scholarly journals Characterization of an Aminoacylase from the Hyperthermophilic Archaeon Pyrococcus furiosus

2001 ◽  
Vol 183 (14) ◽  
pp. 4259-4268 ◽  
Author(s):  
Sherry V. Story ◽  
Amy M. Grunden ◽  
Michael W. W. Adams
Keyword(s):  
Amino Acids ◽  
Recombinant Protein ◽  
Pyrococcus Furiosus ◽  
Protein A ◽  
Genome Database ◽  
Hyperthermophilic Archaeon ◽  
Protein Renaturation ◽  
Cell Extracts ◽  
Growth Substrates ◽  
Catalytically Active

ABSTRACT Aminoacylase was identified in cell extracts of the hyperthermophilic archaeon Pyrococcus furiosus by its ability to hydrolyze N-acetyl-l-methionine and was purified by multistep chromatography. The enzyme is a homotetramer (42.06 kDa per subunit) and, as purified, contains 1.0 ± 0.48 g-atoms of zinc per subunit. Treatment of the purified enzyme with EDTA resulted in complete loss of activity. This was restored to 86% of the original value (200 U/mg) by treatment with ZnCl2 (and to 74% by the addition of CoCl2). After reconstitution with ZnCl2, the enzyme contained 2.85 ± 0.48 g-atoms of zinc per subunit. Aminoacylase showed broad substrate specificity and hydrolyzed nonpolarN-acylated l amino acids (Met, Ala, Val, and Leu), as well as N-formyl-l-methionine. The high Km values for these compounds indicate that the enzyme plays a role in the metabolism of protein growth substrates rather than in the degradation of cellular proteins. Maximal aminoacylase activity withN-acetyl-l-methionine as the substrate occurred at pH 6.5 and a temperature of 100°C. The N-terminal amino acid sequence of the purified aminoacylase was used to identify, in theP. furiosus genome database, a gene that encodes 383 amino acids. The gene was cloned and expressed in Escherichia coli by using two approaches. One involved the T7lac promoter system, in which the recombinant protein was expressed as inclusion bodies. The second approach used the Trx fusion system, and this produced soluble but inactive recombinant protein. Renaturation and reconstitution experiments with Zn2+ ions failed to produce catalytically active protein. A survey of databases showed that, in general, organisms that contain a homolog of theP. furiosus aminoacylase (≥50% sequence identity) utilize peptide growth substrates, whereas those that do not contain the enzyme are not known to be proteolytic, suggesting a role for the enzyme in primary catabolism.

scholarly journals Purification and Characterization of the Alanine Aminotransferase from the Hyperthermophilic Archaeon Pyrococcus furiosus and Its Role in Alanine Production

2000 ◽  
Vol 182 (9) ◽  
pp. 2559-2566 ◽  
Author(s):  
Donald E. Ward ◽  
Servé W. M. Kengen ◽  
John van der Oost ◽  
Willem M. de Vos
Keyword(s):  
Molecular Mass ◽  
Alanine Aminotransferase ◽  
Pyrococcus Furiosus ◽  
Sodium Dodecyl ◽  
Northern Analysis ◽  
Transcriptional Level ◽  
Significant Activity ◽  
Genome Database ◽  
Hyperthermophilic Archaeon ◽  
Cell Extracts

ABSTRACT Alanine aminotransferase (AlaAT) was purified from cell extracts of the hyperthermophilic archaeon Pyrococcus furiosusby multistep chromatography. The enzyme has an apparent molecular mass of 93.5 kDa, as estimated by gel filtration, and consists of two identical subunits of 46 kDa, as deduced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the gene sequence. The AlaAT displayed a broader substrate specificity than AlaATs from eukaryal sources and exhibited significant activity with alanine, glutamate, and aspartate with either 2-oxoglutarate or pyruvate as the amino acceptor. Optimal activity was found in the pH range of 6.5 to 7.8 and at a temperature of over 95°C. The N-terminal amino acid sequence of the purified AlaAT was determined and enabled the identification of the gene encoding AlaAT (aat) in theP. furiosus genome database. The gene was expressed inEscherichia coli, and the recombinant enzyme was purified. The pH and temperature dependence, molecular mass, and kinetic parameters of the recombinant were indistinguishable from those of the native enzyme from P. furiosus. Thek cat/Km values for alanine and pyruvate formation were 41 and 33 s−1mM−1, respectively, suggesting that the enzyme is not biased toward either the formation of pyruvate, or alanine. Northern analysis identified a single 1.2-kb transcript for the aatgene. In addition, both the aat and gdh(encoding the glutamate dehydrogenase) transcripts appear to be coregulated at the transcriptional level, because the expression of both genes was induced when the cells were grown on pyruvate. The coordinated control found for the aat and gdhgenes is in good agreement with these enzymes acting in a concerted manner to form an electron sink in P. furiosus.

