Extremely Thermostable Serine-type Protease from Aquifex pyrophilus
MOLECULAR CLONING, EXPRESSION, AND CHARACTERIZATION*

In-Geol ChoiDagger §, Won-Gi Bang§, Sung-Hou KimDagger , and Yeon Gyu YuDagger parallel

From the Dagger  Structural Biology Center, Korea Institute of Science and Technology, Seoul, 136-791 Korea, the § Department of Agricultural Chemistry, Korea University Seoul, 136-701 Korea, and the  Department of Chemistry, University of California, Berkeley, California 94720

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

A gene encoding a serine-type protease has been cloned from Aquifex pyrophilus using a sequence tag containing the consensus sequence of proteases as a probe. Sequence analysis of the cloned gene reveals an open reading frame of 619 residues that has three canonical residues (Asp-140, His-184, and Ser-502) that form the catalytic site of serine-type proteases. The size of the mature form (43 kDa) and its localization in the cell wall fraction indicate that both the NH2- and COOH-terminal sequences of the protein are processed during maturation. When the cloned gene is expressed in Escherichia coli, it is weakly expressed as active and processed forms. The pH optimum of this protease is very broad, and its activity is completely inactivated by phenylmethylsulfonyl fluoride. The half-life of the protein is 6 h at 105 °C, suggesting that it is one of the most heat-stable proteases. The cysteine residues in the mature form may form disulfide bonds that are responsible for the strong stability of this protease, because the thermostability of the protein is significantly reduced in the presence of reducing reagent.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Hyperthermophiles are organisms that grow optimally at 80 °C or higher temperatures. Ever since they were identified at various geothermal locations, there has been interest in their evolutionary significance as well as their ability to thrive at temperatures in which normal proteins would be denatured. From the ability to grow autotrophically at high temperature and their position in the deepest branch of the phylogenetic tree, hyperthermophiles are presumed to be the closest living descendants of a universal ancestor of life (1, 2). Except for two genera of eubacteria, Aquifex and Thermotoga, all identified hyperthermophiles belong to Archaea. Most of them are grouped as autotrophs that use inorganic materials such as carbon and nitrogen sources or heterotrophs that exploit organic materials as nutrients (3). To degrade exogenous organic compounds, heterotrophic hyperthermophiles have various hydrolytic enzymes such as proteases and glycosidases. Autotrophic organisms also contain proteases that may be involved in cellular processes. The proteases from hyperthermophiles have been studied for their biotechnological applications as well as for their metabolic significance (4). Among the existing types of proteases such as serine-, thiol-, or acid-type proteases, the majority of them belong to a family of serine-type proteases (5-9). Some of them are processed from a preprotein to a mature form and are localized at the cell wall or are secreted. These proteases show heat stability or are resistant to a high concentration of denaturing agents. The half-life measured at around 100 °C varied from a few minutes for archaelysin (5) to several hours for pyrolysin (6).

Aquifex pyrophilus is a microaerobic eubacterium that grows optimally at 85 °C. This organism is also regarded as the most ancient bacterium because of its autotrophic nature and its deep location in the phylogenetic tree (10). Several genes homologous to proteases have been identified from the genomic sequence of Aquifex aeolicus (11). In addition, sequence tags similar to protease-like genes such as the lon protease in Escherichia coli allowed the recognition of the signal peptidase and the serine-type protease from the sequence analysis of the genomic DNA of A. pyrophilus (12). The role of the serine-type protease in A. pyrophilus may not be involved in the degradation of extracellular peptides because these compounds are not utilized as carbon or nitrogen sources.

To characterize the serine-type protease of A. pyrophilus, we have cloned the entire gene and determined its DNA sequence. The cloned gene belongs to the subtilisin family and is localized in the cell wall fraction of A. pyrophilus as a mature form of 43 kDa. When the gene is transformed into E. coli, it is expressed as active and processed forms, although the expression level is low. This protein shows extreme stability at high temperatures and at a broad range of pH values.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials, Strains, and Cultivation-- Restriction enzymes and T4 DNA ligase were obtained from Promega. Sequencing primers were purchased from Bioneer (Seoul, Korea). A. pyrophilus was obtained from the German Collection of Microorganism and Cell Culture (Deutsch Sammlung von Mikroorganismen, Braunschweig, Germany) and was grown in a modified SME medium (13) at 85 °C as described by Huber et al. (14). The cells were inoculated into 20 ml of liquid medium in a 120-ml bottle that was filled with 3 bars of hydrogen, carbon dioxide, and oxygen at a ratio of 79.5:20:0.5 and grown for 12 h with moderate shaking. Cells were harvested by centrifugation at 10,000 × g for 10 min, washed with 10 mM Tris-HCl, pH 8.0, and stored at -80 °C until further usage.

