Proteases and Glycosyl Hydrolases from Hyperthermophilic Microorganisms

Bauer, Michael; Halio, Sheryl B.; Kelly, Robert M.

doi:10.1016/s0065-3233(08)60364-2

Cited by 28 publications

(15 citation statements)

References 130 publications

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“…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).…”

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

et al. 2006

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“…480 glycosyl hydrolases with 52 different EC numbers were classified in 45 families (10). The earliest studies along these lines revealed that a number of ␣-specific glycosyl hydrolases were produced by heterotrophic hyperthermophiles, and more recently ␤-specific glycosyl hydrolases have also been identified (11)(12)(13)(14).…”

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Cloning and Characterization of Thermostable Endoglucanase (Cel8Y) from the Hyperthermophilic Aquifex aeolicus VF5

Kim

Park

Lim

et al. 2000

Biochemical and Biophysical Research Communications

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“…Autotrophic organisms also contain proteases that may be involved in cellular processes. The proteases from hyperthermophiles have been studied for their biotechnological applications as well as for their metabolic significance (4). Among the existing types of proteases such as serine-, thiol-, or acid-type proteases, the majority of them belong to a family of serine-type proteases (5)(6)(7)(8)(9).…”

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Extremely Thermostable Serine-type Protease fromAquifex pyrophilus

Choi

Bang

Kim

et al. 1999

Journal of Biological Chemistry

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A gene encoding a serine-type protease has been cloned from Aquifex pyrophilus using a sequence tag containing the consensus sequence of proteases as a probe. Sequence analysis of the cloned gene reveals an open reading frame of 619 residues that has three canonical residues (Asp-140, His-184, and Ser-502) that form the catalytic site of serine-type proteases. The size of the mature form (43 kDa) and its localization in the cell wall fraction indicate that both the NH 2 -and COOHterminal sequences of the protein are processed during maturation. When the cloned gene is expressed in Escherichia coli, it is weakly expressed as active and processed forms. The pH optimum of this protease is very broad, and its activity is completely inactivated by phenylmethylsulfonyl fluoride. The half-life of the protein is 6 h at 105°C, suggesting that it is one of the most heat-stable proteases. The cysteine residues in the mature form may form disulfide bonds that are responsible for the strong stability of this protease, because the thermostability of the protein is significantly reduced in the presence of reducing reagent.

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Proteases and Glycosyl Hydrolases from Hyperthermophilic Microorganisms

Cited by 28 publications

References 130 publications

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

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

Cloning and Characterization of Thermostable Endoglucanase (Cel8Y) from the Hyperthermophilic Aquifex aeolicus VF5

Extremely Thermostable Serine-type Protease fromAquifex pyrophilus

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