Molecular characterization of glycolysis in Pyrococcus furiosus

In the last few decades microorganisms have been isolated from rather unknown and hostile locations, such as those with high salt concentrations, an extreme pH, or low or high temperatures. Microorganisms isolated from these environments are referred to as extremophiles (1). The most extensively studied group of these extremophiles are the hyperthermophiles, microorganisms that have an optimum temperature for growth above 80 °C (2). Except for two bacterial genera, the Thermotageles and Aquifex , all hyperthermophiles isolated to date belong to the domain of the archaea. The archaea compose together with the bacteria and eucarya the three domains of life (3).Pyrococcus furiosus is a hyperthermophilic archaeon, with an optimal growth temperature of 100 °C that grows heterotrophically on a variety of substrates including peptides and saccharides. For its growth on saccharides it uses a modified version of the Embden-Meyerhof pathway, that involves novel enzymes and unique control mechanisms. The research described in this thesis has mainly focussed on the molecular and biochemical characterization of enzymes involved in the upper part of glycolysis in P. furiosus and related organisms (Fig. 10.1).A brief outline of this study is giving in Chapter 1 . In Chapter 2 sugar metabolism in archaea is reviewed. Recent studies on various modifications in the Entner-Doudoroff and Embden-Meyerhof pathways are discussed, and potential scenarios on the evolution of sugar metabolism are proposed. Figure 10.1 Classical Embden-Meyerhof (EM) pathway vs modified EM-pathway.Classical EM-pathway is operative in bacteria and eucarya. Modifications (*) in the EM-pathway are found in archaea. Enzymes that were under investigation in this thesis are indicated in blocks .Chapter 3 describes the first characterization of an ADP-dependent phosphofructokinase (ADP-PFK). Attempts to purify the ADP-PFK from P. furiosus cell extracts were not successful, because of the difficult purification procedure of this enzyme, which tends to stick to other proteins. Alternative approaches based on anticipated homology with the ADP-dependent glucokinase (ADP-GLK) have resulted in the identification of the gene encoding the ADP-PFK on the P. furiosus genome. The gene encoding the ADP-PFK was functionally expressed in Escherichia coli using a well-established expression system. The production of the ADP-PFK in the mesophilic E. coli allowed for a simple purification procedure consisting of a heat-treatment of the cell extract followed by a single chromatographic step. The purified enzyme was able to phosphorylate fructose-6-phosphate into fructose-1,6-bisphosphate with ADP as phosphoryl group donor. Classical PFKs use ATP or PP i as potential phosphoryl group donor, indicating that the P. furiosus enzyme differs from its canonical counterparts. The enzyme was not regulated by any of the known allosteric modulators of ATP-PFKs, implying that the P. furiosus glycolysis does not possess a typical site of regulation.Sequence analysis on the primary structure of the ADP-PFKs showed no significant sequence similarity with the classical monophyletic PFKs (PFKA). However, high similarity (21% identity) was observed with the ADP-dependent glucokinase (ADP-GLK) from P. furiosus , suggesting that both ADP-dependent sugar kinases are phylogenetically related, and belong to the same enzyme family. Orthologs of the ADP-PFK were identified in genome databases of the closely related P. horikoshii and P. abyssi . Also the paralogous ADP-GLK was present in these Pyrococci. Furthermore, orthologs of the ADP-PFK were identified in the hyperthermophilic methanogen Methanococcus jannaschii and the mesophilic methanogen Methanosarcina mazei ( Chapter 4 ). Based on a combination of genomic comparison and activity measurements it is concluded that ADP-PFKs are not restricted to the Thermococcales , but are present in mesophilic methanogens as well. Interestingly, uncharacterized homologs (presumably ADP-dependent) of this unusual kinase are present in several higher eucarya, including human, mouse and fly. The gene encoding the ADP-PFK from M. jannaschii was expressed in E. coli , and the enzyme was subsequently purified. The biochemical characteristics of the first ADP-PFK from a chemolithoautotrophic archaeon were compared to those of the ADP-PFK from the heterotrophic archaea P. furiosus and Thermococcus zilligii ( Chapter 4 ).In Chapter 5 an ATP-dependent galactokinase (catalyzing the first step of the Leloir pathway) from P. furiosus is described. Therefore, both ADP-dependent sugar kinases and an ATP-dependent sugar kinase appear co-exist in this hyperthermophile. The three dimensional structure of the P. furiosus galactokinase has recently been solved in close collaboration with the group of Prof. David Rice (Sheffield, England). Despite the ADP-dependent sugar kinases, the ATP-dependent galactokinase shares two conserved motifs and a high degree of overall similarity ( ± 32 % identity) to the canonical galactokinases. The galactokinase and the ADP-GLK from P. furiosus were produced in E. coli , and their characteristics were compared to each other and to their canonical counterparts. The kinetic and physical parameters of the heterologously produced ADP-GLK were in good agreement with those of the native ADP-GLK, indicating that the enzyme was successfully produced and folded in E. coli . The affinity for ATP of the galactokinase was extremely high at 90 °C ( K m for ATP of 0.008 mM) compared to the classical galactokinase from mesophiles. However, the affinity for galactose was comparable to that of the canonical enzymes. It was suggested that the extremely high affinity of the galactokinase for ATP might reflect an adaptation to a relative low intracellular ATP concentration in P. furiosus . This might also explain the presence of the ADP-dependent sugar kinases in P. furiosus . Both the ATP-dependent galactokinase and the ADP-GLK showed a high catalytic efficiency for their phosphoryl group donor at 90 °C, compared to their