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 PP(i)-dependent PFK, have been distinguished in bacteria and eucarya, as well as in some archaea. Hyperthermophilic 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. Here, we report the presence of ADP-PFKs in glycogen-producing members of the orders Methanococcales and Methanosarcinales, including both mesophilic and thermophilic representatives. To verify the substrate specificities of the methanogenic kinases, the gene encoding the ADP-PFK from Methanococcus jannaschii was functionally expressed in Escherichia 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 low K(m) for fructose 6-phosphate (9.6 microM), 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.

The phosphoglucose isomerase from the hyperthermophilic archaeon Pyrococcus furiosus is a unique glycolytic enzyme that belongs to the cupin superfamily.

Pyrococcus furiosus uses a variant of the Embden-Meyerhof pathway during growth on sugars. All but one of the genes that encode the glycolytic enzymes of P. furiosus have previously been identified, either by homology searching of its genome or by reversed genetics. We here report the isolation of the missing link of the pyrococcal glycolysis, the phosphoglucose isomerase (PGI), which was purified to homogeneity from P. furiosus and biochemically characterized. The P. furiosus PGI, a dimer of identical 23.5-kDa subunits, catalyzes the reversible isomerization of glucose 6-phosphate to fructose 6-phosphate, with K(m) values of 1.99 and 0.63 mm, respectively. An optimum pH of 7.0 has been determined in both directions, and at its optimum temperature of 90 degrees C the enzyme has a half-life of 2.4 h. The N-terminal sequence was used for the identification of the pgiA gene in the P. furiosus genome. The pgiA transcription start site has been determined, and a monocistronic messenger was detected in P. furiosus during growth on maltose and pyruvate. The pgiA gene was functionally expressed in Escherichia coli BL21(DE3). The deduced amino acid sequence of this first archaeal PGI revealed that it is not related to its bacterial and eukaryal counterparts. In contrast, this archaeal PGI shares similarity with the cupin superfamily that consists of a variety of proteins that are generally involved in sugar metabolism in both prokaryotes and eukaryotes. As for the P. furiosus PGI, distinct phylogenetic origins have previously been reported for other enzymes from the pyrococcal glycolytic pathway. Apparently, convergent evolution by recruitment of several unique enzymes has resulted in the unique Pyrococcus glycolysis.

Archaeal fructose-1,6-bisphosphate aldolases constitute a new family of archaeal type class I aldolase.

Fructose-1,6-bisphosphate (FBP) aldolase activity has been detected previously in several Archaea. However, no obvious orthologs of the bacterial and eucaryal Class I and II FBP aldolases have yet been identified in sequenced archaeal genomes. Based on a recently described novel type of bacterial aldolase, we report on the identification and molecular characterization of the first archaeal FBP aldolases. We have analyzed the FBP aldolases of two hyperthermophilic Archaea, the facultatively heterotrophic Crenarchaeon Thermoproteus tenax and the obligately heterotrophic Euryarchaeon Pyrococcus furiosus. For enzymatic studies the fba genes of T. tenax and P. furiosus were expressed in Escherichia coli. The recombinant FBP aldolases show preferred substrate specificity for FBP in the catabolic direction and exhibit metal-independent Class I FBP aldolase activity via a Schiff-base mechanism. Transcript analyses reveal that the expression of both archaeal genes is induced during sugar fermentation. Remarkably, the fbp gene of T. tenax is co-transcribed with the pfp gene that codes for the reversible PP(i)-dependent phosphofructokinase. As revealed by phylogenetic analyses, orthologs of the T. tenax and P. furiosus enzyme appear to be present in almost all sequenced archaeal genomes, as well as in some bacterial genomes, strongly suggesting that this new enzyme family represents the typical archaeal FBP aldolase. Because this new family shows no significant sequence similarity to classical Class I and II enzymes, a new name is proposed, archaeal type Class I FBP aldolases (FBP aldolase Class IA).

Molecular and biochemical characterization of the ADP-dependent phosphofructokinase from the hyperthermophilic archaeon Pyrococcus furiosus.

Pyrococcus furiosus uses a modified Embden-Meyerhof pathway involving two ADP-dependent kinases. Using the N-terminal amino acid sequence of the previously purified ADP-dependent glucokinase, the corresponding gene as well as a related open reading frame were detected in the genome of P. furiosus. Both genes were successfully cloned and expressed in Escherichia coli, yielding highly thermoactive ADP-dependent glucokinase and phosphofructokinase. The deduced amino acid sequences of both kinases were 21.1% identical but did not reveal significant homology with those of other known sugar kinases. The ADP-dependent phosphofructokinase was purified and characterized. The oxygen-stable protein had a native molecular mass of approximately 180 kDa and was composed of four identical 52-kDa subunits. It had a specific activity of 88 units/mg at 50 degrees C and a pH optimum of 6.5. As phosphoryl group donor, ADP could be replaced by GDP, ATP, and GTP to a limited extent. The K(m) values for fructose 6-phosphate and ADP were 2.3 and 0.11 mM, respectively. The phosphofructokinase did not catalyze the reverse reaction, nor was it regulated by any of the known allosteric modulators of ATP-dependent phosphofructokinases. ATP and AMP were identified as competitive inhibitors of the phosphofructokinase, raising the K(m) for ADP to 0.34 and 0.41 mM, respectively.