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PubMed1994

Occurrence of tertiary and quaternary branched polyamines in thermophilic archaebacteria.

Polyamines of thermophilic archaebacteria were analysed by high-performance liquid chromatography and gas chromatography. Thermoplasma acidophilum and Thermoplasma volcanium ubiquitously contained spermidine and spermine. Four species of Sulfolobus, S. acidocaldarius, S. solfataricus, S. metallicus and S. shibatae, two species of Acidianus, A. brierleyi and A. infernus, and Metallosphaera sedula, contained norspermidine and norspermine in addition to spermidine and spermine, but quantitative distribution profiles were species-specific. A tertiary tetra-amine, N4-aminopropylspermidine, and a quaternary penta-amine, N4-bis(aminopropyl)spermidine, were detected as a major polyamine in three species of Thermococcus, T. celer, T. litoralis and T. stetteri, and two Pyrococcus species, P. furiosus and P. woesei. This is the first report on the occurrence of branched polyamines in archaebacteria.

Archaea↗
PubMed2005

Discovery and characterization of a Coenzyme A disulfide reductase from Pyrococcus horikoshii. Implications for this disulfide metabolism of anaerobic hyperthermophiles.

We have cloned NADH oxidase homologues from Pyrococcus horikoshii and P. furiosus, and purified the recombinant form of the P. horikoshii enzyme to homogeneity from Escherichia coli. Both enzymes (previously referred to as NOX2) have been shown to act as a coenzyme A disulfide reductases (CoADR: CoA-S-S-CoA + NAD(P)H + H+-->2CoA-SH + NAD(P)+). The P. horikoshii enzyme shows a kcat app of 7.2 s(-1) with NADPH at 75 degrees C. While the enzyme shows a preference for NADPH, it is able to use both NADPH and NADH efficiently, with both giving roughly equal kcats, while the Km for NADPH is roughly eightfold lower than that for NADH. The enzyme is specific for the CoA disulfide, and does not show significant reductase activity with other disulfides, including dephospho-CoA. Anaerobic reductive titration of the enzyme with NAD(P)H proceeds in two stages, with an apparent initial reduction of a nonflavin redox center with the first reduction resulting in what appears to be an EH2 form of the enzyme. Addition of a second of NADPH results in the formation of an apparent FAD-NAD(P)H complex. The behavior of this enzyme is quite different from the mesophilic staphylococcal version of the enzyme. This is only the second enzyme with this activity discovered, and the first from a strict anaerobe, an Archaea, or hyperthermophilic source. P. furiosus cells were assayed for small molecular mass thiols and found to contain 0.64 micromol CoA.g dry weight(-1) (corresponding to 210 microM CoA in the cell) consistent with CoA acting as a pool of disulfide reducing equivalents.

Amino Acid Sequence↗
PubMed2001

Structure and expression of an amylopullulanase gene from Bacillus stearothermophilus TS-23.

An amylopullulanase gene (apuTS) from Bacillus stearothermophilus TS-23 was cloned and characterized. apuTS consisted of an open reading frame of 6054 bp encoding a protein of 2018 amino acids with a calculated M(r) of 223811. The deduced amino acid sequence revealed four highly conserved regions that are common among amylolytic enzymes. In the C-terminal region, a six-amino-acid sequence (Pro-Gly-Ser-Gly-Thr-Thr) is repeated nine times. It shared the highest degree of homology with the amylopullulanase of Bacillus sp. XAL601. The enzyme also had moderate homology with amylopullulanases from thermophilic anaerobic bacteria. Low levels of homology were observed between the ApuTS of B. stearothermophilus TS-23 and amylopullulanases of Pyrococcus abyssi Orsay, P. furiosus and Bacillus sp. KSM1378. When the intact coding region of apuTS was expressed in Escherichia coli under the control of the lac promoter, the product was degenerate, as revealed by amylase activity staining after SDS/PAGE. The largest active polypeptide had an M(r) of about 220000, while the smallest one had an M(r) of about 105000. Upstream of the apuTS gene, a gene orfX was fortuitously cloned. The putative OrfX protein was weakly related to the myosin heavy chain. It was predicted to contain a central, 179-residue-long, coiled-coil domain.