The E. coli strain used for plasmid DNA amplification was DH5aH (supE44 Delta lacU169(phi 80 lacZDelta M15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1). E. coli XL1-blue MRA P2 (Delta (mcrA)18 Delta (mcrCB-hsdSMR-mrr)173 endA1 supE44 thi-l relA1 gyrA96 lac P2 lysogen) was used for lambda  phage propagation, and E. coli BL21(DE3) (E. coli B F- dcm ompT hsdS(rB- mB-) gal lambda (DE3)) was used for protein expression.

DNA Isolation and Library Construction-- A. pyrophilus genomic DNA was purified using a genomic DNA purification kit (Qiagen, Hilden, Germany). For the construction of the genomic lambda  library, 20 µg of genomic DNA from A. pyrophilus was partially digested with HindIII and ligated with 5 µg of lambda  DASHII digested with HindIII. The ligated DNA was packaged using a packaging kit (Stratagene) and amplified in E. coli MRA (P2) in which only phages containing foreign DNA were propagated. About 4,000 primary plaques were obtained and further amplified. DNAs from lambda  clones was isolated using a Qiagen lambda  DNA extraction kit.

Molecular Cloning and Sequence Analysis-- Standard procedures were used for cloning and sequencing as described by Sambrook et al. (15). Southern analysis was performed with an ECL kit from Amersham Pharmacia Biotech (UK). The determination of nucleotide sequence was carried out with an ABI 373 automatic DNA sequencer (Applied Biosystems). The obtained DNA sequences were converted into amino acid sequences in all six possible reading frames and were compared with known proteins in the data base using a sequence comparison program, BLAST (16). For multiple sequence alignment between functionally related proteins, the SEQSEE (17) and MACAW programs (18) were used. The putative membrane-spanning region was assigned using the TMpred program (19).

Fractionation of A. pyrophilus-- Cell paste of A. pyrophilus was suspended in lysis buffer (50 mM Tris-HCl, pH 8.0, 20 µg/ml lysozyme) and incubated for 10 min at 37 °C. The cells were sonicated for 2 min using a Branson Sonifier 450 (Branson Ultrasonic Co.). The crude extract was centrifuged for 20 min at 18,000 × g, and the pellet representing the cell wall fraction was recovered. The proteins in the cell wall fraction were extracted with 0.1% SDS at 95 °C for 20 min. The membrane fraction was prepared from the crude extract using serial centrifugation. The crude extract was centrifuged at 10,000 × g for 10 min, and the supernatant was further centrifuged at 100,000 × g for 1 h. The pellet representing the membrane fraction was resuspended in 50 mM Tris-HCl, pH 8.0. For the preparation of peripheral or integral membrane proteins, the membrane fraction was treated with 1.0 M NaCl or 0.5% Triton X-100 and was centrifuged at 100,000 × g for 1 h.

Construction of Expression Vectors and Expression in E. coli-- Different regions of the open reading frame of the cloned protease gene were amplified by the polymerase chain reaction with 5' and 3' primers harboring NdeI and BamHI restriction sites, respectively. The amplified DNA fragment and the vector, pET21a (Novagen), were treated with NdeI and BamHI, ligated, and transformed into E. coli BL21(DE3) cells. The transformed cells were grown at 37 °C in LB medium containing 100 µg/ml ampicillin with vigorous shaking to an A600 nm of 0.7. Protein expression was induced by adding 1 mM isopropyl-1-thio-beta -D-galactopyranoside for 3 h. Cells were harvested by centrifugation at 4,000 × g for 10 min.

