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Applied and Environmental Microbiology, June 2006, p. 4154-4162, Vol. 72, No. 6
0099-2240/06/$08.00+0     doi:10.1128/AEM.02696-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Ca2+-Dependent Maturation of Subtilisin from a Hyperthermophilic Archaeon, Thermococcus kodakaraensis: the Propeptide Is a Potent Inhibitor of the Mature Domain but Is Not Required for Its Folding

Marian Pulido,1,{dagger} Kenji Saito,1,{dagger} Shun-Ichi Tanaka,1 Yuichi Koga,1 Masaaki Morikawa,1,{ddagger} Kazufumi Takano,1,2 and Shigenori Kanaya1*

Department of Material and Life Science, Graduate School of Engineering, Osaka University,1 PRESTO, JST, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan2

Received 15 November 2005/ Accepted 1 April 2006


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ABSTRACT
 
Subtilisin from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 is a member of the subtilisin family. T. kodakaraensis subtilisin in a proform (T. kodakaraensis pro-subtilisin), as well as its propeptide (T. kodakaraensis propeptide) and mature domain (T. kodakaraensis mat-subtilisin), were independently overproduced in E. coli, purified, and biochemically characterized. T. kodakaraensis pro-subtilisin was inactive in the absence of Ca2+ but was activated upon autoprocessing and degradation of propeptide in the presence of Ca2+ at 80°C. This maturation process was completed within 30 min at 80°C but was bound at an intermediate stage, in which the propeptide is autoprocessed from the mature domain (T. kodakaraensis mat-subtilisin*) but forms an inactive complex with T. kodakaraensis mat-subtilisin*, at lower temperatures. At 80°C, approximately 30% of T. kodakaraensis pro-subtilisin was autoprocessed into T. kodakaraensis propeptide and T. kodakaraensis mat-subtilisin*, and the other 70% was completely degraded to small fragments. Likewise, T. kodakaraensis mat-subtilisin was inactive in the absence of Ca2+ but was activated upon incubation with Ca2+ at 80°C. The kinetic parameters and stability of the resultant activated protein were nearly identical to those of T. kodakaraensis mat-subtilisin*, indicating that T. kodakaraensis mat-subtilisin does not require T. kodakaraensis propeptide for folding. However, only ~5% of T. kodakaraensis mat-subtilisin was converted to an active form, and the other part was completely degraded to small fragments. T. kodakaraensis propeptide was shown to be a potent inhibitor of T. kodakaraensis mat-subtilisin* and noncompetitively inhibited its activity with a Ki of 25 ± 3.0 nM at 20°C. T. kodakaraensis propeptide may be required to prevent the degradation of the T. kodakaraensis mat-subtilisin molecules that are activated later by those that are activated earlier.


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INTRODUCTION
 
Subtilisin-like serine proteases (subtilases) are widely distributed in various organisms, including bacteria, archaea, and eucaryotes (48). They are divided into six families (47). Of these, the structures and functions of the subtilisin family (EC 3.4.21.108), which is represented by subtilisin E from Bacillus subtilis (52), subtilisin BPN' from Bacillus amyloliquefaciens (61), and subtilisin Carlsberg from Bacillus licheniformis (25), have been most extensively studied. The crystal structures of these subtilisins have been determined (26, 54, 64). Because subtilisins are commercially valuable enzymes, extensive attempts to improve their activities and stabilities with protein-engineering technology have also been made (10, 55, 60). The subtilisin family includes subtilisins from (hyper)thermophiles (13, 27, 28, 35) and psychrophiles (3, 14, 29, 37). The crystal structures of some of them have been determined (2, 4, 50, 57). These thermostable and thermolabile subtilisins have been regarded not only as good models for studying stability-activity-structure relationships of proteins, but also as potential candidates for various biotechnological applications.

