Cloning, sequencing, and expression of the gene encoding amylopullulanase from Pyrococcus furiosus and biochemical characterization of the recombinant enzyme

The gene encoding the Pyrococcus furiosus hyperthermophilic amylopullulanase (APU) was cloned, sequenced, and expressed in Escherichia coli. The gene encoded a single 827-residue polypeptide with a 26-residue signal peptide. The protein sequence had very low homology (17 to 21% identity) with other APUs and enzymes of the alpha-amylase family. In particular, none of the consensus regions present in the alpha-amylase family could be identified. P. furiosus APU showed similarity to three proteins, including the P. furiosus intracellular alpha-amylase and Dictyoglomus thermophilum alpha-amylase A. The mature protein had a molecular weight of 89,000. The recombinant P. furiosus APU remained folded after denaturation at temperatures of < or = 70 degrees C and showed an apparent molecular weight of 50,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Denaturating temperatures of above 100 degrees C were required for complete unfolding. The enzyme was extremely thermostable, with an optimal activity at 105 degrees C and pH 5.5. Ca2+ increased the enzyme activity, thermostability, and substrate affinity. The enzyme was highly resistant to chemical denaturing reagents, and its activity increased up to twofold in the presence of surfactants.     

Biochemical characterisation of ornithine carbamoyltransferase from Pyrococcus furiosus

Ornithine carbamoyltransferase (OTCase) was purified to homogeneity from the hyperthermophilic archaeon Pyrococcus furiosus. The enzyme is a 400 +/- 20-kDa polymer of a 35-kDa subunit, in keeping with the corresponding gene sequence [Roovers, M., Hethke, C., Legrain, C., Thomm, M. & Glansdorff, N. (1997) Isolation of the gene encoding Pyrococcus furiosus ornithine cabamoyltransferase and study of its expression profile in vivo and in vitro, Eur. J. Biochem. 247, 1038-1045]. In contrast with the dodecameric catabolic OTCase of Pseudomonas aeruginosa, P. furiosus OTCase exhibits no substrate cooperativity. In keeping with other data discussed in the text, this suggests that the enzyme serves an anabolic function. Half-life estimates for the purified enzyme ranged over 21-65 min at 100 degrees C according to the experimental conditions and reached several hours in the presence of ornithine and phosphate. The stability was not markedly influenced by the protein concentration. Whereas comparative examination of OTCase sequences did not point to any outstanding feature possibly related to thermophily, modelling the enzyme on the X-ray structure of P. aeruginosa OTCase (constituted by four trimers assembled in a tetrahedral manner) suggests that the molecule is stabilized, at least in part, by a set of hydrophobic interactions at the interfaces between the trimers. The comparison between P. aeruginosa and P. furiosus OTCases suggests that two different properties, allostery and thermostability, have been engineered starting from a similar quaternary structure of high internal symmetry. Recombinant P. furiosus OTCase synthesised by Escherichia coli proved less stable than the native enzyme. In Saccharomyces cerevisiae, however, an enzyme apparently identical to the native one could be obtained.     

Molecular and biochemical characterization of an endo-beta-1,3- glucanase of the hyperthermophilic archaeon Pyrococcus furiosus

We report here the first molecular characterization of an endo-beta-1,3-glucanase from an archaeon. Pyrococcus furiosus is a hyperthermophilic archaeon that is capable of saccharolytic growth. The isolated lamA gene encodes an extracellular enzyme that shares homology with both endo-beta-1,3- and endo-beta-1,3-1,4-glucanases of the glycosyl hydrolase family 16. After deletion of the N-terminal leader sequence, a lamA fragment encoding an active endo-beta-1,3-glucanase was overexpressed in Escherichia coli using the T7-expression system. The purified P. furiosus endoglucanase has highest hydrolytic activity on the beta-1,3-glucose polymer laminarin and has some hydrolytic activity on the beta-1,3-1,4 glucose polymers lichenan and barley beta-glucan. The enzyme is the most thermostable endo-beta-1,3-glucanase described up to now; it has optimal activity at 100-105 degrees C. In the predicted active site of glycosyl hydrolases of family 16 that show predominantly endo-beta-1,3-glucanase activity, an additional methionine residue is present. Deletion of this methionine did not change the substrate specificity of the endoglucanase, but it did cause a severe reduction in its catalytic activity, suggesting a structural role of this residue in constituting the active site. High performance liquid chromatography analysis showed in vitro hydrolysis of laminarin by the endo-beta-1,3-glucanase proceeds more efficiently in combination with an exo-beta-glycosidase from P. furiosus (CelB). This most probably reflects the physiological role of these enzymes: cooperation during growth of P. furiosus on beta-glucans.     