Antibody Production, Immunoprecipitation, and Western Analysis-- For the preparation of an antibody specific to the cloned gene product, a fragment of the open reading frame representing amino acids 102-494 was expressed in E. coli using the pET system as described above. The expressed protein, recovered from inclusion bodies, was separated by 10% SDS-polyacrylamide gel electrophoresis (PAGE).1 The protein band was excised from the gel, crushed, and injected into a mouse for antibody production. Mouse serum was obtained 4 weeks after injection. For Western analysis, proteins were separated on SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Bio-Rad) in 100 mM CAPS buffer, pH 10.0. The membrane was treated with a 5% skim milk solution and then reacted with 1/2,000 diluted serum in TBST buffer (20 mM Tris-HCl, 150 mM NaCl, 0.1% Tween 20, pH 7.5) for 1 h at room temperature. After washing with TBST buffer, protein bands that bound to the antibody were detected using a chemiluminescent reaction using the ECL Western blotting system (Amersham Pharmacia Biotech, UK). Immunoprecipitation was carried out according to the method described by Kessler (20). The immune serum was added to 50 ml of the cell wall extract and incubated on ice for 1 h with occasional stirring. Then, 50 ml of Staphylococcus aureus formalin fixed cells (Sigma) were added to the mixture and incubated for 1 h at 0 °C. The mixture was centrifuged at 3,000 rpm for 10 min, and protease activity in the supernatant was further analyzed.

Purification of Protease Expressed in E. coli-- E. coli cells that expressed the cloned gene were harvested at 4,000 × g for 10 min and resuspended in lysis buffer without lysozyme (50 mM Tris-HCl, pH 8.0). The cells were disrupted by sonication for 2 min, and the soluble fraction was recovered by centrifugation at 17,000 × g for 20 min. After removing heat-labile proteins in the crude extract by heat treatment for 20 min at 95 °C, the soluble fraction was loaded onto a 1.5 × 15-cm Q-Sepharose column (Amersham Pharmacia Biotech, Sweden) equilibrated with 20 mM Tris-HCl, pH 8.0. A 200-ml linear gradient from 0 to 1.0 M NaCl in equilibrating buffer was used to elute the protease.

Assays-- Either a colorimetric method or a gel-staining method was used to measure protease activity. For a colorimetric method, samples were reacted with 0.5% azocasein as described by Cowan et al. (5) with a slight modification. The reaction mixture consisted of 100 µl of enzyme sample and 500 µl of 0.5% azocasein dissolved in 50 mM potassium phosphate buffer, pH 9.0. After incubation at 85 °C for 30 min, the reaction was stopped by the addition of 500 µl of 15% trichloroacetic acid, and the absorbance value at 440 nm was measured using a UV-visible spectrophotometer (Shimadzu, Kyoto, Japan). The gel staining assay was carried out using 0.1% gelatin-containing gel as described by Kleiner and Stetler-Stevenson (21). Proteins were assayed by the Bradford method (22) using bovine serum albumin as a standard.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Cloning and Sequencing of a Serine-type Protease Gene of A. pyrophilus-- One of the lambda  clones from the genomic DNA library of A. pyrophilus hybridized to the sequence tag (Fig. 1A, AQpBB007) that contained a sequence similar to that found in serine-type proteases (12). Southern analysis indicates that the sequence tag used as a probe is located in the middle of the cloned DNA. The sequence of the 2.1-kilobase region including the sequence tag region has been determined in both strands (Fig. 1B). In the sequenced region, there is only one open reading frame (ORF) larger than 100 amino acids. This ORF contains the serine-type protease consensus sequence present in the sequence tag. The nucleotide sequence and deduced amino acid sequence of the ORF are shown in Fig. 2. Upstream from the beginning of the ORF, consensus bacterial promoter sequences were found. The TATAAT and TTGAAG sequences at 24 and 52 bases upstream from the open reading frame are homologous to the -10 (TATAAT) and -35 (TTGACA) promoter sequences (23). Downstream of the putative promoter sites, there are four ATG codons (at the first, 50th, 102nd, and 121st codon) that are candidates for translation initiation sites of the gene. None of them has a distinct ribosome-binding sequence (AGGAGG) in the adjacent 5'-region, except the purine-rich sequence (AACAAG) located at the 8 bases upstream from the first ATG codon. In addition, the amino acid sequences translated between the first and fourth ATG codon are not conserved among proteases. However, the hydrophobic stretch of 20 amino acids starting right after the first ATG codon has a characteristic bacterial secretion signal. Because the gene product is mostly found in the cell wall fraction (see below), the first ATG codon may serve as a translation initiation site and the hydrophobic sequence may serve as a targeting signal. For these reasons, we have tentatively assigned the first methionine as the start site for translation. This ORF codes for a protein of 619 amino acids (69-kDa protein).