mesophilic counterparts.Chapter 6 describes the purification of a unique phosphoglucose isomerase from P. furiosus , its characterization, isolation of the corresponding gene, and prediction of the structure of the enzyme. The phosphoglucose isomerase was purified from a P. furiosus extract. The N-terminal sequence of the purified enzyme was determined, and the gene, named pgiA , could be identified on the P. furiosus genome. Subsequent expression in E. coli revealed that the gene indeed encoded a phosphoglucose isomerase. The pgiA gene was transcribed as a mono-cistronic messenger, and the transcription start site was mapped. Despite similar substrate specificity and kinetic parameters, no significant sequence similarity was obtained with classical phosphoglucose isomerases. In contrast, the enzyme shares similarity with the CUPIN superfamily (double-stranded beta-helices) that consists of a variety of proteins that are generally involved in sugar binding or protein interaction. This is the first example of a phosphoglucose isomerase that belongs to the CUPIN superfamily, and it is the first characterization of an archaeal phosphoglucose isomerase to date. The novel phosphoglucose isomerase and the two ADP-dependent sugar kinases are examples of an excessive replacement of enzymes in glycolysis, and are a compelling example of convergent evolution.Chapter 7 focuses on two archaeal fructose-1,6-bisphosphate aldolases, i.e fructose-1,6-bisphosphate aldolase from the crenarchaeon Thermoproteus tenax and from the euryarchaeon P. furiosus . The genes encoding these enzymes were identified in the genomes based on sequence similarity with a novel fructose-1,6-bisphosphate aldolase from E. coli . Transcript analyses reveal that the in vivo expression of both genes is induced during sugar fermentation. Subsequently, the genes were expressed in E. coli , and the encoded proteins were purified to homogeneity. Both the archaeal enzymes use a Schiff base mechanism for catalysis similar to the Class I aldolases, in contrast to the Class II aldolases that use metal ions for catalyses. 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 fructose-1,6-bisphosphate aldolase. Because this family shows no overall sequence similarity to classical Class I and II enzymes, a new name is proposed, archaeal type Class I fructose-1,6-bisphosphate aldolase (Class IA). Despite to low sequence similarity between the archaeal type Class I fructose-1,6-bisphosphate aldolases and the classical Class I and Class II aldolases, sequence signatures could be identified resembling the active site region (Lys-191) and the phosphate-binding motif of classical Class I fructose-1,6-bisphosphate aldolases and other members of the (βα)8 barrel superfamilies. This suggests that the archaeal type Class I enzymes are distantly related to the classical Class I fructose-1,6-bisphosphate aldolases, and that they share the same ancestral origin.In Chapter 8 the P. furiosus gluconeogenic fructose-1,6-bisphosphatase is described. The gene was identified in the genome based on the sequence similarity with the recently described Methanococcus jannaschii bi-functional inositol-monophosphatase/fructose-1,6-bisphosphatse. The gene was functionally expressed in E. coli , and the enzyme was subsequently purified to homogeneity. Biochemical characteristics were compared with the homologous gene product from M. jannaschii (MJ0109), revealing distinct characteristics in substrate specificity and inhibitors. The M. jannaschii enzyme is a bi-functional enzyme with high activity on inositol-1-phosphosphate and fructose-1,6-bisphosphate. The P. furiosus enzyme has a more specific substrate specificity with a clear preference for fructose-1,6-bisphosphate. Therefore, the enzyme can be regarded as a true fructose-1,6-bisphosphatase. Sequence analysis of the P. furiosus fructose-1,6-bisphosphatase reveals the enzyme to be more similar to inositol monophosphatases than to fructose-1,6-bisphosphatases (type I), both belonging to the sugar phosphatase superfamily, with similar folding and sequence motifs. Because of the higher similarity of the P. furiosus enzyme to the inositol monophosphatases, and because of its specific preference for fructose-1,6-bisphosphate, the enzyme was proposed to belong to a new sub-family: the euryarchaeal fructose-1,6-bisphosphatase (type IV). This new sub-family shows limited sequence similarity to classical fructose-1,6-bisphosphatase from bacteria and eucarya (type I), and no significant sequence similarity to the bacterial fructose-1,6-bisphosphatases (type II and III).Preliminary results in promoter architecture of genes encoding glycolytic enzymes are described in Chapter 9 . Promoter elements were identified, and a putative glycolytic regulator binding site (ATCACNNNNNGTGAT, where N are random nucleotides) is observed specifically in P. furiosus promoter sequences of glycolytic-enzyme encoding genes. Complete analysis of the P. furiosus genome revealed that this motif is present in 21 promoter sequences. The majority of the genes encode enzymes involved in sugar metabolism. Further research is needed to reveal the function of this putative binding site.In conclusion, this project has resulted in the identification of unique genes encoding novel enzymes of modified glycolytic pathways in archaea. Key enzymes of the pyrococcal glycolytic pathway were shown to be modified in enzyme catalysis, evolution and regulation. In close collaboration with the group of Prof. David Rice (Sheffield, England) significant progress has been made in crystallization of the ADP-PFK and galactokinase from Pyrococcus. Finally, it is postulated that regulation of the glycolytic flux in P.furiosus might involve modulation of gene expression rather than allosteric regulation of enzyme activities. High throughput screening by transcriptomic and proteomic approaches like DNA micro-arrays and 2D-gelelectrophoresis, and generation of knock-out mutants in Pyrococcus will provide more insight in the actual significance of regulation of gene expression in archaeal central metabolism in the near future.