The carbamate kinase-like carbamoyl phosphate synthetase of the hyperthermophilic archaeon Pyrococcus furiosus, a missing link in the evolution of carbamoyl phosphate biosynthesis

Microbial carbamoyl phosphate synthetases (CPS) use glutamine as nitrogen donor and are composed of two subunits (or domains), one exhibiting glutaminase activity, the other able to synthesize carbamoyl phosphate (CP) from bicarbonate, ATP, and ammonia. The pseudodimeric organization of this synthetase suggested that it has evolved by duplication of a smaller kinase, possibly a carbamate kinase (CK). In contrast to other prokaryotes the hyperthermophilic archaeon Pyrococcus furiosus was found to synthesize CP by using ammonia and not glutamine. We have purified the cognate enzyme and found it to be a dimer of two identical subunits of Mr 32,000. Its thermostability is considerable, 50% activity being retained after 1 h at 100 degrees C or 3 h at 95 degrees C. The corresponding gene was cloned by PCR and found to present about 50% amino acid identity with known CKs. The stoichiometry of the reaction (two ATP consumed per CP synthesized) and the ability of the enzyme to catalyze at high rate a bicarbonate-dependent ATPase reaction however clearly distinguish P. furiosus CPS from ordinary CKs. Thus the CPS of P. furiosus could represent a primeval step in the evolution of CPS from CK. Our results suggest that the first event in this evolution was the emergence of a primeval synthetase composed of subunits able to synthesize both carboxyphosphate and CP; this step would have preceded the duplication assumed to have generated the two subdomains of modern CPSs. The gene coding for this CK-like CPS was called cpkA.     

Characterization of native and recombinant forms of an unusual cobalt-dependent proline dipeptidase (prolidase) from the hyperthermophilic archaeon Pyrococcus furiosus

Proline dipeptidase (prolidase) was purified from cell extracts of the proteolytic, hyperthermophilic archaeon Pyrococcus furiosus by multistep chromatography. The enzyme is a homodimer (39.4 kDa per subunit) and as purified contains one cobalt atom per subunit. Its catalytic activity also required the addition of Co2+ ions (Kd, 0.24 mM), indicating that the enzyme has a second metal ion binding site. Co2+ could be replaced by Mn2+ (resulting in a 25% decrease in activity) but not by Mg2+, Ca2+, Fe2+, Zn2+, Cu2+, or Ni2+. The prolidase exhibited a narrow substrate specificity and hydrolyzed only dipeptides with proline at the C terminus and a nonpolar amino acid (Met, Leu, Val, Phe, or Ala) at the N terminus. Optimal prolidase activity with Met-Pro as the substrate occurred at a pH of 7.0 and a temperature of 100 degrees C. The N-terminal amino acid sequence of the purified prolidase was used to identify in the P. furiosus genome database a putative prolidase-encoding gene with a product corresponding to 349 amino acids. This gene was expressed in Escherichia coli and the recombinant protein was purified. Its properties, including molecular mass, metal ion dependence, pH and temperature optima, substrate specificity, and thermostability, were indistinguishable from those of the native prolidase from P. furiosus. Furthermore, the Km values for the substrate Met-Pro were comparable for the native and recombinant forms, although the recombinant enzyme exhibited a twofold greater Vmax value than the native protein. The amino acid sequence of P. furiosus prolidase has significant similarity with those of prolidases from mesophilic organisms, but the enzyme differs from them in its substrate specificity, thermostability, metal dependency, and response to inhibitors. The P. furiosus enzyme appears to be the second Co-containing member (after methionine aminopeptidase) of the binuclear N-terminal exopeptidase family.     

