New views on an old enzyme: allosteric regulation and evolution of archaeal pyruvate kinases

Abstract

Pyruvate kinases (PKs) synthesize ATP as the final step of glycolysis in the three domains of life. PKs from most bacteria and eukarya are allosteric enzymes that are activated by sugar phosphates; for example, the feed‐forward regulator fructose‐1,6‐bisphosphate, or AMP as a sensor of energy charge. Archaea utilize unusual glycolytic pathways, but the allosteric properties of PKs from these species are largely unknown. Here, we present an analysis of 24 PKs from most archaeal clades with respect to allosteric properties, together with phylogenetic analyses constructed using a novel mode of rooting protein trees. We find that PKs from many Thermoproteales, an order of crenarchaeota, are allosterically activated by 3‐phosphoglycerate (3PG). We also identify five conserved amino acids that form the binding pocket for 3PG. 3PG is generated via an irreversible reaction in the modified glycolytic pathway of these archaea and therefore functions as a feed‐forward regulator. We also show that PKs from hyperthermophilic Methanococcales, an order of euryarchaeota, are activated by AMP. Phylogenetic analyses indicate that 3PG‐activated PKs form an evolutionary lineage that is distinct from that of sugar‐phosphate activated PKs, and that sugar phosphate‐activated PKs originated as AMP‐regulated PKs in hyperthermophilic Methanococcales. Since the phospho group of sugar phosphates and 3PG overlap in the allosteric site, our data indicate that the allostery in PKs first started from a progenitor phosphate‐binding site that evolved in two spatially distinct directions: one direction generated the canonical site that responds to sugar phosphates and the other gave rise to the 3PG site present in Thermoproteales. Overall, our data suggest an intimate connection between the allosteric properties and evolution of PKs.

Unusual Phosphoenolpyruvate (PEP) Synthetase-Like Protein Crucial to Enhancement of Polyhydroxyalkanoate Accumulation in Haloferax mediterranei Revealed by Dissection of PEP-Pyruvate Interconversion Mechanism

“…1B). All characterized members of PYKs with a Glu residue at the equivalent position exhibit K ϩ -dependent activity (39). Similar to K ϩ -dependent PYKs from Bacteria and Eukarya, PYK Hm had a conserved Glu at position 71.…”

An overview of structure, function, and regulation of pyruvate kinases

“…PyKs of hyperthermophilic archaea Thermoproteales (but not thermophilic bacteria) are activated by 3‐phosphoglycerate (3PG) and not by FBP, FDP, or AMP . In the glycolytic pathway of these archaea, glyceraldehyde 3‐phosphate is irreversibly oxidized to 3PG .…”

“…In the glycolytic pathway of these archaea, glyceraldehyde 3‐phosphate is irreversibly oxidized to 3PG . A putative 3PG binding motif was identified in the sequence of PyKs of Thermoproteales, and based on phylogenetic and biochemical analyses it was concluded that these PyKs form an independent lineage . Their sequence contains a Ser residue in the position corresponding to Glu117 in rabbit muscle PyK and these enzymes are K + ‐independent …”

“…44 PyKs of hyperthermophilic archaea Thermoproteales (but not thermophilic bacteria) are activated by 3-phosphoglycerate (3PG) and not by FBP, FDP, or AMP. 36,47,48 In the glycolytic pathway of these archaea, glyceraldehyde 3-phosphate is irreversibly oxidized to 3PG. 49,50 A putative 3PG binding motif was identified in the sequence of PyKs of Thermoproteales, and based on phylogenetic and biochemical analyses it was concluded that these PyKs form an independent lineage.…”

“…49,50 A putative 3PG binding motif was identified in the sequence of PyKs of Thermoproteales, and based on phylogenetic and biochemical analyses it was concluded that these PyKs form an independent lineage. 48 Their sequence contains a Ser residue in the position corresponding to Glu117 in rabbit muscle PyK and these enzymes are K + -independent. 35,41 Thus members of different PyK families use different effectors, and the sequence and architecture of the effector binding sites and their allosteric strategies are linked with their evolution.…”