Purification and characterization of 5′-methylthioadenosine phosphorylase from the hyperthermophilic archaeon Pyrococcus furiosus

Cacciapuoti, Giovanna; Bertoldo, Costanzo; Brio, Assunta; Zappia, Vincenzo; Porcelli, Marina

doi:10.1007/s00792-002-0307-2

Cited by 34 publications

(45 citation statements)

References 41 publications

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“…The structural gene of PfMTAP encodes a protein of 257 residues with a predicted molecular mass of 29 219 Da, which is in good agreement with that of 30 ± 1 kDa estimated by biochemical analyses for the native enzyme [21].…”

Section: Resultssupporting

confidence: 75%

“…IPTG-induced E. coli cells transformed with pET-MTAP produced 1.77 mg of recombinant protein per gram of cells: thus the expression is about 15-fold higher than for MTAP from P. furiosus [21].…”

Section: Resultsmentioning

confidence: 93%

“…PfMTAP can be considered a PNP with unique features [21]. In fact, because of its hexameric quaternary structure, this enzyme belongs to the high-molecular mass class of PNP.…”

mentioning

confidence: 99%

“…The enzyme is also extremely stable to proteolytic cleavage and after incubation with protein denaturants, detergents, organic solvents, and salts even at high temperature [21]. PfMTAP contains 30 cysteine residues (five per subunit).…”

mentioning

confidence: 99%

“…PfMTAP contains 30 cysteine residues (five per subunit). These residues, on the basis of biochemical evidence such as decrease of the thermal stability in the presence of dithiothreitol and different mobility levels of the enzyme on SDS/ PAGE run under reducing and nonreducing conditions, are thought to be involved in intrasubunit disulfide bonds [21].…”

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confidence: 99%

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Methylthioadenosine phosphorylase from the archaeon Pyrococcus furiosus

Cacciapuoti¹,

Moretti

Forte³

et al. 2004

European Journal of Biochemistry

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The extremely heat-stable 5¢-methylthioadenosine phosphorylase from the hyperthermophilic archaeon Pyrococcus furiosus was cloned, expressed to high levels in Escherichia coli, and purified to homogeneity by heat precipitation and affinity chromatography. The recombinant enzyme was subjected to a kinetic analysis including initial velocity and product inhibition studies. The reaction follows an ordered Bi-Bi mechanism and phosphate binding precedes nucleoside binding in the phosphorolytic direction. 5¢-Methylthioadenosine phosphorylase from Pyrococcus furiosus is a hexameric protein with five cysteine residues per subunit. Analysis of the fragments obtained after digestion of the protein alkylated without previous reduction identified two intrasubunit disulfide bridges. The enzyme is very resistant to chemical denaturation and the transition midpoint for guanidinium chloride-induced unfolding was determined to be 3.0 M after 22 h incubation. This value decreases to 2.0 M in the presence of 30 mM dithiothreitol, furnishing evidence that disulfide bonds are needed for protein stability. The guanidinium chloride-induced unfolding is completely reversible as demonstrated by the analysis of the refolding process by activity assays, fluorescence measurements and SDS/PAGE. The finding of multiple disulfide bridges in 5¢-methylthioadenosine phosphorylase from Pyrococcus furiosus argues strongly that disulfide bond formation may be a significant molecular strategy for stabilizing intracellular hyperthermophilic proteins.Keywords: disulfide bonds; hyperthermostability; 5¢-methyl thioadenosine phosphorylase; purine nucleoside phosphorylase; Pyrococcus furiosus.Hyperthermophilic enzymes which retain their structure and function near the boiling point of water have been, over the past decade, the object of extensive studies on protein stabilization, folding and evolutionary aspects [1][2][3][4]. Moreover, their unique structure-function properties of high thermostability are potentially significant for developing biotechnological applications [5,6]. Thus, there is a great deal of interest in studies on the biochemical adaptation of hyperthermophiles whose enzymes provide unique models for the study and understanding of the evolution of enzymes in terms of structure, specificity and catalytic properties.Much work has been done to identify the structural determinants of the enhanced stability of hyperthermophilic proteins. Several mechanisms of thermal stabilization have been proposed, among which additional networks of salt bridges and hydrogen bonds, improved packing density and enhanced secondary structure are the most cited [2][3][4][7][8][9]. In spite of this, no general rules have been established to date, and it has been concluded that each protein evolves individually through a limited number of factors that occur at different levels, also involving the amino acid sequence and the quaternary structure of the proteins.In recent years, growing attention has been paid to the presence of disulfide bonds in intracellular hy...

