Fructose-1,6-bisphosphate (FBP) aldolase activity has been detected previously in several Archaea. However, no obvious orthologs of the bacterial and eucaryal Class I and II FBP aldolases have yet been identified in sequenced archaeal genomes. Based on a recently described novel type of bacterial aldolase, we report on the identification and molecular characterization of the first archaeal FBP aldolases. We have analyzed the FBP aldolases of two hyperthermophilic Archaea, the facultatively heterotrophic Crenarchaeon Thermoproteus tenax and the obligately heterotrophic Euryarchaeon Pyrococcus furiosus. For enzymatic studies the fba genes of T. tenax and P. furiosus were expressed in Escherichia coli. The recombinant FBP aldolases show preferred substrate specificity for FBP in the catabolic direction and exhibit metal-independent Class I FBP aldolase activity via a Schiff-base mechanism. Transcript analyses reveal that the expression of both archaeal genes is induced during sugar fermentation. Remarkably, the fbp gene of T. tenax is co-transcribed with the pfp gene that codes for the reversible PP i -dependent phosphofructokinase. 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 FBP aldolase. Because this new family shows no significant sequence similarity to classical Class I and II enzymes, a new name is proposed, archaeal type Class I FBP aldolases (FBP aldolase Class IA).Fructose-1,6-bisphosphate (FBP) 1 aldolase (EC 4.1.2.13) catalyzes the reversible aldol condensation of glyceraldehyde 3-phosphate (GAP) and dihydroxyacetone phosphate (DHAP) yielding FBP. The enzyme fulfills an amphibolic function being involved in catabolic (glycolysis) as well as anabolic pathways (gluconeogenesis and Calvin cycle). In spite of this central function in carbohydrate metabolism, up to now no archaeal genes coding for the respective enzyme activities have been analyzed.Two distinct classes of FBP aldolases occur in nature, which differ in their enzymatic mechanisms (1-4). Class I FBP aldolases form a Schiff-base intermediate between the carbonyl substrate (FBP and DHAP) and the ⑀-amino group of the active site lysine residue and are inactivated by borohydride (NaBH 4 ), whereas Class II FBP aldolases depend on divalent metal ions to stabilize the carbanion intermediate and are, therefore, inhibited by EDTA. Class II enzymes of bacterial and eucaryal origin generally form dimers with a subunit molecular mass of ϳ40 kDa, whereas the Class I pendants are heterogeneous. Eucaryal aldolases are homomeric tetramers with a subunit molecular mass of ϳ40 kDa, and for bacterial enzymes oligomeric arrangements from monomer to decamer and subunit molecular masses of 27-40 kDa have been described (5, 6).Sequence comparisons of Class I and II FBP aldolases revealed no detectable sequence homology, suggesting convergent evolut...

The hyperthermophilic, facultatively heterotrophic crenarchaeum Thermoproteus tenax was analyzed using a low-coverage shotgun-sequencing approach. A total of 1.81 Mbp (representing 98.5% of the total genome), with an average gap size of 100 bp and 5.3-fold coverage, are reported, giving insights into the genome of T. tenax. Genome analysis and biochemical studies enabled us to reconstruct its central carbohydrate metabolism. T. tenax uses a variant of the reversible Embden-Meyerhof-Parnas (EMP) pathway and two different variants of the Entner-Doudoroff (ED) pathway (a nonphosphorylative variant and a semiphosphorylative variant) for carbohydrate catabolism. For the EMP pathway some new, unexpected enzymes were identified. The semiphosphorylative ED pathway, hitherto supposed to be active only in halophiles, is found in T. tenax. No evidence for a functional pentose phosphate pathway, which is essential for the generation of pentoses and NADPH for anabolic purposes in bacteria and eucarya, is found in T. tenax. Most genes involved in the reversible citric acid cycle were identified, suggesting the presence of a functional oxidative cycle under heterotrophic growth conditions and a reductive cycle for CO 2 fixation under autotrophic growth conditions. Almost all genes necessary for glycogen and trehalose metabolism were identified in the T. tenax genome.Archaea were recognized as a distinct phylogenetic group more than 25 years ago (75). Their importance as the third major evolutionary line is well established, but our knowledge of their physiological capabilities remains limited. Central metabolic pathways within these organisms are far from fully understood (43,47,72).Thermoproteus tenax was the first hyperthermophilic archaeum described (76). It is able to grow chemolithoautotrophically on H 2 , CO 2 , and S o as well as chemoorganoheterotrophically in the presence of S o and various organic substrates such as glucose, starch, amylose, glycerate, glycerol, ethanol, and malate (12, 76). Physiological and biochemical studies revealed T. tenax as a physiologically versatile organism with numerous archaeon-specific metabolic capabilities, regulation, and thermoadaptive traits.Comparative studies of carbohydrate metabolism in hyperthermophilic archaea indicate that sugars are generally metabolized by variants of the Entner-Doudoroff (ED) and Embden-Meyerhof-Parnas (EMP) pathways. The so-called nonphosphorylative ED pathway (phosphorylation takes place only at the stage of glycerate) is the only pathway that has been identified for sugar degradation in the aerobes Sulfolobus solfataricus (9) and Thermoplasma acidophilum (7), whereas the anaerobes Pyrococcus furiosus (27,28,31,66,69), Thermococcus spp. (31,44,50), Desulfurococcus amylolyticus (20), and Archaeoglobus fulgidus (33) use modified versions of the EMP pathway. In contrast to findings for other hyperthermophilic archaea, T. tenax uses both variants-the ED and EMP pathways-for glucose metabolism, as shown by in vitro studies identifying specific intermediates...