Overexpression and characterization of a prolyl endopeptidase from the hyperthermophilic archaeon Pyrococcus furiosus

The maltose-regulated mlr-2 gene from the hyperthermophilic archaeon Pyrococcus furiosus having homology to bacterial and eukaryal prolyl endopeptidase (PEPase) was cloned and overexpressed in Escherichia coli. Extracts from recombinant cells were capable of hydrolyzing the PEPase substrate benzyloxycarbonyl-Gly-Pro-p-nitroanilide (ZGPpNA) with a temperature optimum between 85 and 90 degrees C. Denaturing gel electrophoresis of purified PEPase showed that enzyme activity was associated with a 70-kDa protein, which is consistent with that predicted from the mlr-2 sequence. However, an apparent molecular mass of 59 kDa was obtained from gel permeation studies. In addition to ZGPpNA (K(Mapp) of 53 microM), PEPase was capable of hydrolyzing azocasein, although at a low rate. No activity was detected when ZGPpNA was replaced by substrates for carboxypeptidase A and B, chymotrypsin, subtilisin, and neutral endopeptidase. N-[N-(L-3-trans-Carboxirane-2-carbonyl)-L-Leu]-agmatine (E-64) and tosyl-L-Lys chloromethyl ketone did not inhibit PEPase activity. Both phenylmethylsulfonyl fluoride and diprotin A inhibited ZGPpNA cleavage, the latter doing so competitively (K(lapp) of 343 microM). At 100 degrees C, the enzyme displayed some tolerance to sodium dodecyl sulfate treatment. Stability of PEPase over time was dependent on protein concentration; at temperatures above 65 degrees C, dilute samples retained most of their activity after 24 h while the activity of concentrated preparations diminished significantly. This decrease was found to be due, in part, to autoproteolysis. Partially purified PEPase from P. furiosus exhibited the same temperature optimum, molecular weight, and kinetic characteristics as the enzyme overexpressed in E. coli. Extracts from P. furiosus cultures grown in the presence of maltose were approximately sevenfold greater in PEPase activity than those grown without maltose. Activity could not be detected in clarified medium obtained from maltose-grown cultures. We conclude that mlr-2, now called prpA, encodes PEPase; the physiological role of this protease is presently unknown.     

Thermostable archaeal O6-alkylguanine-DNA alkyltransferases

Archaea represent some of the most ancient organisms on earth, and they have relatively uncharacterized DNA repair processes. We now show, using an in vitro assay, that extracts of two Crenarchaeota (Sulfolobus acidocaldarius and Pyrobaculum islandicum) and two Euryarchaeota (Pyrococcus furiosus and Thermococcus litoralis) contain the DNA repair protein O6-alkylguanine-DNA alkyltransferase (ATase). The ATase activities found in the archaea were extremely thermostable, with half-lives at 80 degreesC ranging from 0.5 hr (S. acidocaldarius) to 13 hr (T. litoralis). The temperature optima of the four proteins ranged from approximately 75 to approximately 100 degreesC, although activity was seen at 37 degreesC, the temperature optimum of the Escherichia coli and human ATases. In all cases, preincubaton of extracts with a short oligonucleotide containing a single O6-methylguanine residue caused essentially complete loss of ATase activity, suggesting that the alkylphosphotriester-DNA alkyltransferase activity seen in some prokaryotes is not present in Archaea. The ATase from Pyrobaculum islandicum had an apparent molecular mass of 15 kDa, making it the smallest of these proteins so far described. In higher organisms, ATase is responsible for the repair of toxic and mutagenic O6-alkylguanine lesions in alkylated DNA. The presence of ATase in these primitive organisms therefore suggests that endogenous or exogenous exposure to agents that generate appropriate substrates in DNA may be an early event in evolution.     

Sulfide dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus: a new multifunctional enzyme involved in the reduction of elemental sulfur