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Fig. 1.   Probe sequence and restriction map of an A. pyrophilus protease gene. A, the nucleotide sequence of the sequence tag AQpBB007 around the active site. The deduced amino acid sequence from the tag and the corresponding region of aqualysin I (NCBI accession no. 94701) from Thermus aquaticus YT-1 are compared. B, restriction map of the 11-kilobase (kb) fragment containing the A. pyrophilus protease gene. The open reading frame is indicated by a thick line, and the sequence regions are indicated by arrows under the restriction map. The signal sequence and a putative membrane spanning sequence are represented by black boxes, and the predicted mature form is indicated by a gray box. The numbers above the boxes indicate the positions of the amino acid residues. E, EcoRI; H, HindIII; S, SacI.


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Fig. 2.   Nucleotide and amino acid sequence of the cloned protease gene of A. pyrophilus. The numbering is started from the ATG codon at the +1 position. The putative transcription sites are enclosed in a box. Amino acids in black boxes represent the catalytic residues commonly found in a serine-type protease. The putative signal sequence is underlined. The probe sequence is indicated as a dotted line.

Sequence Alignment of the Protease Gene of A. pyrophilus-- The amino acid sequence of the cloned gene was compared with gene sequences deposited in data banks using the BLAST program. The genes that show strong sequence similarity to the cloned gene are serine-type proteases. It has three canonical residues of aspartic acid (Asp-140), histidine (His-184), and serine (Ser-502) that constitute the active site of serine-type proteases. Amino acid sequences around these residues are highly homologous to those of other proteases (Fig. 3A). The cloned gene has three regions of nonhomologous sequences at the NH2 and COOH terminus and between His-184 and Ser-502 (Fig. 3B). The 150 amino acids at the NH2 terminus are assumed to be involved in membrane translocation because the presence of a cluster of hydrophobic amino acids and positive charges following them are well matched to those of a bacterial secretion signal sequence. The COOH-terminal nonhomologous region starts from amino acid 555. Interestingly, this region contains a putative transmembrane sequence (596-615) at the COOH-terminal end (Fig. 3C). The sequence of 150 amino acids between His-184 and Ser-502 does not show any sequence similarity with any proteins in the data base. However, a serine-type protease from Bacillus subtilis, Vpr (24), and a cell envelope-localized serine protease from Lactobacillus lactis, Wg2 (25), also have a nonhomologous sequence between the active site residues, His and Ser. Although these sequences are not homologous, the size of this nonhomologous sequence and the arrangement in the gene are comparable.


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Fig. 3.   Sequence comparison of the cloned gene. A, amino acid (a.a.) alignment of proteases around three canonical residues (*). Identical and homologous residues are indicated by black and gray boxes, respectively. 1:Apy, A. pyrophilus; 2:Bsu, Vpr from B. subtilis (NCBI accession no. 135023); 3:Lla, Wg2 from L. lactis (NCBI accession no. 472836); 4:Sma, STABLE from Staphylothermus marinus (NCBI accession no. 137456); 5:Pfu, pyrolysin from P. furiosus (NCBI accession no. 1556463); 6:Pae, aerolysin from Pyrobaculum aerophilum (NCBI accession no. 2147085); 7:Taq, aqualysin from T. aquaticus (NCBI accession no. 94701), 8:Tvu, thermiatase from Thermoactinomyces vulgaris (NCBI accession no. 494645). B, the schematic alignment of entire protease genes. The regions with sequence similarity are indicated by black boxes. C, the predicted transmembrane regions in cloned gene. The score of the transmembrane region from outside to inside orientation (o-i) is indicated by a solid line, and the score in the reverse direction (i-o) is denoted by a dotted line. The sequence region that has a score higher than 500 is considered a transmembrane region.