Amino Acid Sequence↗
PubMed2001

Constant relative rate of protein evolution and detection of functional diversification among bacterial, archaeal and eukaryotic proteins.

BACKGROUND: Detection of changes in a protein's evolutionary rate may reveal cases of change in that protein's function. We developed and implemented a simple relative rates test in an attempt to assess the rate constancy of protein evolution and to detect cases of functional diversification between orthologous proteins. The test was performed on clusters of orthologous protein sequences from complete bacterial genomes (Chlamydia trachomatis, C. muridarum and Chlamydophila pneumoniae), complete archaeal genomes (Pyrococcus horikoshii, P. abyssi and P. furiosus) and partially sequenced mammalian genomes (human, mouse and rat). RESULTS: Amino-acid sequence evolution rates are significantly correlated on different branches of phylogenetic trees representing the great majority of analyzed orthologous protein sets from all three domains of life. However, approximately 1% of the proteins from each group of species deviates from this pattern and instead shows variation that is consistent with an acceleration of the rate of amino-acid substitution, which may be due to functional diversification. Most of the putative functionally diversified proteins from all three species groups are predicted to function at the periphery of the cells and mediate their interaction with the environment. CONCLUSIONS: Relative rates of protein evolution are remarkably constant for the three species groups analyzed here. Deviations from this rate constancy are probably due to changes in selective constraints associated with diversification between orthologs. Functional diversification between orthologs is thought to be a relatively rare event. However, the resolution afforded by the test designed specifically for genomic-scale datasets allowed us to identify numerous cases of possible functional diversification between orthologous proteins.

Animals↗
PubMed2000

Cloning and expression of alpha-amylase from the hyperthermophilic archaeon Pyrococcus woesei in the moderately halophilic bacterium Halomonas elongata.

An extracellular alpha-amylase gene from the hyperthermophilic archaeon Pyrococcus woesei has been cloned and sequenced. The 1.4-kb protein-coding sequence is identical to that of the corresponding alpha-amylase gene of the closely related species P. furiosus. By using a shuttle cloning vector for halophilic bacteria, the P. woesei alpha-amylase was expressed in the moderate halophile Halomonas elongata, under the control of a native H. elongata promoter. The hyperthermophilic amylase activity expressed in the halophilic host was recovered completely in the crude membrane fraction of cell homogenates, suggesting the formation of inclusion bodies or that the secretion machinery of H. elongata may fail to recognize and release the pyrococcal alpha-amylase to the extracellular medium. However, thermal stability, metal ion interactions, optimal temperature and pH values for the crude and purified recombinant alpha-amylase were comparable with those of the native pyrococcal enzyme. The P. woesei amylase activity expressed in H. elongata was consistently detected in the cells upon growth on a wide range of NaCl concentrations (0.7-2.5 mol l-1). To our knowledge, this is the first report on the expression of an archaeal gene (P. woesei alpha-amylase) in a moderate halophilic host which serves as a cell factory able to grow under extreme salt conditions and with very simple nutritional requirements.

Base Sequence↗
PubMed2006

Improvement of the enzymatic activity of the hyperthermophilic cellulase from Pyrococcus horikoshii.

A hyperthermophilic beta-1,4 endoglucanase (EGPh) from the hyperthermophilic archaeon Pyrococcus horikoshii exhibits a strong hydrolyzing activity toward crystalline cellulose. The characteristic features of EGPh are: (1) it appears to have disulfide bonds, which is rare among anaerobic hyperthermophilic archaeon proteins, and (2) it lacks a carbohydrate-binding domain, which is necessary for effective hydrolysis of cellulose. We first examined the relationship between the disulfide bonds and the catalytic activity by analyzing various cysteine mutations. The activities of the mutated enzymes toward carboxy methyl cellulose (CMC) increased without any loss in thermostability. Second, we prepared a fusion enzyme so that the thermostable chitin-binding domain of chitinase from P. furiosus was joined to the C-terminus of EGPh and its variants. These fusion enzymes showed stronger activities than did the wild-type EGPh toward both CMC and crystalline cellulose (Avicel).

