Comparative Analysis of Pyruvate Kinases from the Hyperthermophilic Archaea Archaeoglobus fulgidus, Aeropyrum pernix, and Pyrobaculum aerophilum and the Hyperthermophilic Bacterium Thermotoga maritima

Abstract

Pyruvate kinases (PK, EC 2.7.1.40) from three hyperthermophilic archaea (Archaeoglobus fulgidus strain 7324, Aeropyrum pernix, and Pyrobaculum aerophilum) and from the hyperthermophilic bacterium Thermotoga maritima were compared with respect to their thermophilic, kinetic, and regulatory properties. PKs from the archaea are 200-kDa homotetramers composed of 50-kDa subunits. The enzymes required divalent cations, Mg 2؉ and Mn 2؉ being most effective, but were independent of K ؉ . Temperature optima for activity were 85°C (A. fulgidus) and above 98°C (A. pernix and P. aerophilum). The PKs were highly thermostable up to 110°C (A. pernix) and showed melting temperatures for thermal unfolding at 93°C (A. fulgidus) or above 98°C (A. pernix and P. aerophilum). All archaeal PKs exhibited sigmoidal saturation kinetics with phosphoenolpyruvate (PEP) and ADP indicating positive homotropic cooperative response with both substrates. Classic heterotropic allosteric regulators of PKs from eukarya and bacteria, e.g. fructose 1,6-bisphosphate or AMP, did not affect PK activity of hyperthermophilic archaea, suggesting the absence of heterotropic allosteric regulation. PK from the bacterium T. maritima is also a homotetramer of 50-kDa subunits. The enzyme was independent of K ؉ ions, had a temperature optimum of 80°C, was highly thermostable up to 90°C, and had a melting temperature above 98°C. The enzyme showed cooperative response to PEP and ADP. In contrast to its archaeal counterparts, the T. maritima enzyme exhibited the classic allosteric response to the activator AMP and to the inhibitor ATP. Sequences of hyperthermophilic PKs showed significant similarity to characterized PKs from bacteria and eukarya. Phylogenetic analysis of PK sequences of all three domains indicates a distinct archaeal cluster that includes the PK from the hyperthermophilic bacterium T. maritima.Hyperthermophilic prokaryotes, with an optimal growth temperature higher than 80°C, are considered to represent the phylogenetically most ancestral organisms (1). Recent comparative studies of the hexose degradation pathways in hyperthermophilic archaea and in the hyperthermophilic bacterium Thermotoga revealed that the classic Embden-Meyerhof (EM) 1 pathway is operative only in Thermotoga, whereas in all archaea, the EM pathway exists in modified versions. The modified EM pathways contain, e.g. unusual glucokinases (GLK) and 6-phosphofructokinases (PFK) such as ADP-dependent GLK and ADP-dependent PFK in Pyrococcus, Thermococcus, and Archaeoglobus; unusual ATP-dependent archaeal GLKs of the ROK (Regulators, ORFs, Kinases) protein family; non-regulatory ATP-dependent PFKs in Desulfurococcus and Aeropyrum; and pyrophosphate-dependent PFK in Thermoproteus. In addition, the modified EM pathways contain novel enzymes of glyceraldehyde 3-phosphate (GAP) oxidation to 3-phosphoglycerate, such as GAP:ferredoxin oxidoreductase and non-phosphorylative glyceraldehyde-3-phosphate dehydrogenase, which replace GAP dehydrogenase and phosphoglycerate kinase in the conventiona...

Carbohydrate Metabolism in Archaea: Current Insights into Unusual Enzymes and Pathways and Their Regulation

“…acidophilum enzyme, archaeal PKs do not depend on the presence of monovalent cations like K ϩ or NH 4 ϩ , as described for many other PKs from Bacteria and Eukarya. This independence was attributed to the substitution of a conserved glutamate, which is highly conserved in the K ϩ sites of PKs stimulated by monovalent cations (232,236). This glutamate is also present in the K ϩ -dependent Tpl.…”

“…From phylogenetic analyses, it has been concluded that the PK family is subdivided into two distinct branches (232,235,236), and it appears that this clustering is due to K ϩ dependence or independence (236). The archaeal PK sequences show a high degree of similarity with their bacterial and eukaryotic counterparts, including the prosite consensus patterns for PKs (232,235). PKs, as deduced from the crystal structures available from bacterial and eukaryotic sources and also recently from the crenarchaeon Pyb.…”

“…The three classical points of allosteric control in Bacteria and Eukarya, i.e., hexokinase, PFK, and PK, are absent in Thermococcales (and also generally in Archaea). The ADP-dependent kinases and also most archaeal PKs possess no allosteric properties toward classical effectors of bacterial and eukaryotic enzymes (232,235). Instead, the only allosterically controlled enzyme in the modified EMP pathway of Thermococcales identified so far is GAPN.…”

“…pernix, and Pyb. aerophilum (232)(233)(234)(235). As for their bacterial and eukaryotic counterparts, the archaeal PKs represent 200-kDa homotetrameric proteins composed of 50-kDa subunits and require divalent metal ions (Mg 2ϩ and Mn 2ϩ ) for activity.…”

“…Allosteric regulation by these classical effectors appears to be absent in most archaeal PKs; again, Tpl. acidophilum PK seems to be an exception, being activated by AMP (232,235). The PK structure from Pyb.…”