ADP-dependent phosphofructokinases in mesophilic and thermophilic methanogenic archaea

Phosphofructokinase (PFK) is a key enzyme of the glycolytic pathway in all domains of life. Two related PFKs, ATP-dependent and PPi-dependent PFK, hav e been distinguished in bacteria and eucarya, as well as in some archaea. H yperthermophilic archaea of the order Thermococcales, including Pyrococcus and Thermococcus spp., have recently been demonstrated to possess a unique ADP-dependent PFK (ADP-PFK) that appears to be phylogenetically distinct. H ere, we report the presence of ADP-PFKs in glycogen-producing members of th e orders Methanococcales and Methanosarcinales, including both mesophilic a nd thermophilic representatives. To verify the substrate specificities of t he methanogenic kinases, the gene encoding the ADP-PFK from Methanococcus j annaschii was functionally expressed in Escherichia coli, and the produced enzyme was purified and characterized in detail. Compared to its counterpar ts from the two members of the order Thermococcales, the M. jannaschii ADP- PFK has an extremely low K-m for fructose 6-phosphate (9.6 muM), and it acc epts both ADP and acetyl-phosphate as phosphoryl donors. Phylogenetic analy sis of the ADP-PFK reveals it to be a key enzyme of the modified Embden-Mey erhof pathway of heterotrophic and chemolithoautotrophic archaea. Interesti ngly, uncharacterized homologs of this unusual kinase are present in severa l eucarya.