SummaryThe interconversion of phosphoenolpyruvate and pyruvate represents an important control point of the Embden-Meyerhof-Parnas (EMP) pathway in Bacteria and Eucarya, but little is known about this site of regulation in Archaea. Here we report on the coexistence of phosphoenolpyruvate synthetase (PEPS) and the first described archaeal pyruvate, phosphate dikinase (PPDK), which, besides pyruvate kinase (PK), are involved in the catalysis of this reaction in the hyperthermophilic crenarchaeote Thermoproteus tenax . The genes encoding T. tenax PEPS and PPDK were cloned and expressed in Escherichia coli , and the enzymic and regulatory properties of the recombinant gene products were analysed. Whereas PEPS catalyses the unidirectional conversion of pyruvate to phosphoenolpyruvate, PPDK shows a bidirectional activity with a preference for the catabolic reaction. In contrast to PK of T. tenax , which is regulated on transcript level but exhibits only limited regulatory potential on protein level, PEPS and PPDK activities are modulated by adenosine phosphates and intermediates of the carbohydrate metabolism. Additionally, expression of PEPS is regulated on transcript level in response to the offered carbon source as revealed by Northern blot analyses. The combined action of the differently regulated enzymes PEPS, PPDK and PK represents a novel way of controlling the interconversion of phosphoenolpyruvate and pyruvate in the reversible EMP pathway, allowing short-term and long-term adaptation to different trophic conditions. Comparative genomic analyses indicate the coexistence of PEPS, PPDK and PK in other Archaea as well, suggesting a similar regulation of the carbohydrate metabolism in these organisms.

The phosphorylation of glucose by different sugar kinases plays an essential role in Archaea because of the absence of a phosphoenolpyruvate-dependent transferase system characteristic for Bacteria. In the genome of the hyperthermophilic Archaeon Thermoproteus tenax a gene was identified with sequence similarity to glucokinases of the so-called ROK family (repressor protein, open reading frame, sugar kinase). The T. tenax enzyme, like the recently described ATP-dependent "glucokinase" from Aeropyrum pernix, shows the typical broad substrate specificity of hexokinases catalyzing not only phosphorylation of glucose but also of other hexoses such as fructose, mannose, or 2-deoxyglucose, and thus both enzymes represent true hexokinases. The T. tenax hexokinase shows strikingly low if at all any regulatory properties and thus fulfills no important control function at the beginning of the variant of the EmbdenMeyerhof-Parnas pathway in T. tenax. Transcript analyses reveal that the hxk gene of T. tenax is cotranscribed with an upstream located orfX, which codes for an 11-kDa protein of unknown function. Growth-dependent studies and promoter analyses suggest that post-transcriptional RNA processing might be involved in the generation of the monocistronic hxk message, which is observed only under heterotrophic growth conditions. Data base searches revealed T. tenax hexokinase homologs in some archaeal, few eukaryal, and many bacterial genomes. Phylogenetic analyses confirm that the archaeal hexokinase is a member of the so-called ROK family, which, however, should be referred to as ROK group because it represents a group within the bacterial glucokinase fructokinase subfamily II of the hexokinase family. Thus, archaeal hexokinases represent a second major group of glucose-phosphorylating enzymes in Archaea beside the recently described archaeal ADP-dependent glucokinases, which were recognized as members of the ribokinase family. The distribution of the two types of sugar kinases, differing in their cosubstrate as well as substrate specificity, within Archaea is discussed on the basis of physiological constraints of the respective organisms.

Background: Previous research has called for improving psychological interventions and developing new treatments for prisoners. Animal-assisted prison-based programmes have increasingly been used as an approach, but there is a lack of studies investigating the effectiveness of such programmes. Objective: To investigate the effects of a dog-assisted social- and emotional-competence training on the socioemotional competences of prisoners compared to treatment as usual. Methods: In a controlled trial, we investigated 62 prisoners that participated in either a 6-month dog-assisted psychotherapeutic programme or the standard treatment. We assessed social and emotional competences before and after the training and at a 4-month follow-up. Data were analysed with linear models. Results: The prisoners’ self-assessed social and emotional competences did not differ. The psychotherapists rated the prisoners’ emotional competences in the intervention group higher at the follow-up but not after the training. The psychotherapists did not rate the prisoners’ social competences in the intervention group differently but did find them to have higher self-regulation at follow-up and lower aggressiveness after the training than the control group. Conclusions: This study indicates that dog-assisted programmes with a therapeutic aim might be beneficial for prisoners. However, the inconsistent results indicate that more research is needed to determine the potential and limits of animal-assisted programmes in forensic settings.