Bacterial subtilisins are synthesized in a precursor form called pre-pro-subtilisin, in which a presequence (signal peptide) and a prosequence (propeptide) are attached to the N terminus of the mature domain (47). They are secreted in a proform (pro-subtilisin) with the assistance of a signal peptide and activated upon autoprocessing and degradation of the propeptide (45). Propeptide binds to its cognate mature domain with high affinity and thereby inhibits its activity (24, 31). According to the crystal structure of the complex between the mature domain of subtilisin and propeptide (19, 26), the C-terminal tyrosine residue of the propeptide binds to the active site of the enzyme in a product-like manner. Thus, degradation of the propeptide is required to produce active subtilisin (65). It has been proposed that propeptides of bacterial subtilisins function not only as inhibitors of their cognate mature domains, but also as intramolecular (12, 15, 17, 30, 31, 40, 43, 44, 46, 58, 65-68) and intermolecular (40, 68) chaperones that facilitate folding of the mature domains. The mature domains alone are not folded into an active form but are folded into an inactive form with molten-globular structure in the absence of propeptides (15, 44). The requirement for a propeptide for maturation of its cognate mature domain has also been reported, not only for other members of the subtilase family (5, 7, 34), but also for other proteases (33, 38, 39, 49, 51, 62). However, it remains to be determined whether archaeal subtilisins mature in a similar manner.

T. kodakaraensis subtilisin from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 is a member of the subtilisin family (28). It consists of a putative signal peptide (Met–93-Ala–70), a propeptide (Gly–69-Leu–1), and a mature domain (Gly1-Gly329) (Fig. 1). The putative mature domain contains three insertion sequences compared to those of bacterial subtilisins. This domain without three insertion sequences, which consists of 271 amino acid residues, is similar to bacterial subtilisins in size and shows amino acid sequence identities of 45 to 46% to subtilisin E, subtilisin BPN', and subtilisin Carlsberg. Nevertheless, it has been proposed that T. kodakaraensis subtilisin exhibits enzymatic activity in a proform, because activity staining of the gel following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) indicated that T. kodakaraensis pro-subtilisin, which was overproduced in Escherichia coli in inclusion bodies, solubilized by 8 M urea, and refolded, exhibited enzymatic activity (28). However, it remains to be determined whether a propeptide of T. kodakaraensis subtilisin is removed from T. kodakaraensis pro-subtilisin during activity staining of the gel. If the propeptide were removed from T. kodakaraensis pro-subtilisin during activity staining of the gel, the mature form of the protein would exhibit activity at the position to which T. kodakaraensis pro-subtilisin migrates in the gel. In this report, we show that T. kodakaraensis pro-subtilisin, which was refolded in the absence of Ca2+, was activated upon autoprocessing and degradation of its propeptide in the presence of Ca2+ at high temperatures. Overproduction, purification, and characterization of its propeptide and mature domain indicated that the propeptide inhibits the activity of the mature form of T. kodakaraensis subtilisin but is not required for its folding. Based on these results, we discuss the maturation process of T. kodakaraensis subtilisin.


Figure 1
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  		FIG. 1. Schematic representations of the primary structures of pre-pro-subtilisin E (top) and pre-T. kodakaraensis pro-subtilisin (bottom). The hatched box represents a signal peptide, the gray box represents a propeptide, the dark box represents a mature domain, and the open box represents an insertion sequence. The signal peptide, propeptide, and mature domain of pre-T. kodakaraensis pro-subtilisin are putative. The locations of the active-site residues and the regions of the recombinant proteins analyzed in this study are also shown.


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MATERIALS AND METHODS
 
Cells and plasmids.
E. coli BL21-CodonPlus(DE3)-RIL [F– ompT hsdS(rB– mB–) dcm+ Tetr gal {lambda}(DE3) endA Hte (argU ileY leuW Camr)] was from Stratagene. Plasmid pET25b was from Novagen. Subtilisin E was kindly donated by Takara Shuzo Co., Ltd.

Overproduction and purification.
T. kodakaraensis pro-subtilisin (Gly–69-Pro329) was overproduced in E. coli in inclusion bodies, solubilized in the presence of 8 M urea, refolded, and purified as described previously (28). The pET25b derivatives for overproduction of T. kodakaraensis propeptide (Gly–69-Leu–1) and T. kodakaraensis mat-subtilisin (Gly1-Gly329) were constructed as previously described for that of T. kodakaraensis pro-subtilisin. PCR primers were designed so that the initiation codon for translation was attached to the 5' termini of the genes encoding T. kodakaraensis propeptide and T. kodakaraensis mat-subtilisin. Overproducing strains were constructed by transforming E. coli BL21-CodonPlus(DE3)-RIL with these pET25b derivatives. For overproduction, these transformants were grown at 37°C in Luria-Bertani medium containing 50 µg/ml ampicillin. When the absorbance at 660 nm of the culture reached around 0.5, 0.5 mM isopropyl-ß-D-thiogalactopyranoside (IPTG) was added to the culture medium and cultivation was continued for an additional 4 h. The cells were then harvested and subjected to the following purification procedures.

