ABSTRACT
The hyperthermophilic archaeon Pyrococcus furiosus grows optimally at 100(deg)C by the fermentation of peptides and carbohydrates. From this organism, we have purified to homogeneity an intracellular protease, previously designated PfpI (P. furiosus protease I) (S. B. Halio, I. I. Blumentals, S. A. Short, B. M. Merrill, and R. M. Kelly, J. Bacteriol. 178:2605-2612, 1996). The protease contains a single subunit with a molecular mass of approximately 19 kDa and exists in at least two functional conformations, which were purified separately. The predominant form from the purification (designated PfpI-C1) is a hexamer with a molecular mass of 124 (plusmn) 6 kDa (by gel filtration) and comprises about 90% of the total activity. The minor form (designated PfpI-C2) is trimeric with a molecular mass of 59 (plusmn) 3 kDa. PfpI-C1 hydrolyzed both basic and hydrophobic residues in the P1 position, indicating trypsin- and chymotrypsin-like specificities, respectively. The temperature optimum for Ala-Ala-Phe-7-amido-4-methylcoumarin (AAF-MCA) hydrolysis was (symbl)85(deg)C both for purified PfpI-C1 and for proteolytic activity in P. furiosus cell extract. In contrast, the temperature optimum for PfpI prepared by incubating a cell extract of P. furiosus at 98(deg)C in 1% sodium dodecyl sulfate for 24 h at 95 to 100(deg)C (I. I. Blumentals, A. S. Robinson, and R. M. Kelly, Appl. Environ. Microbiol. 56:1255-1262, 1990), designated PfpI-H, was (symbl)100(deg)C. Moreover, the half-life of activity of PfpI-C1 at 98(deg)C was less than 30 min, in contrast to a value of more than 33 h measured for PfpI-H. PfpI-C1 appears to be a predominant serine-type protease in cell extracts but is converted in vitro, probably in part by deamidation of Asn and Gln residues, to a more thermally stable form (PfpI-H) by prolonged heat treatment. The deamination hypothesis is supported by the differences in the measured pI values of PfpI-C1 (6.1) and PfpI-H (3.8). High levels of potassium phosphate (>0.5 mM) were found to extend the half-life of PfpI-C1 activity towards AAF-MCA by up to 2.5-fold at 90(deg)C, suggesting that compatible solutes play an important role in the in vivo function of this protease.
ABSTRACT
A previously identified intracellular proteolytic activity in the hyperthermophilic archaeon Pyrococcus furiosus (I. I. Blumentals, A. S. Robinson, and R. M. Kelly, Appl. Environ. Microbiol. 56:1992-1998, 1990) was found to be a homomultimer consisting of 18.8-kDa subunits. Dissociation of this native P. furiosus protease I (PfpI) into a single subunit was seen by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) but only after trichloroacetic acid precipitation; heating to 95 degrees C in the presence of 2% SDS and 80 mM dithiothreitol did not dissociate the protein. The gene (pfpI) coding for this protease was located in genomic digests by Southern blotting with probes derived from the N-terminal amino acid sequence. pfpI was cloned, sequenced, and expressed in active form in Escherichia coli as a fusion protein with a histidine tag. The recombinant protease from E. coli showed maximum proteolytic activity at 95 degrees C, and its half-life was 19 min at this temperature. This level of stability was significantly below that previously reported for the enzyme purified by electroelution of a 66-kDa band from SDS-PAGE after extended incubation of cell extracts at 98 degrees C in 1% SDS (>30 h). The pfpI gene codes for a polypeptide of 166 amino acid residues lacking any conserved protease motifs; no protease activity was detected for the 18.8-kDa PfpI subunit (native or recombinant) by substrate gel assay. Although an immunological relationship of this protease to the eukaryotic proteasome has been seen previously, searches of the available databases identified only two similar amino acid sequences: an open reading frame of unknown function from Staphylococcus aureus NCTC 8325 (171 amino acid residues, 18.6 kDa, 41% identity) and an open reading frame also of unknown function in E. coli (172 amino acid residues, 18.8 kDa, 47% identity). Primer extension experiments with P. furiosus total RNA defined the 5' end of the transcript. There are only 10 nucleotides upstream of the start of translation; therefore, it is unlikely that there are any pre- or pro-regions associated with PfpI which could have been used for targeting or assembly of this protease. Although PfpI activity appears to be the dominant proteolytic activity in P. furiosus cell extracts, the physiological function of PfpI is unclear.
