Corné H. Verhees

The unique features of glycolytic pathways in Archaea

An early divergence in evolution has resulted in two prokaryotic domains, the Bacteria and the Archaea. Whereas the central metabolic routes of bacteria and eukaryotes are generally well-conserved, variant pathways have developed in Archaea involving several novel enzymes with a distinct control. A spectacular example of convergent evolution concerns the glucose-degrading pathways of saccharolytic archaea. The identification, characterization and comparison of the glycolytic enzymes of a variety of phylogenetic lineages have revealed a mosaic of canonical and novel enzymes in the archaeal variants of the Embden-Meyerhof and the Entner-Doudoroff pathways. By means of integrating results from biochemical and genetic studies with recently obtained comparative and functional genomics data, the structure and function of the archaeal glycolytic routes, the participating enzymes and their regulation are re-evaluated.

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 ofP. 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 °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 theK m for ADP to 0.34 and 0.41 mm, respectively.

Biochemical adaptations of two sugar kinases from the hyperthermophilic archaeon Pyrococcus furiosus.

The hyperthermophilic archaeon Pyrococcus furiosus possesses a modified Embden-Meyerhof pathway, including an unusual ADP-dependent glucokinase (ADP-GLK) and an ADP-dependent phosphofructokinase. In the present study, we report the characterization of a P. furiosus galactokinase (GALK) and its comparison with the P. furiosus ADP-GLK. The pyrococcal genes encoding the ADP-GLK and GALK were functionally expressed in Escherichia coli, and the proteins were subsequently purified to homogeneity. Both enzymes are specific kinases with an optimal activity at approx. 90 degrees C. Biochemical characterization of these enzymes confirmed that the ADP-GLK is unable to use ATP as the phosphoryl group donor, but revealed that GALK is ATP-dependent and has an extremely high affinity for ATP. There is a discussion about whether the unusual features of these two classes of kinases might reflect adaptations to a relatively low intracellular ATP concentration in the hyperthermophilic archaeon P. furiosus.

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.

Molecular and Biochemical Characterization of a Distinct Type of Fructose-1,6-Bisphosphatase from Pyrococcus furiosus

The Pyrococcus furiosus fbpA gene was cloned and expressed in Escherichia coli, and the fructose-1,6-bisphosphatase produced was subsequently purified and characterized. The dimeric enzyme showed a preference for fructose-1,6-bisphosphate, with a K(m) of 0.32 mM and a V(max) of 12.2 U/mg. The P. furiosus fructose-1,6-bisphosphatase was strongly inhibited by Li(+) (50% inhibitory concentration, 1 mM). Based on the presence of conserved sequence motifs and the substrate specificity of the P. furiosus fructose-1,6-bisphosphatase, we propose that this enzyme belongs to a new family, class IV fructose-1,6-bisphosphatase.

Crystal Structure of Pyrococcus furiosus Phosphoglucose Isomerase IMPLICATIONS FOR SUBSTRATE BINDING AND CATALYSIS

Phosphoglucose isomerase (PGI) catalyzes the reversible isomerization between d-fructose 6-phosphate and d-glucose 6-phosphate as part of the glycolytic pathway. PGI from the Archaea Pyrococcus furiosus (Pfu) was crystallized, and its structure was determined by x-ray diffraction to a 2-Å resolution. Structural comparison of this archaeal PGI with the previously solved structures of bacterial and eukaryotic PGIs reveals a completely different structure. Each subunit of the homodimeric Pfu PGI consists of a cupin domain, for which the overall structure is similar to other cupin domain-containing proteins, and includes a conserved transition metal-binding site. Biochemical data on the recombinant enzyme suggests that Fe2+ is bound to Pfu PGI. However, as catalytic activity is not strongly influenced either by the replacement of Fe2+ by a range of transition metals or by the presence or absence of the bound metal ion, we suggest that the metal may not be directly involved in catalysis but rather may be implicated in substrate recognition.