For purification of T. kodakaraensis mat-subtilisin, cells were suspended in 20 mM Tris-HCl (pH 9.0), disrupted by sonication, and centrifuged at 15,000 x g for 30 min. The pellet was dissolved in 20 mM Tris-HCl (pH 9.0) containing 8 M urea, dialyzed against 20 mM Tris-HCl (pH 9.0) to remove the urea, and centrifuged at 15,000 x g for 30 min. The resultant supernatant was applied to a Hi-Trap Q (5 ml; Pharmacia Biotech) equilibrated with the same buffer. The refolded protein of T. kodakaraensis mat-subtilisin was eluted from the column as a single peak at an NaCl concentration of approximately 0.2 M by linearly increasing the NaCl concentration from 0 to 0.5 M in the same buffer.

For purification of T. kodakaraensis propeptide, cells were suspended in 20 mM Tris-HCl (pH 7.5), disrupted by sonication, and centrifuged at 15,000 x g for 30 min. The supernatant was dialyzed against 20 mM Tris-HCl (pH 7.5) and centrifuged at 15,000 x g for 30 min. The resultant supernatant was applied to a Hi-Trap SP HP (5 ml; Pharmacia Biotech) equilibrated with the same buffer. Proteins were eluted with a linear gradient from 0 to 0.5 M NaCl in 20 mM Tris-HCl (pH 7.5). T. kodakaraensis propeptide was eluted as a single peak at an NaCl concentration of approximately 0.2 M and was pooled and dialyzed against 20 mM Tris-HCl (pH 9.0). After centrifugation at 15,000 x g for 30 min, the supernatant was applied to a Hi-Trap Q HP (5 ml; Pharmacia Biotech) equilibrated with 20 mM Tris-HCl (pH 9.0). A linear gradient of 0 to 1.0 M NaCl in the same buffer was used to elute the bound proteins. T. kodakaraensis propeptide was eluted at 0.2 M NaCl. After dialysis against 20 mM Tris-HCl (pH 9.0), followed by centrifugation, the protein was precipitated by adding 80% ammonium sulfate. The pellet was dissolved in 20 mM Tris-HCl (pH 9.0) and dialyzed against the same buffer.

The purities of the proteins were analyzed by Tricine-SDS-PAGE using a 15% polyacrylamide gel (http://hincklab.uthscsa.edu/html/protocols/tricine_sds.shtml), followed by staining with Coomassie brilliant blue (CBB). The N-terminal amino acid sequence of the protein was determined by a Procise automated sequencer (Perkin-Elmer model 491).

Protein concentration.
The concentrations of the recombinant proteins were determined from the UV absorption at 280 nm with A2800.1% values (absorbance of a 1.0-mg/ml solution at 280 nm) of 1.25 for T. kodakaraensis pro-subtilisin, 1.47 for T. kodakaraensis mat-subtilisin, and 0.21 for T. kodakaraensis propeptide. These values were calculated by using absorption coefficients of 1,526 M–1 cm–1 for tyrosine and 5,225 M–1 cm–1 for tryptophan at 280 nm (20). The concentration of T. kodakaraensis mat-subtilisin*, which was produced from T. kodakaraensis pro-subtilisin upon autoprocessing, was estimated from the intensity of the band visualized with CBB staining following 15% Tricine-SDS-PAGE with the Scion Image program, using T. kodakaraensis mat-subtilisin purified from E. coli as the standard.

Autoprocessing of T. kodakaraensis pro-subtilisin.
Autoprocessing of T. kodakaraensis pro-subtilisin was analyzed by 15% Tricine-SDS-PAGE. T. kodakaraensis pro-subtilisin (0.3 µM) was incubated at 80°C in 1 ml of 50 mM CAPS (N-cyclohexyl-3-aminopropanesulfonic acid)-NaOH (pH 9.5) containing 5 mM CaCl2, unless otherwise stated. At appropriate intervals, 112 µl of trichloroacetic acid (100% [wt/vol]) was added to this solution to precipitate the proteins. The resultant pellets were washed with 70% acetone, dissolved in the Tricine-SDS-PAGE sample buffer, and subjected to Tricine-SDS-PAGE. The amount of T. kodakaraensis mat-subtilisin* produced from T. kodakaraensis pro-subtilisin upon autoprocessing was estimated as described above.