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Section: Resultssupporting

confidence: 75%

Section: Resultsmentioning

confidence: 93%

“…PfMTAP can be considered a PNP with unique features [21]. In fact, because of its hexameric quaternary structure, this enzyme belongs to the high-molecular mass class of PNP.…”

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Methylthioadenosine phosphorylase from the archaeon Pyrococcus furiosus

Cacciapuoti¹,

Moretti

Forte³

et al. 2004

European Journal of Biochemistry

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Enzymatic Synthesis of Nucleoside Analogues by Nucleoside Phosphorylases

Kamel

Yehia

Neubauer

et al. 2018

Enzymatic and Chemical Synthesis of Nucleic Acid Derivatives

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S‐adenosylhomocysteine hydrolase from the archaeon Pyrococcus furiosus: Biochemical characterization and analysis of protein structure by comparative molecular modeling

et al. 2005

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S-adenosylhomocysteine hydrolase (AdoHcyHD) is an ubiquitous enzyme that catalyzes the breakdown of S-adenosylhomocysteine, a powerful inhibitor of most transmethylation reactions, to adenosine and L-homocysteine. AdoHcyHD from the hyperthermophilic archaeon Pyrococcus furiosus (PfAdoHcyHD) was cloned, expressed in Escherichia coli, and purified. The enzyme is thermoactive with an optimum temperature of 95 degrees C, and thermostable retaining 100% residual activity after 1 h at 90 degrees C and showing an apparent melting temperature of 98 degrees C. The enzyme is a homotetramer of 190 kDa and contains four cysteine residues per subunit. Thiol groups are not involved in the catalytic process whereas disulfide bond(s) could be present since incubation with 0.8 M dithiothreitol reduces enzyme activity. Multiple sequence alignment of hyperthermophilic AdoHcyHD reveals the presence of two cysteine residues in the N-terminus of the enzyme conserved only in members of Pyrococcus species, and shows that hyperthermophilic AdoHcyHD lack eight C-terminal residues, thought to be important for structural and functional properties of the eukaryotic enzyme. The homology-modeled structure of PfAdoHcyHD shows that Trp220, Tyr181, Tyr184, and Leu185 of each subunit and Ile244 from a different subunit form a network of hydrophobic and aromatic interactions in the central channel formed at the subunits interface. These contacts partially replace the interactions of the C-terminal tail of the eukaryotic enzyme required for tetramer stability. Moreover, Cys221 and Lys245 substitute for Thr430 and Lys426, respectively, of the human enzyme in NAD-binding. Interestingly, all these residues are fairly well conserved in hyperthermophilic AdoHcyHDs but not in mesophilic ones, thus suggesting a common adaptation mechanism at high temperatures.

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Purification and characterization of 5′-methylthioadenosine phosphorylase from the hyperthermophilic archaeon Pyrococcus furiosus

Cited by 34 publications

References 41 publications

Methylthioadenosine phosphorylase from the archaeon Pyrococcus furiosus

Methylthioadenosine phosphorylase from the archaeon Pyrococcus furiosus

Enzymatic Synthesis of Nucleoside Analogues by Nucleoside Phosphorylases

S‐adenosylhomocysteine hydrolase from the archaeon Pyrococcus furiosus: Biochemical characterization and analysis of protein structure by comparative molecular modeling

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