Characterization of native and recombinant forms of an unusual cobalt-dependent proline dipeptidase (prolidase) from the hyperthermophilic archaeon Pyrococcus furiosus

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 degrees 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, the Km values for the substrate Met-Pro were comparable for the native and recombinant forms, although the recombinant enzyme exhibited a twofold greater Vmax 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.

FIG. 1

SDS–12% polyacrylamide gel electrophoresis of purified N- and R-prol. Lane 1, molecular mass markers (kilodaltons): myosin (200), β-galactosidase (116), phosphorylase b (97), bovine serum albumin (66.3), glutamic dehydrogenase (55.4), lactate dehydrogenase (36.5), carbonic anhydrase (31); lane 2, R-prol; lane 3, N-prol.

FIG. 2

The 1,047-bp gene encoding P. furiosus prolidase and the deduced amino acid sequence (348 amino acids) are shown. A putative TATA box is indicated in boldface. The ribosomal binding site is underlined, and the translation start site is marked by an arrow.

FIG. 3

The effects of pH (A) and temperature (B) on the activities of N-prol (squares) and R-prol (circles). The assay mixtures contained prolidase (0.015 μg), Met-Pro (4 mM), and CoCl₂ (1.2 mM). For determination of the effects of pH, the following buffers (each at 50 mM) were used: sodium acetate, pH 5.0; bis-Tris-HCl, pH 6.0; MOPS, pH 7.0; EPPS, pH 8.0; CHES (2-[N-cyclohexylamino]-ethanesulfonic acid), pH 9.0; and CAPS, pH 10.0. The assays were carried out at 100°C. For determination of the effects of temperature, the buffer used was 50 mM MOPS, pH 7.0. An N-prol activity level of 100% corresponds to 600 U/mg while 100% R-prol activity corresponds to 1,250 U/mg (with Met-Pro as substrate and measured at 100°C).

FIG. 4

The effects of Co²⁺ and Mn²⁺ ions on the activities of N- and R-prol. The assay mixtures contained 0.02 μg of N-prol (solid symbols) or R-prol (open symbols), Met-Pro (4 mM), and various concentrations of either CoCl₂ (squares) or MnCl₂ (circles).

FIG. 5

Alignment of the amino acid sequence of P. furiosus prolidase with other prolidases (Prol), Alteromonas OPAA, and E. coli methionine aminopeptidase (MAP). The GenBank accession numbers for the prolidases other than the one sequenced in this work are as follows: P46545, L. delbrueckii prolidase; P15034, E. coli prolidase; U56398, Alteromonas OPAA; and P07906, E. coli methionine aminopeptidase. Identical residues are designated by the gray shading while similar residues are boxed. The five residues that are ligands to the binuclear cobalt site in the subunit of E. coli methionine aminopeptidase are indicated by asterisks.