Characterization of a Novel Zinc-Containing, Lysine-Specific Aminopeptidase from the Hyperthermophilic ArchaeonPyrococcus furiosus

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 Co 2؉ 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.Aminopeptidases are exopeptidases that catalyze the removal of amino acid residues at the N termini of peptides and proteins. Intracellular proteolytic degradation by aminopeptidases is necessary for modulation of protein concentrations, maintenance of amino acid pools, and removal of damaged proteins (17). In addition, aminopeptidases have more specific functions, including activation (10) and inactivation (21) of biologically active peptides and the removal of N-terminal methionyl residues of newly synthesized proteins. The breakdown of proteinaceous substrates, whether imported into the cell or generated from damaged proteins, are further degraded by di-, tri-, and carboxypeptidases, as well as by aminopeptidases (17). Classification of aminopeptidases has been generally based upon their substrate specificities, such as preference for a neutral, acidic, or basic amino acid in the P1 position of the amino terminus of peptides (44). In recent years, more focus has been placed on classifying peptidases based on structural analyses (35). Aminopeptidases are widely distributed among eukaryotes and prokaryotes, although only a few have been isolated from archaea (37). About two-thirds of all aminopeptidases are represented by the metal-dependent peptidases in which zinc is the most frequently associated metal (34).Hyperthermophilic archaea are potentially rich sources of peptidase-type enzymes, because most of them are capable of using protein-based substrates as ...

Metallo-aminopeptidase inhibitors

“…In this respect, a wide variety of structurally diverse aminopeptidases have been recently isolated and characterized from a range of bacterial species. These include: aminopeptidase P isolated from common strain of Escherichia coli [38], aminopeptidase M from pathogenic Mycobacterium tuberculosis [39] and leucine aminopeptidase from Helicobacter pylori [40], cold-active aminopeptidase from psychrotropic Colwellia psychrerythraea [41], a thermophilic enzyme from Geobacillus thermoleovorans [42], an extracellular zinc metalloprotease and putative virulence factor involved in pathogenicity of the fish pathogen Vibrio anguillarum [43], and a lysine-specific enzyme from unusually resistant, hyperthermophilic archaeon Pyrococcus furiosus [44]. This clearly indicates that bacterial aminopeptidases are widely distributed and are of vital importance.…”

Transcriptional and Biochemical Analysis of Starch Metabolism in the Hyperthermophilic Archaeon Pyrococcus furiosus

“…A His-affinity tag of -AAALEHHHHHH was placed at the C terminus of PF0272, while the other proteins were expressed by using a modified form of the N-terminal His-tagged vector, pET24d. The vector was modified to have an N-terminal His tag (MAH HHHHHGS-) and BamHI and NotI sites were used for cloning (41). In brief, recombinant proteins were purified by using a column (5 ml) of Histrap nickelaffinity resin using an AKTA explorer (GE Healthcare, Piscataway, NJ).…”

A Novel Aminopeptidase with Highest Preference for Lysine

“…Its molecular mass, substrate specificity, inhibitor specificity, and sensitivity to metals differ from the microbial lysine-specific aminopeptidases (6, 10À14). The enzymes might be phylogenetically related but of diverse biological functions (10,11,14). The abundance of KAP in various tissues suggests that it could participate in the final stages of neuropeptide-processing mechanisms and/or in particular inflammatory processes and tumor developments (1,9).…”

“…Aspergillus niger KAP belongs to M1 family of metallo-aminopeptidases (12,13). However, hyperthermophilic archaeon KAP belongs to the M18 family (14). In continuation of our search in neuropeptide metabolism, we studied the brain KAP, a possible enzyme designated for neuropeptide processing.…”