Characterization of an aminoacylase from the hyperthermophilic archaeon Pyrococcus furiosus

Aminoacylase was identified in cell extracts of the hyperthermophilic archa eon Pyrococcus furiosus by its ability to hydrolyze N-acetyl-L-methionine a nd was purified by multistep chromatography. The enzyme is a homotetramer ( 42.06 kDa per subunit) and, as purified, contains 1.0 +/- 0.48 g-atoms of z inc per subunit. Treatment of the purified enzyme with EDTA resulted in com plete loss of activity. This was restored to 86% of the original value (200 U/mg) by treatment with ZnCl2 (and to 74% by the addition of CoCl2). After reconstitution with ZnCl2 the enzyme contained 2.85 +/- 0.48 g-atoms of zi nc per subunit. Aminoacylase showed broad substrate specificity and hydroly zed nonpolar N-acylated L amino acids (Met, Ala, Val, and Leu), as well as N-formyl-L-methionine. The high K-m values for these compounds indicate tha t the enzyme plays a role in the metabolism of protein growth substrates ra ther than in the degradation of cellular proteins. Maximal aminoacylase act ivity with N-acetyl-L-methionine as the substrate occurred at pH 6.5 and a temperature of 100 degreesC. The N-terminal amino acid sequence of the puri fied aminoacylase was used to identify, in the P. furiosus genome database, a gene that encodes 383 amino acids. The gene was cloned and expressed in Escherichia coli by using two approaches. One involved the T7 lac promoter system, in which the recombinant protein was expressed as inclusion bodies. The second approach used the Trx fusion system, and this produced soluble but inactive recombinant protein. Renaturation and reconstitution experimen ts with Zn2+ ions failed to produce catalytically active protein. A survey of databases showed that, in general, organisms that contain a homolog of t he P. furiosus aminoacylase (greater than or equal to 50% sequence identity ) utilize peptide growth substrates, whereas those that do not contain the enzyme are not known to be proteolytic, suggesting a role for the enzyme in primary catabolism.