Identification and Localization of the Cloned Gene Product in A. pyrophilus-- To confirm whether the cloned gene is expressed in A. pyrophilus, Western analysis was performed on the crude extract of A. pyrophilus with an antibody raised against a truncated protein of the cloned gene. A 43-kDa protein from the crude extract of A. pyrophilus was strongly detected by the antibody along with two faint bands migrating at 55 and 64 kDa. These bands are mainly observed in the cell wall fraction, and only small portions are detected in the cytosolic fraction (Fig. 4A). These results indicate that the cloned gene is expressed in A. pyrophilus as a precursor form, translocated to the cell wall, and processed to a mature form of 43 kDa. The 64- and 55-kDa bands are probably intermediate forms of the processing step. When the proteases in the extract of A. pyrophilus were observed by the gel staining assay, at least three positive proteases appeared in the cell wall fraction. Among them, the 43-kDa protease showed the highest proteolytic activity (Fig. 4B). Treatment of the gel with 1 mM phenylmethylsulfonyl fluoride abolished most of the protease activity of the 43-kDa protein,2 indicating that the 43-kDa protease is a serine-type protease. To examine whether the 43-kDa protein observed in the Western analysis and in the gel-staining activity assay is the same protein, immunoprecipitation analysis was performed. The 43-kDa protease selectively cross-reacted with the antibody specific to the cloned gene product and was precipitated by S. aureus cells (Fig. 4C). These results show that the 43-kDa protein encoded by the cloned gene is the major serine protease in the cell wall fraction.


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Fig. 4.   Identification of the 43-kDa protein as the product of the cloned gene. Proteins from total cell extract of A. pyrophilus (lane 1), cytosolic fraction (lane 2), and cell wall fraction (lane 3) were analyzed by Western blot (A) or by activity assay after separation in SDS-PAGE containing 0.1% gelatin (B). C, immunoprecipitation. Lane 4, control (no addition of antibody); lane 5, control (addition of bovine serum albumin instead of antibody); lane 6, addition of antibody.

The cloned gene has a putative transmembrane sequence at the COOH terminus. To examine whether the gene product is a membrane protein, the membrane fraction was prepared from the cell extract of A. pyrophilus. Although the 43-kDa protein that cross-reacted with the antibody was mainly observed in the cell wall fraction, a significant amount of the protein was detected in the membrane fraction. To determine whether the protein is a peripheral or integral membrane protein, extracted protein treated with 1.0 M NaCl or 0.5% Triton X-100 was examined. Treatment with high salt did not wash out the protein from the membrane, and only a small fraction of protein was eluted from the membrane by detergent (Fig. 5). Failure of extraction by detergent suggests that the 43-kDa protein is not a membrane protein. The protein observed in the membrane fraction may be because of contamination during cell fractionation.


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Fig. 5.   Localization of Aquifex protease. A, localization of Aquifex protease. The samples were fractionated by ultracentrifugation. Lane 1, cytosolic fraction; lane 2, membrane fraction; lane 3, cell debris. B, extraction of Aquifex protease from the membrane fraction with 1 M NaCl or 0.1% Triton X-100. After incubation for 1 h on ice, insoluble membranes were collected by centrifugation at 100,000 × g for 1 h. Lane 4, insoluble membrane fraction after treatment with 1 M NaCl; lane 5, soluble fraction after treatment with 1 M NaCl; lane 6, insoluble membrane fraction after treatment with 0.1% Triton X-100; lane 7, soluble fraction after treatment with 0.1% Triton X-100.

Expression of the Protease Gene of A. pyrophilus in E. coli-- Attempts to purify the 43-kDa protease from A. pyrophilus were not successful because of low expression and difficulty in cell growth. When the gene was expressed in the E. coli BL21(DE3) cell using a pET21a vector, the expression level was very low, and the host cells stopped growing and lysed. When the coding regions starting from the methionine at the 1st, 50th, or 102nd codon were expressed in E. coli, the proteins were expressed at low levels and showed a toxic effect on the host cells.2 Expression of the coding region from the methionine at the 50th codon showed the lowest toxic effect, and the expressed protein was characterized. The expressed protein was not distinctively observed in Coomassie-stained PAGE (Fig. 6A); however, it was observed as multiple bands whose sizes were in the range of 34-55 kDa by Western analysis (Fig. 6B). The size of the expressed protein was reduced after heat treatment (Fig. 6B). This result indicated that the cloned protease expressed in E. coli undergoes self-processing.