Pyrococcus furiosus is an anaerobic archaeon that grows optimally at 100 degrees C by the fermentation of carbohydrates yielding acetate, CO2, and H2 as the primary products. If elemental sulfur (S0) or polysulfide is added to the growth medium, H2S is also produced. The cytoplasmic hydrogenase of P. furiosus, which is responsible for H2 production with ferredoxin as the electron donor, has been shown to also catalyze the reduction of polysulfide to H2S (K. Ma, R. N. Schicho, R. M. Kelly, and M. W. W. Adams, Proc. Natl. Acad. Sci. USA 90:5341-5344, 1993). From the cytoplasm of this organism, we have now purified an enzyme, sulfide dehydrogenase (SuDH), which catalyzes the reduction of polysulfide to H2S with NADPH as the electron donor. SuDH is a heterodimer with subunits of 52,000 and 29,000 Da. SuDH contains flavin and approximately 11 iron and 6 acid-labile sulfide atoms per mol, but no other metals were detected. Analysis of the enzyme by electron paramagnetic resonance spectroscopy indicated the presence of four iron-sulfur centers, one of which was specifically reduced by NADPH. SuDH has a half-life at 95 degrees C of about 12 h and shows a 50% increase in activity after 12 h at 82 degrees C. The pure enzyme has a specific activity of 7 mumol of H2S produced.min-1.mg of protein-1 at 80 degrees C with polysulfide (1.2 mM) and NADPH (0.4 mM) as substrates. The apparent Km values were 1.25 mM and 11 microM, respectively. NADH was not utilized as an electron donor for polysulfide reduction. P. furiosus rubredoxin (K(m) = 1.6 microM) also functioned as an electron acceptor for SuDH, and SuDH catalyzed the reduction of NADP with reduced P. furiosus ferredoxin (K(m) = 0.7 microM) as an electron donor. The multiple activities of SuDH and its proposed role in the metabolism of S(o) and polysulfide are discussed.     

Citrate synthase from the hyperthermophilic Archaeon, Pyrococcus furiosus

The gene encoding the enzyme citrate synthase has been cloned and sequenced from the hyperthermophilic Archaeon Pyrococcus furiosus, and the derived amino acid sequence has been phylogenetically compared with citrate synthases from archaeal, bacterial and eukaryal organisms. The gene has been over-expressed in Escherichia coli to produce an active enzyme that has then been characterized with respect to its kinetic, oligomeric and hyperthermostable properties. A structurally-based sequence alignment was made to the citrate synthase from the thermophilic Archaeon Thermoplasma acidophilum, the crystal structure of which we have determined recently. From this alignment, a homology-modelled structure for the P.furiosus citrate synthase was generated and analysed.     

Cloning and sequencing of a gene from the archaeon Pyrococcus furiosus with high homology to a gene encoding phosphoenolpyruvate synthetase from Escherichia coli

A gene from the hyperthermophilic archaeon Pyrococcus furiosus, strain Vc1 (DSM 3638), contains an 817-amino-acid open reading frame which shows 42% identity to the phosphoenolpyruvate (PEP) synthetase of Escherichia coli. This putative P. furiosus PEP synthetase is slightly larger than the E. coli enzyme, the region between residues 58 and 89 being absent from the latter.     

Telomere length and telomerase activity predict survival in patients with B cell chronic lymphocytic leukemia

The telomere-telomerase hypothesis states that the vast majority of human tumors have a prolonged replicative life span throughout expressing telomerase, which compensates the cell division-associated loss of telomere DNA. The use of telomere length and telomerase expression as new biological markers in cancer patients requires their correlation with disease prognosis. We, therefore, correlated the mean telomere length based on a telomere restriction fragment assay and the activity of telomerase measured with a telomeric repeat amplification protocol with clinical data and overall survival in 58 patients with B cell chronic lymphocytic leukemia (B-CLL). Telomere length showed a highly inverse correlation to telomerase activity. Patients with telomeres below 6.0 kb were associated with high telomerase activity, whereas patients with a telomere length >6.0 kb generally showed low enzyme activity (P <0.001). Patients in Binet A exhibited significantly longer telomeres and had less telomerase activity than did patients in Binet B or Binet C, where significantly shorter telomeres and higher telomerase activity were observed (P=0.031). Short telomere length and high telomerase activity were significantly associated with a shorter median survival (P=0.02 and P <0.001), and telomerase activity was the most significant prognostic factor for overall survival in B-CLL (P <0.001). Our data provide evidence that telomere length, as well telomerase activity, exerts a strong impact on the survival of B-CLL patients and that telomerase activity can be used as a new prognostic marker in this disease.     

