Carbohydrate metabolism

P. torridus and P. oshimae produce extracellular thermo- and acid stable glucoamylases (Serour et al., 2003), meaning that in the case of α-glucan substrates extracellular enzymatic breakdown can occur. In addition, it seems clear that sugar polymers outside the cells of P. torridus could be degraded also, at least partially, by the acidic and hot environment. Inside the cell, oligomeric sugars can be hydrolysed by two predicted intracellular glucoamylases, an α-amylase and an α-glucosidase. The gene coding for one of the glucoamylases has recently been cloned in E. coli and the function of the encoded enzyme confirmed (B. Schepers, personal communication). Also, as a part of the current work, the α-glucosidase gene (malP, ORF 985) was expressed in E.

coli and the activity of the encoded product was shown to be consistent with the annotation (section C.3.1.3).

Despite the fact that the presence of glycogen has been shown in several archaeal species, little is known about its metabolism in this group (König et al., 1982). From genome data alone it is possible only to speculate about the probable role of the ORFs thought to be involved in glycogen turnover. In yeast and mammals the hydrolytic degradation of glycogen is accomplished by a debranching system composed of a polypeptide chain that contains two activities: 6-α-glucosidase and 4-α-glucanotransferase (Bates et al., 1975). The 4-α-4-α-glucanotransferase activity acts on the glycogen phosphorylase limit dextrin chains to expose the single glucose residues, which the 6-glucosidase activity can then hydrolyse. In P. torridus, the intracellular α-amylase (ORF 596) was found in a potential operon of functionally related genes, which is conserved in the Thermoplasmales. Unexpectedly, in P. torridus the 6-phosphofructokinase gene was located upstream of this conserved region. The cluster consists of ORFs for glycogen debranching enzyme (GDE), a glycosyl transferase (GT) and the α-amylase (AmyA). The overlap between the P. torridus ORFs for GDE and GT was found to be 2bp and between those for GT and AmyA – 16 bp. It is interesting to point out that the deduced GDE amino acid sequence showed significant sequence similarity to the N-terminal part of the yeast glycogen debranching enzyme, which is where the 6-α-glucosidase activity is located. The conserved probable operon in several members of the Thermoplasmales and the supposed mode of action of the encoded enzymes on glycogen is shown on Figure 34.

P. torridus F. acidarmanus T. acidophilum T. volcanicum

1 2 3

4 1 2 3

1 2 3

1 2 3

5

5 A 5

B

Fig. 34. A. Organisation of the genes functionally related to glycogen metabolism in P. torridus, F. acidarmanus, T. acidophilum and T. volcanicum. The numbers correspond to ORFs for: 1. glycogen debranching enzyme (GDE), 2. glycosil transferase (GT), 3. α-amylase (AmyA), 4. 6-phosphofructokinase and 5.

glucoamylase.

B. Mode of glycogen degradation by the enzymes encoded in the cluster.

The circles represent glucose residues

As has been described in the Results (section C.1.3.8), P. torridus most likely catabolises glucose via a non-phosphorylated variant of the Entner-Doudoroff (ED) pathway. The identification of the gluconate dehydratase gene, which previously had not been identified in other archaeal genomes was based on strong similarities to galactonate dehydratase genes of enterobacteria. In support, orthologs of this ORF are clustered with the KDG aldolase gene in S. solfataricus and with the glucose dehydrogenase gene in T. acidophilum.

Concerning the EMP pathway, it was assumed that it is functional despite the lack of a candidate gene for a fructose-1,6-bisphosphate aldolase. It has been argued previously that alternative enzymes are utilised to obtain the missing activity in T.

acidophilum (Ruepp et al., 2000). Interestingly, P. torridus possesses, in contrast to T.

acidophilum and S. solfataricus, a phosphofructokinase gene which would be unnecessary unless its reaction product is further cleaved in an aldolase reaction or vice versa in the gluconeogenic orientation of the pathway. Recently, Lamble and coworkers