Activity staining of gel.
SDS-PAGE was carried out using a 12% polyacrylamide gel containing 0.1% gelatin. Samples (0.1 µg each) were boiled for 5 min in the presence of 3 M urea before they were loaded onto the gel. After electrophoresis, the gel was washed with 25% (vol/vol) Triton X-100 at room temperature for 30 min, incubated in 50 mM CAPS-NaOH (pH 9.5) containing 5 mM CaCl2 at 20°C for 12 h or 80°C for 4 h, and stained with CBB. Protease bands were visualized as clear zones due to the hydrolysis of gelatin.

Enzymatic activity.
The enzymatic activities of T. kodakaraensis subtilisin (28) and subtilisin E (56) were determined under the conditions previously reported by using azocasein (Sigma) as a substrate at various temperatures. The reaction mixture (300 µl) contained 50 mM CAPS-NaOH (pH 9.5), 5 mM CaCl2, and 2% azocasein (for T. kodakaraensis subtilisin) or 50 mM Tris-HCl (pH 8.5), 1 mM CaCl2, and 2% azocasein (for subtilisin E). The enzymatic reaction was initiated by adding an appropriate amount of the enzyme and terminated by adding 200 µl of 15% trichloroacetic acid (final concentration, 6%). The reaction time was usually 20 min. After centrifugation at 15,000 x g for 15 min, an aliquot of the supernatant (40 µl) was withdrawn, mixed with 10 µl of 2 M NaOH, and measured for absorption at 440 nm (A440). One unit of enzymatic activity was defined as the amount of the enzyme that increased the A440 value of the assay reaction mixture by 0.1 in 1 min.

The enzymatic activity was also determined by using N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (Suc-AAPF-pNA) (Sigma) as a substrate at various temperatures. The reaction mixture (100 µl) contained 50 mM CAPS-NaOH (pH 9.5), 5 mM CaCl2, and 2 mM Suc-AAPF-pNA. The amount of p-nitroaniline released from the substrate was determined from the absorption at 410 nm with an absorption coefficient of 8,900 M–1 cm–1 by automatic UV spectrophotometer (Beckman model DU640). One unit of enzymatic activity was defined as the amount of the enzyme that produced 1 µmol of p-nitroaniline per min. The specific activity was defined as the enzymatic activity per milligram of protein. For kinetic analyses, the concentration of Suc-AAPF-pNA was varied from 0.5 to 5 mM. The enzymatic reaction followed Michaelis-Menten kinetics, and the Km and kcat values were determined from Lineweaver-Burk plots.

Nucleotide sequence accession numbers.
The GenBank accession numbers of the sequences are K01988 for pre-pro-subtilisin E and AB056701 for pre-T. kodakaraensis pro-subtilisin.


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RESULTS
 
Autoprocessing of T. kodakaraensis pro-subtilisin.
Refolded T. kodakaraensis pro-subtilisin was incubated at 80°C in the presence of Ca2+. Under these conditions, T. kodakaraensis pro-subtilisin was rapidly degraded into two peptides with molecular masses of 38 and 10 kDa (Fig. 2). These masses were identical to those of T. kodakaraensis propeptide (Gly–69-Leu–1) and T. kodakaraensis mat-subtilisin (Gly1-Gly329) separately overproduced in E. coli and purified (see below for details). The N-terminal amino acid sequences of the 10- and 38-kDa peptides were determined to be GEQNT and GKPSW, respectively, which were identical to those of T. kodakaraensis propeptide and T. kodakaraensis mat-subtilisin, respectively. These results indicate that T. kodakaraensis pro-subtilisin is autoprocessed at Leu–1-Gly1 to produce T. kodakaraensis propeptide* and T. kodakaraensis mat-subtilisin* (marked by asterisks to distinguish them from those separately overproduced in E. coli and purified). The amount of T. kodakaraensis mat-subtilisin* reached the maximum within ~5 min, which was estimated to be approximately 30% of that of T. kodakaraensis pro-subtilisin from the intensity of the band visualized with CBB staining. It was not significantly changed upon further incubation regardless of whether T. kodakaraensis pro-subtilisin was completely degraded or not, suggesting that approximately 70% of T. kodakaraensis pro-subtilisin was not autoprocessed into T. kodakaraensis propeptide* and T. kodakaraensis mat-subtilisin* but was fully degraded to small fragments. Moreover, T. kodakaraensis pro-subtilisin was not autoprocessed and remained intact when it was incubated at 80°C in the presence of 1 mM EDTA instead of 5 mM CaCl2. This result indicates that the Ca2+ ion is required for autoprocessing of T. kodakaraensis pro-subtilisin. It is unlikely that T. kodakaraensis pro-subtilisin was degraded by a contaminating E. coli protease at such a high temperature.


