Comprehensive DNA Microarray Analysis of Bacillus subtilis Two-Component Regulatory Systems

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Abstract
Phosphofructokinase (PFK) is a key enzyme of the glycolytic pathway in all domains of life. Two related PFKs, ATP-dependent and PPi-dependent PFK, have been distinguished in bacteria and eucarya, as well as in some archaea. Hyperthermophilic archaea of the order Thermococcales, including Pyrococcus andThermococcus spp., have recently been demonstrated to possess a unique ADP-dependent PFK (ADP-PFK) that appears to be phylogenetically distinct. Here, we report the presence of ADP-PFKs in glycogen-producing members of the orders Methanococcalesand Methanosarcinales, including both mesophilic and thermophilic representatives. To verify the substrate specificities of the methanogenic kinases, the gene encoding the ADP-PFK fromMethanococcus jannaschii was functionally expressed inEscherichia coli, and the produced enzyme was purified and characterized in detail. Compared to its counterparts from the two members of the order Thermococcales, the M. jannaschii ADP-PFK has an extremely lowKm for fructose 6-phosphate (9.6 μM), and it accepts both ADP and acetyl-phosphate as phosphoryl donors. Phylogenetic analysis of the ADP-PFK reveals it to be a key enzyme of the modified Embden-Meyerhof pathway of heterotrophic and chemolithoautotrophic archaea. Interestingly, uncharacterized homologs of this unusual kinase are present in several eucarya.