Sequence, expression in Escherichia coli, and analysis of the gene encoding a novel intracellular protease (PfpI) from the hyperthermophilic archaeon Pyrococcus furiosus

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. 56:1992Microbiol. 56: -1998Microbiol. 56: , 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؇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؇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؇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.Hyperthermophilic microorganisms (those that grow above 90ЊC with an optimum temperature of at least 80ЊC), which are mostly archaea (53), have been investigated for clues to evolutionary processes as well as to uncover biological strategies underlying life at elevated temperatures. Most of the focus has been on characterization of new isolates (3, 45) and on the mechanisms responsible for heightened levels of biomolecular thermostability (1). Although just as important, the physiology of this novel group of organisms has been less studied (20). This is not surprising since hyperthermophiles are generally difficult to culture, in addition to the fact that no genetic systems are available for directed ...

The 1.1-Å resolution crystal structure of DJ-1, the protein mutated in autosomal recessive early onset Parkinson's disease

“…Protease activity was assayed in the same manner as that used for PfpI (28,29). A 0.1% azocasein solution in 50 mM sodium phosphate buffer (pH 7.3) was incubated with Ϸ0.25 mg͞ml protein [DJ-1, YDR533p, YOR391p, or papain (a cysteine protease control)] at 37°C for 30 min.…”

“…ThiJ itself catalyzes the phosphorylation of hydroxymethylpyrimidine (HMP) to HMP monophosphate (25); such thiamine derivatives are thought to protect cells against singlet oxygen and other types of ROS (26,27). However DJ-1 is also Ϸ30% identical on average with members of the PfpI family of putative intracellular cysteine proteases (28), and many of the same residues are conserved between both the ThiJ and PfpI families. The three-dimensional structure of PfpI was determined as part of a structural genomics initiative (29); it shows a dimer of trimeric subunits with the active sites located at subunit interfaces.…”

Familial Parkinson's Disease-associated L166P Mutation Disrupts DJ-1 Protein Folding and Function

“…Protease-Since DJ-1 also shares significant sequence and structural homology with the PfpI family of intracellular proteases (23,35), we sought to determine whether DJ-1 possesses intrinsic protease activity. We first assessed the proteolytic activity of DJ-1 by using the same in-gel protease assay that was used to demonstrate the protease activity of PfpI and PH1704 (34,35).…”

“…DJ-1 is an evolutionarily conserved, 189-amino acid protein that exhibits significant sequence homology with the PfpI family of intracellular proteases (23) and with the ThiJ family of bacterial proteins involved in thiamin synthesis (24). We and other groups have recently solved the crystal structure of human DJ-1, which shows that DJ-1 adopts a helix-strand-helix sandwich structure similar to the bacterial protease PH1704 and Escherichia coli chaperone protein Hsp31 (25)(26)(27)(28)(29).…”

Enhanced production of subtilisin of Pyrococcus furiosus expressed in Escherichia coli using auto-inducing medium

“…Cell extracts from P. furiosus were found to contain five proteases, some of which were found resistant to denaturation by sodium dodecyl sulfate, 8 M urea, 80 mM dithiothreitol and 5% β-mercaptoethanol (Halio et al, 1996;Hicks, 1998). A subtilisin-like cell envelope-associated protease was isolated from P. furiosus.…”