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Fig. 6.   Western analysis of the expressed protein in E. coli. A, Coomassie-stained PAGE. B, Western analysis in SDS-PAGE containing 0.1% gelatin. M, molecular mass markers; lane 1, crude extract of E. coli harboring pET 21a; lane 2, crude extract of E. coli harboring the expression vector before induction; lane 3, crude extract after induction with 1 mM isopropyl-1-thio-beta -D-galactopyranoside; lane 4, crude extract after heat treatment at 95 °C for 20 min.

Characteristics of the Protease-- To examine the properties of the cloned protease, the protein expressed in E. coli was partially purified by anion exchange chromatography and characterized. In addition, the thermostability and pH optimum of the native protease in the cell wall fraction of A. pyrophilus were also investigated. The activity of the native protease was highest at 85 °C and pH 7-9 (Fig. 7, A and B). When protease activity of the expressed protein in E. coli was investigated using azocasein as a substrate, it also showed a pattern similar to the native protease. The maximum activity was measured at 85-95 °C, and the activity gradually decreased as the temperature decreased (Fig. 8A). The optimum pH for the protease activity was obtained at pH 9 (Fig. 8B). At the same pH, the protease seemed slightly more active in phosphate buffer than in Tris-HCl buffer. The stability of the expressed protein was examined by measuring the decrease in activity after incubation at high temperature. When the protein was incubated at 85 °C, it was slowly inactivated, and its half-life was calculated to be 90 h (Fig. 9A). At 105 °C, the activity of the protease decreased by 50% after incubation for 6 h (Fig. 9B). These results indicate that the expressed protein is one of the highest thermostable proteases.


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Fig. 7.   Characteristics of the protease activity from protein extract of the cell wall fraction of A. pyrophilus. A, the effect of temperature. B, the effect of pH. After the same amount of protein was separated in SDS-PAGE containing 0.1% gelatin, each lane was incubated at different temperatures or pH values for 30 min.


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Fig. 8.   Characteristics of the protease expressed in E. coli. The partially purified protease was assayed for its protease activity using azocasein as a substrate at different pH values at 85 °C (A) or different incubation temperatures at pH 8.0 (B) for 30 min. Sodium phosphate buffer (100 mM) was used at pH 6-7 (bullet ), 100 mM Tris-HCl buffer at pH 7-9 (black-square), and 100 mM CAPS buffer at pH 9-11 (black-triangle).


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Fig. 9.   Thermostability of the expressed protease. Activity assay of the partially purified protease was performed using azocasein. The protein sample was incubated at 85 (A) or 105 °C (B) for the indicated time, and the residual activity was measured.


    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Sequence Characterization-- The cloned protease gene has characteristics similar to those in the subtilisin family, the second largest class of serine-type proteases (26). Although a canonical ribosome-binding sequence that has been found in a manganese-superoxide dismutase gene of A. pyrophilus (27) has not been found in the cloned gene, the first methionine codon of the open reading frame was proposed as the initiation site of translation because it was located downstream of the putative promoter sequences. The size of the native protein was smaller than the calculated size based on the deduced amino acid sequence, indicating that the gene was translated as a precursor protein and processed to a mature form. Many subtilisin family proteases are translated as the prepro-form containing a secretion signal sequence. The signal sequence (presequence) was processed after translocation through the periplasmic membrane, and the following sequence (prosequence) was further processed to become the mature form (28). In some cases, the prosequences were located at both the NH2 and COOH termini of the protein and were cleaved during the maturation process (29). The 21 hydrophobic amino acids at the NH2 terminus of the open reading frame have characteristics found in signal sequences (30). Besides the signal sequence, portions of the NH2- and COOH-terminal sequences should be processed. The distance between the conserved sequences containing aspartate (Asp-140) and serine (Ser-502) is about 385 residues, whose calculated size is slightly smaller than the size of the mature form. Hence, the 110-130 residues at the NH2 terminus and the 80-100 residues at the COOH terminus of the precursor form should be processed to generate a 43-kDa protein. It is interesting that a putative membrane spanning sequence is present at the presumed COOH terminus prosequence (Fig. 3C). A transmembrane sequence at the COOH terminus of a protease that anchors the protein to the membrane has been found in the Wg2 protease from L. lactis (25). The hydrophobic sequence at the COOH terminus may serve as a membrane anchor. In that case, the protein may be attached to the membrane transiently because most of the gene product was present as a processed form in the cell wall fraction.