Archaeal Proteins↗
PubMed1999

Gene analysis and enzymatic properties of thermostable beta-glycosidase from Pyrococcus kodakaraensis KOD1.

A beta-glycosidase with broad substrate specificity was identified from a hyperthermophilic archaeon, Pyrococcus kodakaraensis KOD1. The gene encoding beta-glycosidase (Pk-gly) consists of 1449 nucleotides corresponding to a polypeptide of 483 amino acids. The protein showed similarity with other beta-glycosidases from family-1 glycosyl hydrolases, in particular, it showed high identity to beta-mannosidase from P. furiosus (55.7%), beta-glycosidase from Sulfolobus solfataricus (42.7%) and beta-glucosidase from P. furiosus (41.9%). The cloned gene was expressed in Escherichia coli and the recombinant protein was purified. The beta-glycosidase showed optimal activity at pH 6.5 and at an extremely high temperature of 100 degrees C, and had a half-life of 18 h at 90 degrees C. The beta-glycosidase hydrolyzed various pNp-beta-glycopyranosides, with kcat K(m) values in the order of pNp-beta-glucopyranoside = pNp-beta-mannopyranoside > pNp-beta-galactopyranoside > pNp-beta-xylopyranoside. pNp-beta-mannopyranoside was the substrate exhibiting the lowest K(m) value [0.254 mM] with a kcat K(m) ratio comparable to that of pNp-beta-glucopyranoside. This substrate specificity was distinct from previously reported beta-glycosidases. We observed that the region in PK-Gly corresponding to the fifth alpha-helix and beta-strand region of beta-glycosidase from S. solfataricus, which constitutes a large portion of the channel for substrate incorporation, displayed a chimeric structure, with the N-terminal region similar to beta-glycosidases and the C-terminal region similar to beta-mannosidases. An exo-type hydrolytic activity and transglycosylation activity were also observed towards cellooligomers.

Journal Article↗
PubMed2004

Analysis of the function of a hyperthermophilic endoglucanase from Pyrococcus horikoshii that hydrolyzes crystalline cellulose.

A hyperthermophilic beta-1,4 endoglucanase was identified in Pyrococcus horikoshii, a hyperthermophilic archaeon. In order to clarify the function of the protein in detail, structural and catalytic site studies were performed using protein engineering. By removing some of the C-terminal sequence of the ORF of the endoglucanase (PH1171), two types of recombinant proteins were expressed from one ORF, using Escherichia coli. One exhibited endoglucanase activity, and the other did not. An SD-like sequence was identified in the ORF of the endoglucanase. By removing the SD-like sequence without changing the amino acid sequence of the endoglucanase, one recombinant endoglucanase was prepared effectively from E. coli. From the analysis of the N- and C-terminal regions of the ORF, this endoglucanase appears to be a secreted and membrane-binding enzyme of P. horikoshii. A mutation analysis of the endoglucanase, using the synthetic substrate, indicated that Glu342 is a candidate for the active center and plays a critical role in the activity of the enzyme. Additional catalytic amino acid residues were not found. These results indicate that the catalytic residue of the enzyme is different from that of typical family 5 endoglucanase, even though it has a high homology to the endoglucanase from Acidothermus celluloliticus. The activity of the enzyme, using carboxy methylcellulose and crystalline cellulose as the substrates, was increased, but not for a synthetic low-molecular substrate when a carbohydrate-binding module of chitinase from P. furiosus was added to the C-terminal region.

Amino Acid Sequence↗