Our research interests focus on

Comparative analyses of novel glycolytic pathways (glucose) of extremophilic archaea

3-Phosphoglycerate, a novel allosteric activator of pyruvate kinases from Thermoproteales

Fructose transport and degradation in haloarchaea

Novel pentose degradation pathways and its transcriptional regulation in archaea

Novel enzymes of acetate formation in archaea: Acetyl-CoA Synthetase (ADP forming)

Novel thermophilic enzymes for industrial application

Comparative analyses of novel glycolytic pathways (glucose) of extremophilic archaea

Hyperthermophilic archaea degrade glucose via modified Embden-Meyerhof (EM) pathways, which differ from the classical EM pathway by several novel enzymes and catalytic reactions, and unusual energetics (ATP yields). Since the hyperthermophilic bacterium Thermotoga utilizes the classical EM pathway, it is concluded that the modifications in archaeal glycolysis are a result of a domain specific evolution rather than an adaptation to a hyperthermophilic life style.
Enzymes of the classical EM pathway are shown in red, modified enzymes of archaeal glycolysis are marked by different colors
Enzymes of the classical EM pathway are shown in red, modified enzymes of archaeal glycolysis are marked by different colors.

Pyruvate kinases from Archaea

Pyruvate kinases (PK) catalyze the final step of glycolysis in all three domains of life. Most PKs display sigmoidal kinetics with respect to PEP and/or ADP indicating cooperative binding of substrate. In addition, PKs from bacteria and eukarya are allosterically activated by sugar phosphates, e.g. fructose 1,6-bisphosphate (FBP), or by AMP as shown for PK of the hyperthermophilic bacterium Thermotoga maritima. In our studies we found that PKs from hyperthermophilic archaea are not activated by any of the classical allosteric effectors. The structure of PK from the hyperthermophilic archaeon Pyrobaculum aerophilum was solved, explaining why FBP cannot act as an allosteric activator of PK activity. Instead, we identified 3-phosphoglycerate (3PG) as a novel type of allosteric activator.

Allosteric activation of PK from Pyrobaculum by 3-phosphoglycerate (3PG)
Allosteric activation of PK from Pyrobaculum by 3-phosphoglycerate (3PG).
Allosteric domain of PK structure of Pyrobaculum with bound 3PG
Allosteric domain of PK structure of Pyrobaculum with bound 3PG

In bacteria and eukarya glucose is degraded via classical glycolysis (Embden-Meyerhof EM pathway) to pyruvate. This classical pathway is coordinated regulated inter alia by the product of the irreversible enzyme phosphofructokinase FBP. In contrast, in the archaeon P. aerophilum glucose is degraded via a modified version of the EM pathway in particular GAP oxidation to 3PG is catalyzed by the irreversible one-step reaction of glycerinaldehyde-3-phosphate oxidoreductase (GAPOR), whereas in bacteria and eukarya GAP is oxidized by a reversible two-step reaction catalyzed by GAPDH and GK. Regulation of P. aerophilum PK by a carboxylate molecule (3PG) rather than a sugar phosphate (FBP) may reflect a step in the evolution of glycolysis that predates the dominance of sugars in metabolism.

Classical Embden-Meyerhof pathway and its modification in the hyperthermophilic archaeon P. aerophilum. This schematic shows (A) the modified EM pathway of P. aerophilum and (B) the classical EM pathway of eukarya and bacteria. Irreversible enzymes that operate in each pathway are designated by red arrows, and intermediates that activate PK are shown in red boxes

Classical Embden-Meyerhof pathway and its modification in the hyperthermophilic archaeon P. aerophilum. This schematic shows (A) the modified EM pathway of P. aerophilum and (B) the classical EM pathway of eukarya and bacteria. Irreversible enzymes that operate in each pathway are designated by red arrows, and intermediates that activate PK are shown in red boxes

 

Solomons et al. (2013) 3-Phosphoglycerate is an allosteric activator of pyruvate kinase from the hyperthermophilic archaeon Pyrobaculum aerophilum. Biochemistry 52: 5865-75

