The Endopolysaccharide Metabolism of the Hyperthermophilic Archeon Thermococcus hydrothermalis : Polymer Structure and Biosynthesis

Gruyer, Sebastien; Legin, Estelle; Bliard, Christophe; Ball, Steven; Duchiron, Francis

doi:10.1007/s00284-001-0029-1

Cited by 19 publications

(19 citation statements)

References 17 publications

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“…At least in the Thermococcales, therefore, the previous absence of a BE ortholog gene in the genome sequence was a "missing link" preventing an understanding of glycogen biosynthesis. BE activity was previously reported in T. hydrothermalis cells grown on maltose (11). After partial purification, the authors reported that a 65-kDa protein showed BE activity, although analysis to identify the protein sequence was not performed.…”

Section: Discussionmentioning

confidence: 99%

“…Archaeal species in the order Thermococcales seem to synthesize glycogen-like glucans ubiquitously: (i) an ␣-glucan with a branched structure was found in Thermococcus hydrothermalis (11), and a similar glycogen-like glucan was detected in T. kodakaraensis KOD1 (36) and Thermococcus celer (23); (ii) glycogen synthase (GS) genes are present in all the completely sequenced genomes of Thermococcales; and (iii) GS proteins of Pyrococcus furiosus and Pyrococcus abyssi contain carbohydrate polymerization activities (15,45). At least in the Thermococcales, therefore, the previous absence of a BE ortholog gene in the genome sequence was a "missing link" preventing an understanding of glycogen biosynthesis.…”

Section: Discussionmentioning

confidence: 99%

“…In the cases of P. furiosus and P. abyssi, glycogen can be synthesized through UDP-glucose, because orthologous genes encoding glucose-1-phosphate uridylyltransferase (EC 2.7.7.9) are present in these organisms (PF0770 and PF1356 in P. furiosus and PAB0771 in P. abyssi), and GS of the Thermococcales catalyzes the transfer of glucosyl units from both ADP-glucose and UDP-glucose (11,15,45). Nevertheless, as glucose-1-phosphate uridylyltransferase ortholog genes are not ubiquitously present in the Thermococcales, we suggest that the Thermococcales contain a gene(s), as yet unidentified, encoding glucose-1-phosphate adenylyltransferase.…”

Section: Discussionmentioning

confidence: 99%

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A Novel Branching Enzyme of the GH-57 Family in the Hyperthermophilic Archaeon Thermococcus kodakaraensis KOD1

et al. 2006

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Branching enzyme (BE) catalyzes formation of the branch points in glycogen and amylopectin by cleavage of the ␣-1,4 linkage and its subsequent transfer to the ␣-1,6 position. We have identified a novel BE encoded by an uncharacterized open reading frame (TK1436) of the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. TK1436 encodes a conserved protein showing similarity to members of glycoside hydrolase family 57 (GH-57 family). At the C terminus of the TK1436 protein, two copies of a helix-hairpin-helix (HhH) motif were found. TK1436 orthologs are distributed in archaea of the order Thermococcales, cyanobacteria, some actinobacteria, and a few other bacterial species. When recombinant TK1436 protein was incubated with amylose used as the substrate, a product peak was detected by high-performance anion-exchange chromatography, eluting more slowly than the substrate. Isoamylase treatment of the reaction mixture significantly increased the level of short-chain ␣-glucans, indicating that the reaction product contained many ␣-1,6 branching points. The TK1436 protein showed an optimal pH of 7.0, an optimal temperature of 70°C, and thermostability up to 90°C, as determined by the iodine-staining assay. These properties were the same when a protein devoid of HhH motifs (the TK1436⌬H protein) was used. The average molecular weight of branched glucan after reaction with the TK1436⌬H protein was over 100 times larger than that of the starting substrate. These results clearly indicate that TK1436 encodes a structurally novel BE belonging to the GH-57 family.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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A Novel Branching Enzyme of the GH-57 Family in the Hyperthermophilic Archaeon Thermococcus kodakaraensis KOD1

et al. 2006

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“…The nature of the glucan that serves as the primer for PF0272 in vivo is not known. Possible sources of the primer are precursors or breakdown products of glycogen or other endopolysaccharides (17).…”

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confidence: 99%

Transcriptional and Biochemical Analysis of Starch Metabolism in the Hyperthermophilic Archaeon Pyrococcus furiosus