Purification and molecular characterization of the tungsten-containing formaldehyde ferredoxin oxidoreductase from the hyperthermophilic archaeon Pyrococcus furiosus: the third of a putative five-member tungstoenzyme family

Pyrococcus furiosus is a hyperthermophilic archaeon which grows optimally near 100 degreesC by fermenting peptides and sugars to produce organic acids, CO2, and H2. Its growth requires tungsten, and two different tungsten-containing enzymes, aldehyde ferredoxin oxidoreductase (AOR) and glyceraldehyde-3-phosphate ferredoxin oxidoreductase (GAPOR), have been previously purified from P. furiosus. These two enzymes are thought to function in the metabolism of peptides and carbohydrates, respectively. A third type of tungsten-containing enzyme, formaldehyde ferredoxin oxidoreductase (FOR), has now been characterized. FOR is a homotetramer with a mass of 280 kDa and contains approximately 1 W atom, 4 Fe atoms, and 1 Ca atom per subunit, together with a pterin cofactor. The low recovery of FOR activity during purification was attributed to loss of sulfide, since the purified enzyme was activated up to fivefold by treatment with sulfide (HS-) under reducing conditions. FOR uses P. furiosus ferredoxin as an electron acceptor (Km = 100 microM) and oxidizes a range of aldehydes. Formaldehyde (Km = 15 mM for the sulfide-activated enzyme) was used in routine assays, but the physiological substrate is thought to be an aliphatic C5 semi- or dialdehyde, e.g., glutaric dialdehyde (Km = 1 mM). Based on its amino-terminal sequence, the gene encoding FOR (for) was identified in the genomic database, together with those encoding AOR and GAPOR. The amino acid sequence of FOR corresponded to a mass of 68.7 kDa and is highly similar to those of the subunits of AOR (61% similarity and 40% identity) and GAPOR (50% similarity and 23% identity). The three genes are not linked on the P. furiosus chromosome. Two additional (and nonlinked) genes (termed wor4 and wor5) that encode putative tungstoenzymes with 57% (WOR4) and 56% (WOR5) sequence similarity to FOR were also identified. Based on sequence motif similarities with FOR, both WOR4 and WOR5 are also proposed to contain a tungstobispterin site and one [4Fe-4S] cluster per subunit.     

A novel DNA polymerase family found in Archaea

One of the most puzzling results from the complete genome sequence of the methanogenic archaeon Methanococcus jannaschii was that the organism may have only one DNA polymerase gene. This is because no other DNA polymerase-like open reading frames (ORFs) were found besides one ORF having the typical alpha-like DNA polymerase (family B). Recently, we identified the genes of DNA polymerase II (the second DNA polymerase) from the hyperthermophilic archaeon Pyrococcus furiosus, which has also at least one alpha-like DNA polymerase (T. Uemori, Y. Sato, I. Kato, H. Doi, and Y. Ishino, Genes Cells 2:499-512, 1997). The genes in M. jannaschii encoding the proteins that are homologous to the DNA polymerase II of P. furiosus have been located and cloned. The gene products of M. jannaschii expressed in Escherichia coli had both DNA polymerizing and 3'-->5' exonuclease activities. We propose here a novel DNA polymerase family which is entirely different from other hitherto-described DNA polymerases.     

Unusual fatty acid compositions of the hyperthermophilic archaeon Pyrococcus furiosus and the bacterium Thermotoga maritima

The fatty acid compositions of the hyperthermophilic microorganisms Thermotoga maritima and Pyrococcus furiosus were studied and compared. A total of 37 different fatty acids were identified in T. maritima, including the novel 13,14-dimethyloctacosanedioic acid. In contrast, a total of 18 different fatty acids were characterized, as minor components, in P. furiosus, and these included saturated, monounsaturated, and dicarboxylic acids. This is the first report of fatty acids from an archaeon.     

Carotid endarterectomy and abdominal aoritc aneurysm repair: are these reasonable treatments for patients over age 80?

Due to the aging of America, increased numbers of very elderly patients require peripheral vascular surgery. From April 1980 to November 1997, 191 patients age 80 years or older had carotid endarterectomy (CEA) and/or abdominal aortic aneurysm (AAA) repair at Loma Linda University Medical Center. The total perioperative stroke and death rate in the CEA group was 2.9 per cent. Mean postoperative cumulative survival in this group was 8.4 years. The cumulative stroke-free survival rate was 95.5 per cent for all yearly postoperative intervals up to 12 years. The perioperative mortality rate was 10.7 per cent in the nonruptured AAA group and 53.8 per cent in the ruptured AAA group (P < 0.00001). Mean cumulative survival was 8.6 years in the nonruptured AAA group and 1.1 years in the ruptured AAA group (P = 0.0001). These data support the conclusion that CEA and nonemergent AAA repair in octo- and nonagenarians are safe and effective in prolonging stroke-free and rupture-free survival. The utility of ruptured AAA repair in this age-group is less clear.     