Johnsen et al. (2003) Comparative analysis of pyruvate kinases from the hyperthermophilic archaea Archaeoglobus fulgidus, Aeropyrum pernix, and Pyrobaculum aerophilum and the hyperthermophilic bacterium Thermotoga maritima: Unusual regulatory properties in hpyerthermophilic archaea. J Biol Chem 278: 25417-27

 

Fructose transport and degradation in Haloarchaea

In most bacteria fructose uptake is mediated by a phosphoenolpyruvate (PEP) dependent phosphotransferase system (PTS), which phosphorylates fructose during transport to fructose-1-phosphate. In general, PTS are composed of five components, two cytoplasmic proteins, protein kinase enzyme I (EI) and histidine protein (HPr), and the substrate-specific enzyme II (EII). EII consists of two soluble components (EIIA and EIIB) and a transmembrane component (EIIC) that carries out both the transport and concomitant phosphorylation of the substrate across the membrane. The transfer of the phosphoryl group from PEP to sugar proceeds via the transient phosphorylation of EI, HPr, EIIA, and EIIB. So far, PTS-like sugar uptake systems have not been reported in the archaeal domain.
Our studies indicate that fructose uptake and degradation in the haloarchaeon Haloferax volcanii involve a bacterial type PTS, forming fructose-1-phosphate, a specific fructose-1-phosphate kinase (1-PFK) and a metal dependent class II fructose-1,6-bisphosphate aldolase (FBA). The studies included transcriptional analyses, construction and analyses of knock-out mutants and expression and characterization of enzymes involved. This is the first report of the functional involvement of a bacterial type PTS in sugar transport in archaea. The high similarity of bacterial and haloarchaeal PTS and the absence of PTS in all other archaea and eukarya indicate lateral gene transfer of PTS from bacteria to the haloarchaea.

Gene Cluster

Fructose transport and degradation in Haloarchaea

Pickl et al. (2012) Fructose degradation in the haloarchaeon Haloferax volcanii involves a bacterial phosphoenolpyruvate dependent phosphotransferase system, fructose-1-phosphate kinase, and class II fructose-1,6-bisphosphate aldolase. J Bacteriol 194:3088-97

Novel pentose degradation pathways in archaea

D-Xylose and L-arabinose, major components of the abundant hemicellulose, are degraded in most bacteria to xylose-5-phosphate as an intermediate. In contrast we have shown, that halophilic archaea, e. g. Haloferax volcanii, degrade these two pentoses via novel oxidative pathways. The studies included (I) genome analyses, (II) transcriptional analyses, (III) construction and analyses of knock-out mutants and (IV) expression and characterization of enzymes involved. Further, we have identified a novel transcriptional regulator, XacR, that belongs to the IclR family of bacterial regulators. XacR was characterized as an activator of D-xylose/L-arabinose degrading genes and as a repressor of its own synthesis. XacR homologues were found only in haloarchaea but are absent in any other archaeal genome. Since XacR homologues were also absent in the eukaryal domain, but are highly abundant in bacteria, it can be concluded that the haloarchaeal XacR originated from IclR from bacteria via lateral gene transfer. Pentose degradation pathways were also characterized in the thermoacidophilic archaea Sulfolobus solfataricus and S. acidocaldarius. In these archaea D-xylose/L-arabinose are degraded by a variant of the oxidative pathways described in haloarchaea. (funded by the DFG).

Genomumgebung

ArchaeaBacteria

Johnsen et al. (2015) XacR – a novel transcriptional regulator of D-xylose and L-arabinose catabolism in the haloarchaeon Haloferax volcanii. Environ Microbiol 17:1663-1676
Nunn et al. (2010) Metabolism of pentose sugars in the hyerthermophilic archaea Sulfolobus solfataricus and Sulfolobus acidocaldarius. J Biol Chem 285:33701-9
Johnsen et al. (2009) D-Xylose degradation pathway in the halophilic archaeon Haloferax volcanii. J Biol Chem 284:27290-303

Novel thermophilic enzymes in biotechnology

In an european project, funded by EU/BMBF, novel thermophilic enzymes for application in biotechnology are being studied. The project concentrates on the identification and the biochemical characterization of novel transferase classes, including transaminases, transketolases, prenyltransferases, methyl- and hydroxymethyl-transferases. These enzymes are important catalysts, e. g. for the synthesis of chiral compounds in pharmaceutical industry.