et al. 2006

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Pyrococcus furiosus utilizes starch and its degradation products, such as maltose, as primary carbon sources, but the pathways by which these ␣-glucans are processed have yet to be defined. For example, its genome contains genes proposed to encode five amylolytic enzymes (including a cyclodextrin glucanotransferase [CGTase] and amylopullulanase), as well as two transporters for maltose and maltodextrins (Mal-I and Mal-II), and a range of intracellular enzymes have been purified that reportedly metabolize maltodextrins and maltose. However, precisely which of these enzymes are involved in starch processing is not clear. In this study, starch metabolism in P. furiosus was examined by biochemical analyses in conjunction with global transcriptional response data for cells grown on a variety of glucans. In addition, DNA sequencing led to the correction of two key errors in the genome sequence, and these change the predicted properties of amylopullulanase (now designated PF1935*) and CGTase (PF0478*). Based on all of these data, a pathway is proposed that is specific for starch utilization that involves one transporter (Mal-II [PF1933 to PF1939]) and only three enzymes, amylopullulanase (PF1935*), 4-␣-glucanotransferase (PF0272), and maltodextrin phosphorylase (PF1535). Their expression is upregulated on starch, and together they generate glucose and glucose-1-phosphate, which then feed into the novel glycolytic pathway of this organism. In addition, the results indicate that several hypothetical proteins encoded by three gene clusters are also involved in the transport and processing of ␣-glucan substrates by P. furiosus.Pyrococcus furiosus is an obligately anaerobic, heterotrophic, hyperthermophilic archaeon that was isolated from a shallow hydrothermal vent near Vulcano Island, Italy (12). The organism grows optimally near 100°C and is able to utilize a range of sugars as a primary carbon source (4,11,28,29). These include cellobiose (28, 43), laminarin (43), chitin (16), maltose (29), barley glucan (3), and starch (29). However, the means by which these compounds are metabolized by P. furiosus are not well understood. In attempting to define the pathway by which starch, and one of its degradation products, maltose, serve as carbon sources, there are two complicating factors. First, the genome sequence of P. furiosus (36) contains an array of genes that encode putative glycosyl hydrolases and glycosyl transferases. With respect to starch, they include five enzymes that potentially hydrolyze ␣-glucans. Two of them (PF0477 and PF1935) have putative signal peptides and are presumably extracellular, while the other three (PF0272, PF0478, and PF1939) lack a signal peptide and are presumably intracellular. They are annotated as amylases (PF0272 and PF0477), amylopullulanases (PF1934 and PF1935), and cyclodextrin glucanotransferases (PF0478). The question is, are all of these enzymes involved in degrading starch? The pathway for utilizing maltose and maltodextrins, primary products of starch breakdown, is well defined in ...

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“…While other routes to alanine formation are possible, such routes are not apparent from the P. furiosus genome sequence. To reduce the amount of carbon entering the glycolytic pathway, P. furiosus, like other Thermococcales, can produce glycogen (21) and extracellular polysaccharides, which could in turn provide the basis for biofilm formation (40,41).…”

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confidence: 99%

Impact of Substrate Glycoside Linkage and Elemental Sulfur on Bioenergetics of and Hydrogen Production by the Hyperthermophilic Archaeon Pyrococcus furiosus

Chou

Shockley

Conners

et al. 2007

Appl Environ Microbiol

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Glycoside linkage (cellobiose versus maltose) dramatically influenced bioenergetics to different extents and by different mechanisms in the hyperthermophilic archaeon Pyrococcus furiosus when it was grown in continuous culture at a dilution rate of 0.45 h ؊1 at 90°C. In the absence of S 0 , cellobiose-grown cells generated twice as much protein and had 50%-higher specific H 2 generation rates than maltose-grown cultures. Addition of S 0 to maltose-grown cultures boosted cell protein production fourfold and shifted gas production completely from H 2 to H 2 S. In contrast, the presence of S 0 in cellobiose-grown cells caused only a 1.3-fold increase in protein production and an incomplete shift from H 2 to H 2 S production, with 2.5 times more H 2 than H 2 S formed. Transcriptional response analysis revealed that many genes and operons known to be involved in ␣-or ␤-glucan uptake and processing were up-regulated in an S 0 -independent manner. Most differentially transcribed open reading frames (ORFs) responding to S 0 in cellobiose-grown cells also responded to S 0 in maltose-grown cells; these ORFs included ORFs encoding a membrane-bound oxidoreductase complex (MBX) and two hypothetical proteins (PF2025 and PF2026). However, additional genes (242 genes; 108 genes were up-regulated and 134 genes were down-regulated) were differentially transcribed when S 0 was present in the medium of maltose-grown cells, indicating that there were different cellular responses to the two sugars. These results indicate that carbohydrate characteristics (e.g., glycoside linkage) have a major impact on S 0 metabolism and hydrogen production in P. furiosus. Furthermore, such issues need to be considered in designing and implementing metabolic strategies for production of biofuel by fermentative anaerobes.Because of problems with sufficient access to petroleum and natural gas resources and the emerging threat of global warming, there is increasing interest in alternative energy options to supplement or replace fossil fuels (43). One prospect that has received considerable attention is the conversion of renewable resources (i.e., biomass) to ethanol using biological routes (20). While availability of bioprocess-based ethanol could offset current demands to some extent, problems with significant CO 2 emissions upon energy conversion would remain (37). A longer-term option being considered is the production of molecular hydrogen from biomass using fermentative, anaerobic microorganisms (38). For example, many mesophilic Clostridium and Enterobacter species can grow on fermentable sugars and produce hydrogen as a by-product of energy metabolism (8,11,16,28,38).Studies suggest that biohydrogen production rates may be enhanced at higher temperatures (9, 23). In fact, the production and consumption of molecular hydrogen drive the microbial physiology and bioenergetics of many hyperthermophilic bacteria and archaea inhabiting hydrothermal environments (1). The potential of these microorganisms for biofuel processes has not gone unnoticed (25)....

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The Endopolysaccharide Metabolism of the Hyperthermophilic Archeon Thermococcus hydrothermalis : Polymer Structure and Biosynthesis

Cited by 19 publications

References 17 publications

A Novel Branching Enzyme of the GH-57 Family in the Hyperthermophilic Archaeon Thermococcus kodakaraensis KOD1

A Novel Branching Enzyme of the GH-57 Family in the Hyperthermophilic Archaeon Thermococcus kodakaraensis KOD1

Transcriptional and Biochemical Analysis of Starch Metabolism in the Hyperthermophilic Archaeon Pyrococcus furiosus

Impact of Substrate Glycoside Linkage and Elemental Sulfur on Bioenergetics of and Hydrogen Production by the Hyperthermophilic Archaeon Pyrococcus furiosus

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