Cyanamide-induced granulocytopenia

BACKGROUND: The appropriate management of patients who are older than 80 years of age and who present with an abdominal aortic aneurysm (AAA) remains controversial. While it appears that elective repair can be performed safely, appropriate management of these patients in the emergency situation is unclear. The purpose of the present study was to examine the results obtained in treating this elderly group in the elective and emergency setting, by operation and conservative techniques at St George Hospital, Kogarah. METHODS: Between January 1987 and December 1994 85 patients older than 80 years of age were treated for AAA. These patients were divided into four groups: I, elective presentation/no surgery; II, elective presentation/elective surgical repair; III, emergency presentation/surgical repair; and IV, emergency presentation/conservative treatment. We examined age, sex, size of AAA, mode of presentation, type of treatment, length of survival and cause of death. RESULTS: The mean age of the total group (n = 85) of patients was 84 years (range: 80-94). The mean AAA diameter for this group was 5.6 cm (95% CI: 5.2-6 cm). The diameters for group I (n = 40), II (n = 22), III (n = 16) and IV (n = 7) were 4.9 cm (4.4-5.5, 95% CI), 5.7 (4.9-6.5 CI), 7.0 (6.1-7.7 CI) and 6.2 (5.2-7.2 CI), respectively. The median survival for groups I, II, III and IV was 18, 38.5, 0.25 and 0 months, respectively. Group II had a longer survival than any other group (P = 0.015), and group IV had a shorter survival than the total group (P = 0.001). However, the length of survival was no different for III versus IV (P = 0.146). Deaths in each group were due to the following reasons. I: cardiopulmonary events (14), rupture (3), malignancy/sepsis (3); II: cardiopulmonary events (3), rupture (thoracic aneurysm) (2), malignancy (I); III: rupture (10), malignancy (I); and (IV): rupture (6), malignancy (1). CONCLUSIONS: Elective surgical repair offers the best management option for AAA in patients older than 80 years of age. Death may still occur from progression of aneurysmal disease at other sites. An aggressive surgical approach to the management of haemodynamically unstable patients in this age group is of questionable benefit.     

Pyrrolidone carboxyl peptidase from the hyperthermophilic Archaeon Pyrococcus furiosus: cloning and overexpression in Escherichia coli of the gene, and its application to protein sequence analysis

A gene for a pyrrolidone carboxyl peptidase (Pcp: EC 3.4.19.3, pyroglutamyl peptidase), which removes amino-terminal pyroglutamyl residues from peptides and proteins, has been cloned from the hyperthermophilic Archaeon Pyrococcus furiosus using its cosmid protein library, sequenced, and expressed in Escherichia coli. The DNA sequence encodes a protein containing 208 amino acid residues with methionine at the N-terminus. Analysis of the recombinant protein expressed in E. coli, including amino acid sequence analysis from the N-terminus by automated Edman degradation and ionspray mass spectrometric analysis of the peptides generated by enzymatic digestions with lysylendopeptidase and Staphylococcus aureus V8 protease, showed its primary structure to be completely identical with that deduced from its cDNA sequence. Comparison of the amino acid sequence of P. furiosus Pcp (P.f.Pcp) with those of bacterial Pcps revealed that a high degree of sequence identity (more than 40%) and conservation of the amino acid residues comprising the catalytic triad, Cys142, His166, and Glu79. On the other hand, a unique short stretch sequence (positions around 175-185) that is absent in bacterial Pcps was found in P.f.Pcp. A similar stretch has also been reported recently in the amino acid sequence of Pcp from the hyperthermophilic Archaeon Thermococcus litoralis [Littlechild et al., in abstracts of the "International Congress on Exthermophiles '98" p. 58 (1998)]. To elucidate their contribution to the hyperthermostability of these enzymes, further structural studies are required.