Purification and Characterization of Two Functional Forms of Intracellular Protease PfpI from the Hyperthermophilic Archaeon Pyrococcus furiosus

Halio, Sheryl B.; Bauer, Michael; Mukund, Swarnalatha; Adams, Michael W. W.; Kelly, Robert M.

doi:10.1128/aem.63.1.289-295.1997

Cited by 42 publications

(30 citation statements)

References 43 publications

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“…4). Preferred amino acids of the P1 site for DJ-1 protease are Val, Ile and Ala, which are non-polar amino acids, and these phenomena are similar to those for Pfpi [28]. Optimal pH for DJ-1 protease was 5.5-6.0 and its activity decreased at more alkaline pH (Fig.…”

Section: Discussionmentioning

confidence: 63%

Identification of the recognition sequence and target proteins for DJ‐1 protease

et al. 2013

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a b s t r a c t DJ-1, the product of familial Parkinson's disease gene and an oncogene, is a cysteine protease which plays a role in anti-oxidative stress reaction. In this study, we identified the recognition sequence for DJ-1 protease by using recombinant DJ-1 and a peptide library. Protease activity of DJ-1 lacking Cterminal a-helix (DJ-1DH9) was stronger than that of full-sized DJ-1, and the most susceptible sequence digested by DJ-1DH9 was valine-lysine-valine-alanine (VKVA) under the optimal conditions of pH 5.5 and 0 mM NaCl. Divalent ions, especially Cu 2+ , were inhibitory to DJ-1's protease activity. c-abl oncogene 1 product (ABL1) and kinesin family member 1B (KIF1B) containing VKVA were digested by DJ-1DH9. Structured summary of protein interactions:DJ-1 cleaves KIF1B by enzymatic study (View interaction) DJ-1 cleaves ABLI by enzymatic study (View interaction)

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Section: Discussionmentioning

confidence: 63%

Identification of the recognition sequence and target proteins for DJ‐1 protease

et al. 2013

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“…Nevertheless, this value seems to be still too high for a protease. Studies performed on the PfPI (a protease extracted from the hyperthermophilic archaebacteria Pyrococcus furiosus ) showed that PfPI protease has a behavior similar to PPA [14, 15]. PfPI protease has a monomeric form of 18.8 kDa oligomerising into a form of 200 kDa.…”

Section: Resultsmentioning

confidence: 99%

Purification, molecular properties and specificity of a thermoactive and thermostable proteinase from Pyrococcus abyssi, strain st 549, hyperthermophilic archaea from deep‐sea hydrothermal ecosystem

Dib¹,

Chobert²,

Dalgalarrondo³

et al. 1998

FEBS Letters

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A protease was isolated and purified from the supernatant of a culture of hyperthermophilic archaebacteria : Pyrococcus abyssi strain st 549. Purification consisted of three chromatographic steps. The enzyme purification yield was 4% and the purification factor 890. This protease is a seryl-protease hydrolyzing proteins and peptides with a preference for cleavage at the aromatic and hydrophobic residues in P1 and PP1 positions. Its activity is optimal at 95³C and at pH 9. The electrophoretic mobility of the protease observed by zymogram suggests that it can adopt several oligomer forms. Three of them predominate displaying apparent molecular masses of 150, 105 and 60 kDa. Interdependence of the observed bands was revealed by changing the denaturation conditions of the samples (temperature, SDS concentration) before electrophoresis.z 1998 Federation of European Biochemical Societies.

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“…So far, three proteases have been characterized from P. furiosus. These are a membrane-associated serine protease (48), an intracellular protease with trypsin-and chymotrypsin-like activities (28,29), and an intracellular endopeptidase that cleaves at prolyl residues (30). In addition, two proteases have been purified from other members of the family Thermococcales, including a thiol protease from an unclassified species of Pyrococcus (40) and a serine protease from Thermococcus stetteri (33).…”

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

et al. 1998

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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.

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Purification and Characterization of Two Functional Forms of Intracellular Protease PfpI from the Hyperthermophilic Archaeon Pyrococcus furiosus

Abstract: The hyperthermophilic archaeon Pyrococcus furiosus grows optimally at 100؇C by the fermentation of peptides and carbohydrates. From this organism, we have purified to homogeneity an intracellular protease, previously designated PfpI (P. furiosus protease I) (

Cited by 42 publications

References 43 publications

Identification of the recognition sequence and target proteins for DJ‐1 protease

Identification of the recognition sequence and target proteins for DJ‐1 protease

Purification, molecular properties and specificity of a thermoactive and thermostable proteinase from Pyrococcus abyssi, strain st 549, hyperthermophilic archaea from deep‐sea hydrothermal ecosystem

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

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