scholarly journals Characterization of a Novel Zinc-Containing, Lysine-Specific Aminopeptidase from the Hyperthermophilic Archaeon Pyrococcus furiosus

2005 ◽  
Vol 187 (6) ◽  
pp. 2077-2083 ◽  
Author(s):  
Sherry V. Story ◽  
Claudia Shah ◽  
Francis E. Jenney ◽  
Michael W. W. Adams
Keyword(s):  
Metal Ion ◽  
Specific Activity ◽  
Pyrococcus Furiosus ◽  
High Specific Activity ◽  
Native Form ◽  
Genome Database ◽  
Hyperthermophilic Archaeon ◽  
Cell Extracts ◽  
Hydrolysis Of

ABSTRACT Cell extracts of the proteolytic, hyperthermophilic archaeon Pyrococcus furiosus contain high specific activity (11 U/mg) of lysine aminopeptidase (KAP), as measured by the hydrolysis of l-lysyl-p-nitroanilide (Lys-pNA). The enzyme was purified by multistep chromatography. KAP is a homotetramer (38.2 kDa per subunit) and, as purified, contains 2.0 ± 0.48 zinc atoms per subunit. Surprisingly, its activity was stimulated fourfold by the addition of Co2+ ions (0.2 mM). Optimal KAP activity with Lys-pNA as the substrate occurred at pH 8.0 and a temperature of 100°C. The enzyme had a narrow substrate specificity with di-, tri-, and tetrapeptides, and it hydrolyzed only basic N-terminal residues at high rates. Mass spectroscopy analysis of the purified enzyme was used to identify, in the P. furiosus genome database, a gene (PF1861) that encodes a product corresponding to 346 amino acids. The recombinant protein containing a polyhistidine tag at the N terminus was produced in Escherichia coli and purified using affinity chromatography. Its properties, including molecular mass, metal ion dependence, and pH and temperature optima for catalysis, were indistinguishable from those of the native form, although the thermostability of the recombinant form was dramatically lower than that of the native enzyme (half-life of approximately 6 h at 100°C). Based on its amino acid sequence, KAP is part of the M18 family of peptidases and represents the first prokaryotic member of this family. KAP is also the first lysine-specific aminopeptidase to be purified from an archaeon.

scholarly journals Characterization of Native and Recombinant Forms of an Unusual Cobalt-Dependent Proline Dipeptidase (Prolidase) from the Hyperthermophilic Archaeon Pyrococcus furiosus

1998 ◽  
Vol 180 (18) ◽  
pp. 4781-4789 ◽  
Author(s):  
Mousumi Ghosh ◽  
Amy M. Grunden ◽  
Dianne M. Dunn ◽  
Robert Weiss ◽  
Michael W. W. Adams
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Substrate Specificity ◽  
Metal Ion ◽  
Pyrococcus Furiosus ◽  
Cobalt Atom ◽  
Significant Similarity ◽  
Genome Database ◽  
Hyperthermophilic Archaeon ◽  
Cell Extracts

ABSTRACT 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°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, theKm values for the substrate Met-Pro were comparable for the native and recombinant forms, although the recombinant enzyme exhibited a twofold greaterV max 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.

scholarly journals The Elemental Sulfur-Responsive Protein (SipA) from the Hyperthermophilic Archaeon Pyrococcus furiosus Is Regulated by Sulfide in an Iron-Dependent Manner

2010 ◽  
Vol 192 (21) ◽  
pp. 5841-5843 ◽  
Author(s):  
Sonya M. Clarkson ◽  
Elizabeth C. Newcomer ◽  
Everett G. Young ◽  
Michael W. W. Adams
Keyword(s):  
Elemental Sulfur ◽  
Iron Sulfide ◽  
Pyrococcus Furiosus ◽  
Protein A ◽  
Dependent Manner ◽  
Intracellular Iron ◽  
Hyperthermophilic Archaeon

ABSTRACT The gene (sipA) encoding the sulfur-induced protein A (PF2025) is highly upregulated during growth of Pyrococcus furiosus on elemental sulfur (S0). Expression of sipA is regulated by sulfide, the product of S0 reduction, but in an iron-dependent manner. SipA is proposed to play a role in intracellular iron sulfide detoxification.

scholarly journals Maltose Metabolism in the Hyperthermophilic Archaeon Thermococcus litoralis: Purification and Characterization of Key Enzymes