Figure 2
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  		FIG. 2. Autoprocessing of T. kodakaraensis pro-subtilisin at 80°C. T. kodakaraensis pro-subtilisin (0.3 µM) was incubated in 1 ml of 50 mM CAPS-NaOH (pH 9.5) containing 5 mM CaCl2 at 80°C for the indicated times, precipitated by adding 112 µl of trichloroacetic acid (100% [wt/vol]), and subjected to 15% Tricine-SDS-PAGE. The protein was stained with CBB. The sample precipitated at 0 min was not exposed to the buffer containing Ca2+. M, low-molecular-mass marker kit (Pharmacia Biotech). The arrows indicate T. kodakaraensis pro-subtilisin, T. kodakaraensis mat-subtilisin*, and T. kodakaraensis propeptide* from the top to the bottom. The molecular mass of each standard protein is indicated beside the gel.

Activity and stability of T. kodakaraensis mat-subtilisin*.
To examine whether T. kodakaraensis mat-subtilisin* represents the active form of T. kodakaraensis subtilisin, T. kodakaraensis pro-subtilisin (0.3 µM) was incubated at 80°C for 90 min in the presence of Ca2+. Under these conditions, both T. kodakaraensis pro-subtilisin and T. kodakaraensis propeptide* were completely degraded (Fig. 2). The resultant solution containing approximately 0.1 µM of T. kodakaraensis mat-subtilisin* was used to analyze the activity and stability of this protein without further purification.

The temperature dependence of the enzymatic activity of T. kodakaraensis mat-subtilisin* was analyzed by measuring the activity at various temperatures ranging from 20 to 100°C using azocasein as a substrate. For comparative purposes, the temperature dependence of the enzymatic activity of subtilisin E was also analyzed. As shown in Fig. 3, T. kodakaraensis mat-subtilisin* exhibited the highest activity at 90°C. It exhibited comparable activity even at 100°C. In contrast, subtilisin E exhibited the highest activity at 60°C. The maximum specific activity of T. kodakaraensis mat-subtilisin* (3,100 ± 240 units/mg) was sevenfold higher than that of subtilisin E. T. kodakaraensis mat-subtilisin* exhibited two- to threefold-higher activity than subtilisin E even at mild temperatures (20 to 40°C).


Figure 3
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  		FIG. 3. Temperature dependence of enzymatic activity. The temperature dependence of the enzymatic activity of processed T. kodakaraensis mat-subtilisin (solid circles) is shown in comparison with that of subtilisin E (open circles). The enzymatic activities were determined at the temperatures indicated by using azocasein as a substrate, as described in Materials and Methods.

The stability of T. kodakaraensis mat-subtilisin* against irreversible heat inactivation was analyzed by incubating the solution containing T. kodakaraensis mat-subtilisin* mentioned above at 80, 90, and 100°C. At appropriate intervals, an aliquot of the solution was withdrawn and the remaining activity was determined at 80°C using azocasein as a substrate. As shown in Fig. 4, T. kodakaraensis mat-subtilisin* was stable at 80°C for at least 3 h. It lost half of its activity in roughly 9 h at 90°C and in 50 min at 100°C. It has been reported that subtilisin E lost its activity even at 60°C with a half-life of 18 min (56). Thus, T. kodakaraensis mat-subtilisin* was shown to be much more stable than subtilisin E.


Figure 4
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  		FIG. 4. Thermal stability of T. kodakaraensis mat-subtilisin*. Semilog plots of the remaining activity versus incubation time are shown. T. kodakaraensis mat-subtilisin* was incubated at 80°C (open circles), 90°C (solid circles), or 100°C (open squares) in 50 mM CAPS-NaOH (pH 9.5) containing 5 mM CaCl2. An aliquot of the solution was withdrawn at appropriate intervals, and the enzymatic activity was determined by using azocasein as a substrate. The lines were obtained by linear regression of the data.

