Characterization of a novel zinc-containing, lysine-specific aminopeptidase from the hyperthermophilic archaeon Pyrococcus 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 Co2+ ions (0.2 mM). Optimal KAP activity with Lys-pNA as the substrate occurred at pH 8.0 and a temperature of 100 degrees 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 degrees 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.

FIG. 1.

SDS—12% polyacrylamide gel electrophoresis of the lysine aminopeptidase purified from P. furiosus. Lane 1, standard molecular size markers; lane 2, native lysine aminopeptidase (4 μg); lane 3, recombinant lysine aminopeptidase (4 μg).

FIG. 3.

Alignment of the amino acid sequence of P. furiosus lysine aminopeptidase with other aminopeptidases of the M18 family, yeast aminopeptidase I (AAA34738), and mouse aspartyl aminopeptidase (NP_058574). Conserved histidine, aspartate, and glutamate residues are boxed. Residues that are believed to play a role in metal binding are denoted by asterisks.

FIG. 2.

Effects of temperature and pH on P. furiosus lysine aminopeptidase activity. The assay mixture contained lysine aminopeptidase (0.047 mg/ml) and lysine-pNA (10 mM) in 50 mM MOPS, pH 8.0. For the effects of pH, the following buffers (each at 50 mM) were used at the indicated pH: Bis-Tris, pH 6.0, 6.5, and 6.8; MOPS, pH 7.0, 7.2, and 8.0; 2-(N-cyclohexlamino)ethanesulfonic acid (CHES), pH 8.6 and 9.0. For effects of temperature, the buffer used was 50 mM MOPS (pH 8.0). Activity of 100% corresponds to 1,900 and 2,200 U/mg for native (squares) and recombinant (circles) KAP, respectively.