Identification and Molecular Characterization of a Novel Type of α-galactosidase from Pyrococcus furiosus

An α-galactosidase gene from Pyrococcus furiosus was identified, cloned and functionally expressed in Escherichia coli. It is the first α-galactosidase from a hyperthermophilic archaeon described to date. The gene encodes a unique amino acid sequence compared to other α-galactosidases. Highest homology was found with α-amylases classified in family 57 of glycoside hydrolases. The 364 amino acid protein had a calculated mass of 41.6 kDa. The recombinant α-galactosidase specifically catalyzed the hydrolysis of para-nitrophenyl-α-galactopyranoside, and to some extent that of melibiose and raffinose. The enzyme proved to be an extremely thermo-active and thermostable α-galactosidase with a temperature optimum of 115°C and a half-life time of 15 hours at 100°C. The pH optimum is between 5.0 and 5.5. Sequence analysis showed four conserved carboxylic residues. Site-directed mutagenesis was applied to identify the potential catalytic residues. Glu117Ala showed decreased enzyme activity, which could be rescued by the addition of azide or formate. It is concluded that glutamate 117 is the catalytic nucleophile, whereas the acid/base catalyst remains to be identified.

Characterization of ß-glycosyl hydrolases from Pyrococcus furiosus

Publisher Summary Enzymes from bacterial and archaeal hyperthermophiles have been studied extensively for their catalytic properties and stability at extremely high temperatures, as well as their biotechnological potential as biocatalysts at elevated temperatures. Considerable attention is to sugar-converting enzymes from heterotrophic hyperthermophilic archaea, mainly in members of the archaeal orders Sulfolobales—that is, Sulfolobus solfataricus and Thermococcales—that is, Pyrococcus furiosus. The chapter gives an overview of methods that have been instrumental in the molecular and biochemical characterization of enzymes from Pyrococcus species that catalyze the hydrolysis of β-linked sugars, the β-glycosylhydrolases (βGHs). These studies form an essential basis for a next generation of experiments, the engineering of βGHs for optimal exploitation of their potential.

ADP-dependent glucokinase and phosphofructo-kinase from Pyrococcus furiosus

Publisher Summary ADP-dependent glucokinase and phosphofructokinase were demonstrated for the first time by Kengen et al . in the hyperthermophile Pyrococcus furiosus . This anaerobic archaeon grows optimally at 100° by fermenting sugar polymers and polypeptides to mainly acetate and alanine. The catabolism of sugars, such as starch, laminarin, maltose, or cellobiose, has been investigated in detail and has led to the discovery of novel type Embden–Meyerhof pathway, involving several enzymatic steps that are different from the classical ones. In addition to ADP-dependent kinases, the pathway involves a one-step, ferredoxin-dependent conversion of glyceraldehyde-3-phosphate to 3-phosphoglycerate instead of the conventional two-step, NAD-dependent and ATP-generating conversion. Furthermore, a remarkable AMP-dependent, ATP-generating pyruvate kinase has been described, which is different from the normal ADP-dependent pyruvate kinase. Moreover, the available P. furiosus genome sequence shows that for several other glycolytic enzymes, no homologous sequences can be identified, suggesting that these enzymes may be different from the classical ones as well. This chapter describes the procedures for assay and purification of both kinases and the cloning and expression of their genes.

Purification, crystallization and preliminary crystallographic analysis of phosphoglucose isomerase from the hyperthermophilic archaeon Pyrococcus furiosus.

The glycolytic enzyme phosphoglucose isomerase catalyses the reversible isomerization of glucose 6-phosphate to fructose 6-phosphate. The phosphoglucose isomerase from the hyperthermophilic archaeon Pyrococcus furiosus, which shows no sequence similarity to any known bacterial or eukaryotic phosphoglucose isomerase, has been cloned and overexpressed in Escherichia coli, purified and subsequently crystallized by the hanging-drop method of vapour diffusion using 1.6 M sodium citrate as the precipitant at pH 6.5. Multiple-wavelength anomalous dispersive X-ray data have been collected to a maximum resolution of 1.92 A on a single selenomethionine-incorporated crystal. This crystal belongs to space group C2, with approximate unit-cell parameters a = 84.7, b = 42.4, c = 57.3 A, beta = 120.6 degrees and a monomer in the asymmetric unit.