4‐α‐Glucanotransferase from the Hyperthermophilic Archaeon <i>Thermococcus Litoralis</i>

Jeon, Beong Sam; Taguchi, Hayao; Sakai, Hiroshi; Ohshima, Toshihisa; Wakagi, Takayoshi; Matsuzawa, Hiroshi

doi:10.1111/j.1432-1033.1997.00171.x

Cited by 86 publications

(64 citation statements)

References 28 publications

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“…These included enzymes with characterized ␣-amylase and pullulanase activities (10,22,26) as well as a single bacterial example from the anaerobe Dictyoglomus sp. (13).…”

Section: Discussionmentioning

confidence: 99%

“…These enzymes have been classified into two sequence families (13 and 57) based on the presence of distinctive conserved domains (19). A growing number of glycosyl hydrolases, including ␣-amylases and pullulanases, have been characterized from hyperthermophilic archaea (8,21,32,35), including members of family 57 (10,22). However, the lack of suitable genetic methods for hyperthermophilic archaea have precluded studies on the biological significance of these enzymes.…”

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confidence: 99%

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Targeted Disruption of the α-Amylase Gene in the Hyperthermophilic Archaeon Sulfolobus solfataricus

et al. 2003

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Sulfolobus solfataricus secretes an acid-resistant ␣-amylase (amyA) during growth on starch as the sole carbon and energy source. Synthesis of this activity is subject to catabolite repression. To better understand ␣-amylase function and regulation, the structural gene was identified and disrupted and the resulting mutant was characterized. Internal ␣-amylase peptide sequences obtained by tandem mass spectroscopy were used to identify the amyA coding sequence. Anti-␣-amylase antibodies raised against the purified protein immunoprecipitated secreted ␣-amylase activity and verified the enzymatic identity of the sequenced protein. A new gene replacement method was used to disrupt the amyA coding sequence by insertion of a modified allele of the S. solfataricus lacS gene. PCR and DNA sequence analysis were used to characterize the altered amyA locus in the recombinant strain. The amyA::lacS mutant lost the ability to grow on starch, glycogen, or pullulan as sole carbon and energy sources. During growth on a non-catabolite-repressing carbon source with added starch, the mutant produced no detectable secreted amylase activity as determined by enzyme assay, plate assay, or Western blot analysis. These results clarify the biological role of the ␣-amylase and provide additional methods for the directed genetic manipulation of the S. solfataricus genome.Sulfolobus solfataricus is a hyperthermophilic member of the archaea which inhabits acidic geothermal environments. It exhibits diverse modes of metabolism at high temperatures (70 to 90°C), including lithoautotrophy with sulfur and carbon dioxide (5, 23, 37) and chemoheterotrophy with sugars or amino acids (7,15). Despite this metabolic flexibility, mechanisms regulating carbohydrate consumption by this organism are poorly understood. On occasion, plant-derived carbohydrates such as starch and cellulose contribute to the geothermal pool carbon cycle. In the absence of plant life, however, the endogenous microbial community itself may be an important source of reduced carbon compounds. For example, since hyperthermophilic archaea accumulate glycogen as an intracellular storage polymer (24), lysis could make glycogen available for consumption by surviving cells.In hot acid environments polysaccharide degradation occurs rapidly, reflecting high rates of chemical hydrolysis and oxidation. Consequently, successful competition for these carbohydrates necessitates glycosyl hydrolase secretion. Secreted ␣-amylases promote the consumption of exogenous starch by releasing linear maltodextrins for subsequent assimilation. These enzymes have been classified into two sequence families (13 and 57) based on the presence of distinctive conserved domains (19). A growing number of glycosyl hydrolases, including ␣-amylases and pullulanases, have been characterized from hyperthermophilic archaea (8,21,32,35), including members of family 57 (10, 22). However, the lack of suitable genetic methods for hyperthermophilic archaea have precluded studies on the biological significance of these enzym...

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“…These included enzymes with characterized ␣-amylase and pullulanase activities (10,22,26) as well as a single bacterial example from the anaerobe Dictyoglomus sp. (13).…”

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Targeted Disruption of the α-Amylase Gene in the Hyperthermophilic Archaeon Sulfolobus solfataricus

et al. 2003

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“…Therefore, we propose that PF0272 is not an ␣-amylase but a 4-␣-glucanotransferase. In further support of this assignment, the protein shows between 64 and 69% sequence similarity with the 4-␣-glucanotransferases of T. kodakaraensis and T. litoralis, respectively (24,42). The question arises as to the substrate range of PF0272 and its mechanism of action in vivo.…”

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confidence: 92%

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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“…Preparation of a DNA probe by means of PCR and Southern hybridization The genomic DNA of T. litoralis was prepared as described previously [11]. Sense and antisense primers were synthesized based on the strictly conserved amino acid sequences of known PGIs: VRRLSDM and DYGTIAE (residues 25^31 and 160^166 of tlPGI, respectively).…”

Section: Methodsmentioning

confidence: 99%

Characterization of the cupin‐type phosphoglucose isomerase from the hyperthermophilic archaeon Thermococcus litoralisa

Jeong¹,

Fushinobu²,

Ito³

et al. 2003

FEBS Letters

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The gene encoding phosphoglucose isomerase was cloned from Thermococcus litoralis, and functionally expressed in Escherichia coli. The puri¢ed enzyme, a homodimer of 21.5 kDa subunits, was biochemically characterized. The inhibition constants for four competitive inhibitors were determined. The enzyme contained 1.25 mol Fe and 0.24 mol Zn per dimer. The activity was enhanced by the addition of Fe , but inhibited by Zn 2+ and EDTA. Enzymes with mutations in conserved histidine and glutamate residues in their cupin motifs contained no metals, and showed large decreases in k cat . The circular dichroism spectra of the mutant enzymes and the wild type enzyme were essentially the same but with slight di¡erences.

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4‐α‐Glucanotransferase from the Hyperthermophilic Archaeon Thermococcus Litoralis

Cited by 86 publications

References 28 publications

Targeted Disruption of the α-Amylase Gene in the Hyperthermophilic Archaeon Sulfolobus solfataricus

Targeted Disruption of the α-Amylase Gene in the Hyperthermophilic Archaeon Sulfolobus solfataricus

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

Characterization of the cupin‐type phosphoglucose isomerase from the hyperthermophilic archaeon Thermococcus litoralisa

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