The kinetic constants of T. kodakaraensis mat-subtilisin* were determined using Suc-AAPF-pNA as a substrate at 20 and 80°C. The hydrolysis of this substrate with T. kodakaraensis mat-subtilisin* followed Michaelis-Menten kinetics. The Km and kcat values were calculated to be 4.0 ± 0.5 mM and 26 ± 3.0 s–1 (Vmax, 38 ± 4.6 units/mg), respectively, at 20°C and 8.0 ± 1.0 mM and 290 ± 35 s–1 (Vmax, 420 ± 50 units/mg), respectively, at 80°C from the double-reciprocal plot 1/v versus 1/s. Note that the Km value of 8.0 ± 1.0 mM represents the apparent value, because the solubility of the substrate at pH 9.5 is too low to increase its concentration beyond 5 mM. The specific activities of T. kodakaraensis mat-subtilisin* determined at a substrate concentration of 2 mM (Suc-AAPF-pNA) were 13 ± 1.6 units/mg at 20°C and 140 ± 18 units/mg at 80°C.

Temperature dependence of maturation.
To examine whether T. kodakaraensis pro-subtilisin matures slowly at lower temperatures, T. kodakaraensis pro-subtilisin was incubated at 20, 40, 60, and 80°C in the presence of Ca2+. The activity of the aliquot at appropriate intervals was determined at 20°C using Suc-AAPF-pNA as the substrate. The assay was done at this temperature because the maturation of the protein was expected to be too slow to proceed during assay. If the maturation of the protein did not proceed during assay, the activity of T. kodakaraensis pro-subtilisin relative to that determined after incubation at 80°C for 90 min, which represents the maturation yield, would increase until the maturation was completed. As shown in Fig. 5, the maturation of T. kodakaraensis pro-subtilisin was almost fully completed within 30 min at 80°C and 3 h at 60°C, while it was not completed within 4 h at 40°C and below. The maturation of T. kodakaraensis pro-subtilisin was very slow at 20°C, and only a few percent of T. kodakaraensis pro-subtilisin matured upon incubation at 20°C for 4 h.


Figure 5
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  		FIG. 5. Temperature dependence of maturation. T. kodakaraensis pro-subtilisin (0.3 µM) was incubated in 50 mM CAPS-NaOH (pH 9.5) containing 5 mM CaCl2 at 20°C (x), 40°C (open circles), 60°C (solid circles), and 80°C (open squares). At appropriate intervals, an aliquot (10 µl) was withdrawn and the enzymatic activity at 20°C was determined using Suc-AAPF-pNA as a substrate as described in Materials and Methods. The absorbance at 410 nm, which increases as the amount of the mature protein increases, was plotted as a function of time.

To examine whether slow maturation of T. kodakaraensis pro-subtilisin at lower temperatures was due to slow autoprocessing, T. kodakaraensis pro-subtilisin was incubated at 20, 40, 60, and 80°C for 30 min in the presence of Ca2+. The amounts of T. kodakaraensis pro-subtilisin, T. kodakaraensis mat-subtilisin*, and T. kodakaraensis propeptide* after autoprocessing were estimated by Tricine-SDS-PAGE. As shown in Fig. 6, the amounts of T. kodakaraensis mat-subtilisin* produced at 20, 40, and 60°C were comparable to that produced at 80°C. However, as shown in Fig. 5, the maturation yields of T. kodakaraensis pro-subtilisin after 30 min of incubation were 0.3% at 20°C, 1.8% at 40°C, and 12% at 60°C. When the SDS-PAGE profile of T. kodakaraensis pro-subtilisin incubated at 80°C was compared with that at 20, 40, or 60°C, a clear difference was detected in the amounts of T. kodakaraensis pro-subtilisin and T. kodakaraensis propeptide*. T. kodakaraensis pro-subtilisin and T. kodakaraensis propeptide* were completely degraded at 80°C, while they were not completely degraded at 60°C and below. These results indicate that T. kodakaraensis pro-subtilisin* requires not only autoprocessing, but also degradation of propeptide for its maturation. T. kodakaraensis mat-subtilisin* exhibits maximal activity only when T. kodakaraensis propeptide* is completely degraded. At lower temperatures, T. kodakaraensis propeptide* may continue to bind to the mature domain upon autoprocessing and thereby inhibit the activity of the mature domain.


Figure 6
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  		FIG. 6. Autoprocessing of T. kodakaraensis pro-subtilisin at various temperatures. T. kodakaraensis pro-subtilisin was incubated in 50 mM CAPS-NaOH (pH 9.5) containing 5 mM CaCl2 at 20°C (lane 1), 40°C (lane 2), 60°C (lane 3), and 80°C (lane 4) for 30 min, and its autoprocessing was analyzed by 15% Tricine-SDS-PAGE. The protein was stained with CBB. M, low-molecular-mass marker kit (Pharmacia Biotech). The arrows indicate T. kodakaraensis pro-subtilisin, T. kodakaraensis mat-subtilisin*, and T. kodakaraensis propeptide* from the top to the bottom. The molecular mass of each standard protein is indicated beside the gel.

