP-PFK possesses a limited phylogenetic distribution with homologues being found only in the other euryarchaeta Methanococcus jannaschii, Methanosarc in mazei and closely related members of the order Thermococcales. A phyloge netic analysis suggests that a single ancestral gene diverged to form the g lucokinase and PFK lineages of this unique sequence family. Thus, the PFK r eaction, one of the defining enzymatic activities of the Embden-Meyerhof pa thway, can now be represented by three separate sequence families, the well -known PFKA family exemplified by the primary E. coli ATP-PFK (E.C. 2.7.1.1 1) and its associated ATP- and pyrophosphate-dependent PFKs (EC.2.7.1.90), the PFKB family (E. coli PFK 2 encoded by the pfXB gene and its homologues) and the ADP-PFKs of the Euryarchaeota reported here. (C) 2001 Elsevier Sci ence B.V. All rights reserved.