1999 ◽  
Vol 181 (11) ◽  
pp. 3358-3367 ◽  
Author(s):  
Karina B. Xavier ◽  
Ralf Peist ◽  
Marina Kossmann ◽  
Winfried Boos ◽  
Helena Santos
Keyword(s):  
Specific Activity ◽  
Pyrococcus Furiosus ◽  
Maximal Activity ◽  
Consensus Binding Site ◽  
Calculated Molecular Mass ◽  
Thermococcus Litoralis ◽  
Hyperthermophilic Archaeon ◽  
Key Enzymes ◽  
Maltose Metabolism ◽  
Cell Extracts

ABSTRACT Maltose metabolism was investigated in the hyperthermophilic archaeon Thermococcus litoralis. Maltose was degraded by the concerted action of 4-α-glucanotransferase and maltodextrin phosphorylase (MalP). The first enzyme produced glucose and a series of maltodextrins that could be acted upon by MalP when the chain length of glucose residues was equal or higher than four, to produce glucose-1-phosphate. Phosphoglucomutase activity was also detected inT. litoralis cell extracts. Glucose derived from the action of 4-α-glucanotransferase was subsequently metabolized via an Embden-Meyerhof pathway. The closely related organism Pyrococcus furiosus used a different metabolic strategy in which maltose was cleaved primarily by the action of an α-glucosidase, ap-nitrophenyl-α-d-glucopyranoside (PNPG)-hydrolyzing enzyme, producing glucose from maltose. A PNPG-hydrolyzing activity was also detected in T. litoralis, but maltose was not a substrate for this enzyme. The two key enzymes in the pathway for maltose catabolism in T. litoralis were purified to homogeneity and characterized; they were constitutively synthesized, although phosphorylase expression was twofold induced by maltodextrins or maltose. The gene encoding MalP was obtained by complementation in Escherichia coli and sequenced (calculated molecular mass, 96,622 Da). The enzyme purified from the organism had a specific activity for maltoheptaose, at the temperature for maximal activity (98°C), of 66 U/mg. AKm of 0.46 mM was determined with heptaose as the substrate at 60°C. The deduced amino acid sequence had a high degree of identity with that of the putative enzyme from the hyperthermophilic archaeon Pyrococcus horikoshii OT3 (66%) and with sequences of the enzymes from the hyperthermophilic bacteriumThermotoga maritima (60%) and Mycobacterium tuberculosis (31%) but not with that of the enzyme from E. coli (13%). The consensus binding site for pyridoxal 5′-phosphate is conserved in the T. litoralis enzyme.

scholarly journals An Unusual Oxygen-Sensitive, Iron- and Zinc-Containing Alcohol Dehydrogenase from the Hyperthermophilic Archaeon Pyrococcus furiosus

1999 ◽  
Vol 181 (4) ◽  
pp. 1163-1170 ◽  
Author(s):  
Kesen Ma ◽  
Michael W. W. Adams
Keyword(s):  
Alcohol Dehydrogenase ◽  
Specific Activity ◽  
Pyrococcus Furiosus ◽  
Electron Paramagnetic Resonance Spectroscopy ◽  
Physiological Role ◽  
Resonance Spectroscopy ◽  
Hyperthermophilic Archaeon ◽  
Subunit Molecular Weight ◽  
Cell Extracts ◽  
Oxygen Sensitive

ABSTRACT Pyrococcus furiosus is a hyperthermophilic archaeon that grows optimally at 100°C by the fermentation of peptides and carbohydrates to produce acetate, CO2, and H2, together with minor amounts of ethanol. The organism also generates H2S in the presence of elemental sulfur (S0). Cell extracts contained NADP-dependent alcohol dehydrogenase activity (0.2 to 0.5 U/mg) with ethanol as the substrate, the specific activity of which was comparable in cells grown with and without S0. The enzyme was purified by multistep column chromatography. It has a subunit molecular weight of 48,000 ± 1,000, appears to be a homohexamer, and contains iron (∼1.0 g-atom/subunit) and zinc (∼1.0 g-atom/subunit) as determined by chemical analysis and plasma emission spectroscopy. Neither other metals nor acid-labile sulfur was detected. Analysis using electron paramagnetic resonance spectroscopy indicated that the iron was present as low-spin Fe(II). The enzyme is oxygen sensitive and has a half-life in air of about 1 h at 23°C. It is stable under anaerobic conditions even at high temperature, with half-lives at 85 and 95°C of 160 and 7 h, respectively. The optimum pH for ethanol oxidation was between 9.4 and 10.2 (at 80°C), and the apparent Km s (at 80°C) for ethanol, acetaldehyde, NADP, and NAD were 29.4, 0.17, 0.071, and 20 mM, respectively. P. furiosus alcohol dehydrogenase utilizes a range of alcohols and aldehydes, including ethanol, 2-phenylethanol, tryptophol, 1,3-propanediol, acetaldehyde, phenylacetaldehyde, and methyl glyoxal. Kinetic analyses indicated a marked preference for catalyzing aldehyde reduction with NADPH as the electron donor. Accordingly, the proposed physiological role of this unusual alcohol dehydrogenase is in the production of alcohols. This reaction simultaneously disposes of excess reducing equivalents and removes toxic aldehydes, both of which are products of fermentation.