Overproduction and purification of T. kodakaraensis propeptide and T. kodakaraensis mat-subtilisin.
To analyze the role of T. kodakaraensis propeptide*, strains overproducing T. kodakaraensis propeptide (Gly–69-Leu–1) and T. kodakaraensis mat-subtilisin (Gly1-Gly329) were constructed. T. kodakaraensis propeptide was overproduced in E. coli in a soluble form and purified to give a single band on SDS-PAGE (data not shown). T. kodakaraensis mat-subtilisin was overproduced in E. coli in inclusion bodies, solubilized in the presence of 8 M urea, refolded in the absence of Ca2+, and purified to give a single band on SDS-PAGE (see Fig. 9). Determination of the N-terminal amino acid sequences of these proteins indicated that the N-terminal methionine residue was posttranslationally removed from both proteins. The amounts of the proteins purified from 1 liter of culture were roughly 10 mg for T. kodakaraensis propeptide and 5 mg for T. kodakaraensis mat-subtilisin.


Figure 9
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  		FIG. 9. Comparison of the yields of T. kodakaraensis mat-subtilisin* in the presence and absence of T. kodakaraensis propeptide. Refolded T. kodakaraensis mat-subtilisin (0.3 µM) was incubated in 50 mM CAPS-NaOH (pH 9.5) containing 5 mM CaCl2 at 80°C for 30 min in the absence (lane 2) or presence (lane 3) of T. kodakaraensis propeptide (0.3 µM) and subjected to 15% Tricine-SDS-PAGE. The protein was stained with CBB. Lane 1, refolded T. kodakaraensis mat-subtilisin without exposure to the buffer containing Ca2+; M, low-molecular-mass marker kit (Pharmacia Biotech). The molecular mass of each standard protein is indicated beside the gel.

Inhibition of T. kodakaraensis mat-subtilisin* activity by propeptide.
Inhibition of the enzymatic activity of T. kodakaraensis mat-subtilisin* (0.1 nM) with T. kodakaraensis propeptide was analyzed at 20°C using Suc-AAPF-pNA as a substrate. At this temperature, the concentration of T. kodakaraensis propeptide is not significantly changed during the assay. This is because T. kodakaraensis propeptide is not effectively cleaved by T. kodakaraensis mat-subtilisin*. As shown in Fig. 7, the enzymatic activity of T. kodakaraensis mat-subtilisin* was inhibited by T. kodakaraensis propeptide in a concentration-dependent manner and was almost completely inhibited by 50 nM T. kodakaraensis propeptide. To determine the Ki value of T. kodakaraensis propeptide, the enzymatic activity of T. kodakaraensis mat-subtilisin* was kinetically analyzed in the presence or absence of 20 nM T. kodakaraensis propeptide. The kinetic constants were determined from the double-reciprocal plot 1/v versus 1/s as shown in Fig. 8. Compared to the kinetic constants of T. kodakaraensis mat-subtilisin* in the absence of T. kodakaraensis propeptide, the Km value was not significantly changed (4.0 ± 0.5 mM), while the kcat value was decreased from 26 ± 3.0 to 14 ± 1.8 s–1 in the presence of 20 nM T. kodakaraensis propeptide. These results indicate that T. kodakaraensis propeptide noncompetitively inhibits the enzymatic activity of T. kodakaraensis mat-subtilisin* with a Ki value of 25 ± 3.0 nM. A similar result was obtained when the enzymatic activity of T. kodakaraensis mat-subtilisin* was determined in the presence of 10 nM T. kodakaraensis propeptide (data not shown).


Figure 7
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  		FIG. 7. Inhibition of T. kodakaraensis mat-subtilisin* by T. kodakaraensis propeptide. The indicated concentrations of T. kodakaraensis propeptide were premixed with the substrate (Suc-AAPF-pNA) before T. kodakaraensis mat-subtilisin* was added. Hydrolysis of Suc-AAPF-pNA (2 mM) by T. kodakaraensis mat-subtilisin* (0.1 nM) was analyzed at 20°C by monitoring an increase in absorbance at 410 nm, which resulted from a release of p-nitroaniline.