(Lamble et al., 2003) revealed the promiscuity of the KDG-aldolase of S. solfataricus which in its reverse reaction synthesises deoxygalactonate besides 2-keto-3-deoxygluconate from pyruvate and glyceraldehyde. Since also the glucose dehydrogenase of this organism does not differentiate between glucose and galactose, it was assumed that this is true for the complete pathway which in consequence would argue against the use of this pathway (ED) for gluconeogenesis. The biochemical data obtained for the P. torridus glucose dehydrogenase, i.e. its high enzyme activity with galactose as substrate and its high affinity for this sugar (approximately double compared to glucose), suggest that this “promiscuity” is probably present also in P.

torridus. It is therefore proposed by us that a non-classical fructose-1,6-bisphosphate aldolase may be present in P. torridus and that the EMP pathway is used, at least, for gluconeogenesis. The possibility of utilising the EMP for gluconeogenesis is further emphasized by the presence of a putative fructose-1,6-bisphosphatase gene in P.

torridus. This hypothesis is further discussed in the context of the biochemical characteristics of the recombinant P. torridus glucose dehydrogenase. (section D.4.1).

It was shown in the Results that genes coding for all enzyme components of complete TCA and 2-methylcitrate cycles could be detected in the P. torridus genome (section C.1.3.9). Interestingly, it was reported previously that the addition of propionate, lactate, acetate or formate to P. oshimae cells inhibited respiration (van de Vossenberg et al., 1998). Since also a lactate-2-monooxygenase was found which converts lactate to pyruvate, two acetyl-CoA synthetases, parts of a formate hydrogen lyase operon and a formyl-tetrahydrofolate synthetase, it is possible that the tested compounds are not metabolised in substantial amounts but the enzymes and pathways serve mainly as a means of protection against uncoupling of the respiratory chain by organic acids. This uncoupling is probable to occur because at the pH at which P.

torridus grows (0.6-1.0) these weak organic acids are protonated and therefore able to enter the cell, where by deprotonation at the higher pH (4.6) they would “work against”

the respiration.

It must also be noted that the formyl-tetrahydrofolate synthase ORF shows high similarity to bacterial genes and a lactate monooxygenase homolog has so far not been detected in any other archaeal genome. It can therefore be concluded that, for at least some of the organic acid metabolic pathways, their presence in the P. torridus genome is most probably due to horizontal gene transfer.

1.3.4. Overview

It is important to note that many genes which are connected with the abilities of P. torridus to survive in its extremely acidic environment have been obtained by lateral gene transfer. This includes some of the organic acid degradation pathways, the main components of the electron transport chain and mechanisms to deal with oxygen stress.

Below is an overview of the transport, central metabolism and energy production in P.

torridus as deduced from the annotation of the ORFs derived from the genome sequence.

Fig. 35. Overview of the transport, central metabolism and energy production in P. torridus.

Sugar, peptide and amino acid uptake systems are shown in red, drug exporters in pink, trace elements transport systems in green, other and hypothetical transporters in grey. Bold numbers mark the number of each transporter. Protein translocation systems are shown in violet and the components of the respiratory chain in yellow. A total of 93 secondary and 17 primary transporters were found in the genome sequence resulting in an unusual ratio of 5,6:1. So far, no aldolase gene is found. Enzyme activity essays indicate a functional non-physphorylated Entner Doudoroff pathway for glycolysis. Pathways for the respiration of the organic acids acetate, lactate and propanoate were identified. NADH2 and reduced ferredoxin is produced in the P. torridus central carbon metabolism but the final reducing compound of the NADH-oxidoreductase is still unknown as no electron-input module for it was detected.

.

2. Heterologous expression of P. torridus genes

P. torridus lives in an extremely hostile environment and is able to grow at the lowest pH values known for all organisms. It can be expected that these conditions have led to the selection of distinctive changes in the physiology and metabolism of the organism as well as in the structure and function of its biomolecules. Therefore, one of the aims of the current work was to obtain certain enzymes of P. torridus by heterologous expression in order to study their structural and functional characteristics with the ultimate goal to learn how this organism copes with its hot and acid environment.