The integration of a Stat3 specific peptide aptamer into the thioredoxin scaffold protein strongly enhances its inhibitory potency

2011 ◽  
Vol 5 (1) ◽  
pp. 1-9 ◽  
Author(s):  
Hannah Schöneberger ◽  
Astrid Weiss ◽  
Boris Brill ◽  
Natalia Delis ◽  
Corina Borghouts ◽  
...  
Keyword(s):  
Amino Acids ◽  
Recombinant Protein ◽  
Protein A ◽  
Genetic Selection ◽  
Scaffold Protein ◽  
Peptide Sequence ◽  
Yeast Cells ◽  
Tumor Cell Death ◽  
Dimerization Domain ◽  
Specific Peptide

AbstractWe are characterizing peptides which are able to interact with functional domains of oncoproteins and thus inhibit their activity. The yeast two-hybrid system was used to derive a peptide sequence which specifically interacts with the dimerization domain of the transcription factor Stat3. The activated form of Stat3 is required for the survival of many transformed cells and Stat3 inhibition can cause tumor cell death. The genetic selection of specific peptide sequences from random peptide libraries requires the integration into a scaffold protein and the expression in yeast cells. The scaffold protein, a variant of the human thioredoxin protein, has previously been optimized and also allows for effective bacterial expression of the recombinant protein and the cellular uptake of the purified, recombinant protein. We investigated the contributions of the scaffold protein to the inhibitory properties of rS3-PA. For this purpose we compared rS3-PA in which the ligand peptide is embedded within the thioredoxin scaffold protein with a minimal Stat3-interacting peptide sequence. sS3-P45 is a synthetic peptide of 45 amino acids in length and consists only of the Stat3-binding sequence of 20 amino acids, a protein transduction domain (PTD) and a Flag-tag. Both, the recombinant rS3-PA of 19.3 kDa and the synthetic sS3-P45 of 5.1 kDa, were taken up into the cytoplasm of cells by the PTD-mediated transduction process, inhibited Stat3 target gene expression and caused the death of Stat3-dependent tumor cells. Stat3-independent normal cells were unaffected. rS3-PA effectively inhibited Stat3 function at 2 μM, however, sS3-P45 was required at a concentration of 100 μM to exert the same effects. The more potent action of rS3-PA is most probably due to a conformational stabilization of the Stat3-interacting peptide in the context of the scaffold protein.

scholarly journals Identification of residues essential for catalysis and binding of calmodulin in rat brain inositol 1,4,5-trisphosphate 3-kinase

1991 ◽  
Vol 280 (1) ◽  
pp. 125-129 ◽  
Author(s):  
K Takazawa ◽  
C Erneux
Keyword(s):  
Amino Acids ◽  
Rat Brain ◽  
Catalytic Domain ◽  
Protein A ◽  
Restriction Enzymes ◽  
Deletion Mutants ◽  
Amino Acid Residues ◽  
Inositol 1,4,5 Trisphosphate ◽  
Catalytically Active ◽  
Inactive Protein

In order to identify the amino acid residues involved in calmodulin (CaM) binding and catalytic activity, rat brain inositol 1,4,5-trisphosphate (InsP3) 3-kinase was expressed in Escherichia coli as a beta-galactosidase fusion protein [clone C5; Takazawa, Vandekerckhove, Dumont & Erneux (1990) Biochem. J. 272, 107-112]. Three deletion mutants in the plasmid of clone C5 were generated using convenient restriction enzymes. The results show that the removal of 34 amino acids from the C-terminal end of InsP3 3-kinase resulted in an inactive protein which still interacted with CaM-Sepharose in a Ca2(+)-dependent way. The catalytic domain is thus located at the C-terminal end of the protein. A series of 5′ deletion mutants was prepared and used to produce proteins with the same C-terminal end but shortened N-termini, varying in length by over 80 amino acids. Assay of InsP3 3-kinase activity in bacterial extracts indicated that a maximum of 275 amino acids in the C-terminal region may be sufficient for the construction of a catalytically active domain. Affinity chromatography on CaM-Sepharose of 5′ and 3′ deletion mutants revealed that the sequence stretching from Ser-156 to Leu-189 is involved in CaM binding and enzyme stimulation.

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