Physiological Role of the Protease-- The function of the cloned protease in the cell is unclear. The protease may not be used for degradation of exogenous nutritional peptides, because A. pyrophilus is a strict autotroph. The physiological role of this protein may be related to its cellular localization. A. pyrophilus contains a very stable proteinaceous layer, called the S-layer, that is present outside of the outer membrane (14). The S-layer of hyperthermophiles consists of heat-stable proteins, and it is assumed to protect the cell from extreme environments such as high temperature (31). Electron microscopic analysis has shown that a very stable protease has been attached to the middle of the long stem that forms the S-layer (7). We presume that the cloned protease is associated with the S-layer proteins of A. pyrophilus, because the protein is tightly associated to the cell wall where the S-layer is present. However, whether the protease is involved in the protein degradation process or serves as a building block of the S-layer structure is currently unknown.

Factors Contributing to the Thermostability of the Protease-- The cloned protease is extremely stable to heat. The half-life of the expressed protein at 105 °C (6 h) suggests that this enzyme is one of the most heat-stable proteases. Structural analysis has shown that one of the major factors that stabilizes proteins from hyperthermophiles is ionic bonds or salt bridges between charged amino acids (32). In addition, proteins from hyperthermophiles have more charged amino acids than proteins from mesophiles or thermophiles. A higher occurrence of charged amino acids is also observed in the proteins from A. pyrophilus (12). The percentage of aspartate, glutamate, arginine, and lysine of the cloned protease is about 20%. This is similar to the value found in pyrolysin from Pyrococcus furiosus and is much higher than proteases from mesophiles or thermophiles (Table I). These charged residues may be engaged in the formation of salt bridges.

                              
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Table I
Comparison of amino acid composition of Aquifex protease with those of several serine proteases
Pyrolysin was from P. furiosus (NCBI accession no. 1556463), aqualysin I was from T. aquaticus (NCBI accession no. 94701), thermiatase was from T. vulgaris (NCBI accession no. 494645), and subtilisin BPN was from Bacillus amyloliquefaciens (NCBI accession no. 67620).

Another factor that contributes to the stability of the protease is disulfide bonds. There are a total of nine cysteine residues in the precursor form of the cloned protein and eight residues in the predicted mature form. Most of these cysteine residues form disulfide bonds that stabilize the protein because treatment with 1 mM dithiothreitol reduced the heat resistance of the protein at 85 °C to less than 2 h.2 Hence, disulfide bonds contribute to the extremely high stability of the protein. Among 170 subtilisin family proteases, only a few, such as aqualysin I, have cysteine residues that may form disulfide bonds (33). Also, serine-type proteases from hyperthermophiles discovered so far do not have cysteine residues except aerolysin and STABLE (7, 33). Enhancement of thermal stability by introduction of intramolecular disulfide bonds has been reported in phage T4 lysozyme (34) or subtilisin (35); however, stabilization of the protein by disulfide bonds is an unusual case among the serine-type proteases from hyperthermophiles. The identity of the disulfide bonds that contribute to the stability of the cloned protease is of current interest.

    FOOTNOTES

* This work was supported by grants from the Ministry of Science and Technology, Korea and by United States Department of Energy Grant DE-AC03-76SF0098.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

parallel To whom correspondence should be addressed: Structural Biology Center, Korea Inst. of Science and Technology, P.O. Box 131, Cheongryang, Seoul, 136-791 Korea. Tel.: 82-2-958-5936; Fax: 82-2-958-5939; E-mail: ygy{at}kistmail.kist.re.kr.

The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; ORF, open reading frame; CAPS, 3-(cyclohexylamino)propanesulfonic acid.

2 I.-G. Choi, W.-G. Bang, S.-H. Kim, and Y. G. Yu, unpublished results.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

  1. Olson, G. J., Woese, C. R., and Overbeek, R. (1994) J. Bacteriol. 176, 1-6[Medline] [Order article via Infotrieve]
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