Figure 8
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  		FIG. 8. Lineweaver-Burk plot. Suc-AAPF-pNA was hydrolyzed by 0.1 nM T. kodakaraensis mat-subtilisin* in the absence (solid circles) or presence (open circles) of 20 nM T. kodakaraensis propeptide as described in Materials and Methods, and 1/v was plotted versus 1/s.

Enzymatic activity of T. kodakaraensis mat-subtilisin.
The specific activity of refolded T. kodakaraensis mat-subtilisin was determined to be 130 ± 14 units/mg at 80°C using azocasein as a substrate. This value was approximately 5% of that of T. kodakaraensis mat-subtilisin*. However, SDS-PAGE analysis indicated that the amount of T. kodakaraensis mat-subtilisin decreased to approximately 5% upon incubation with Ca2+ at 80°C for 30 min (Fig. 9, lane 2). This amount was not significantly changed upon further incubation for up to 2 h (data not shown). These results suggest that only 5% of the refolded protein of T. kodakaraensis mat-subtilisin is converted to the active form and the other part is completely degraded to small fragments upon Ca2+ binding at 80°C. Therefore, refolded T. kodakaraensis mat-subtilisin was first incubated with Ca2+ at 80°C for 30 min for activation, and then the enzymatic activities of the resultant activated protein were determined at various temperatures using azocasein as a substrate. The concentration of the protein was estimated from SDS-PAGE. The protein exhibited the highest activity at 90°C, like T. kodakaraensis mat-subtilisin* (data not shown). The specific activity of the protein was determined to be 2,700 ± 220 units/mg at 80°C, which was comparable to that of T. kodakaraensis mat-subtilisin* (2,600 ± 210 units/mg). The Km and kcat values of the protein determined at 20 and 80°C using Suc-AAPF-pNA as a substrate were nearly identical to those of T. kodakaraensis mat-subtilisin*. These results indicate that the mature domain of T. kodakaraensis subtilisin does not require propeptide for its correct folding. This domain can be converted to T. kodakaraensis mat-subtilisin* in the absence of T. kodakaraensis propeptide. However, the yield of T. kodakaraensis mat-subtilisin* from refolded T. kodakaraensis mat-subtilisin increased by approximately five times in the presence of an equal molar concentration of T. kodakaraensis propeptide (Fig. 9, lane 3). The resultant yield (~25%) was slightly lower than but comparable to that from T. kodakaraensis pro-subtilisin (~30%). This result indicates that T. kodakaraensis propeptide increases the maturation yield of T. kodakaraensis mat-subtilisin regardless of whether T. kodakaraensis propeptide is added in an isolated form or is present as an N-terminal domain of the refolded protein (T. kodakaraensis pro-subtilisin).

To confirm that T. kodakaraensis mat-subtilisin refolded in the absence of propeptide exhibits Ca2+-dependent activity, T. kodakaraensis mat-subtilisin and T. kodakaraensis pro-subtilisin were subjected to SDS-PAGE, and their protease activities were analyzed by activity staining of the gel at 20 and 80°C. Both proteins exhibited activity, at the positions to which they migrated, only in the presence of Ca2+ (Fig. 10) and not in the absence of Ca2+ (data not shown). However, T. kodakaraensis pro-subtilisin exhibited higher and lower activities than refolded T. kodakaraensis mat-subtilisin at 80 and 20°C, respectively. As mentioned above, T. kodakaraensis pro-subtilisin exhibited activity, because it matured during activity staining of the gel. T. kodakaraensis pro-subtilisin exhibited very weak activity at 20°C, probably because the protein matures very slowly at this temperature. Likewise, as mentioned above, the maximal yield of T. kodakaraensis mat-subtilisin* produced from T. kodakaraensis pro-subtilisin upon incubation at 80°C (~30%) is much higher than that produced from refolded T. kodakaraensis mat-subtilisin (~5%). This may be the reason why T. kodakaraensis pro-subtilisin exhibited higher activity than refolded T. kodakaraensis mat-subtilisin at 80°C in activity staining of the gel.


Figure 10
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  		FIG. 10. Activity staining of gel. Samples (0.1 µg) were subjected to 12% SDS-PAGE using a gel containing 0.1% gelatin. After electrophoresis, the gel was washed with 25% (vol/vol) Triton X-100 at room temperature for 30 min, incubated in 50 mM CAPS-NaOH (pH 9.5) containing 5 mM CaCl2 at 80°C for 4 h (lanes 1 and 2) or 20°C for 12 h (lanes 3 and 4), and stained with CBB. Protease bands were visualized as clear zones due to the hydrolysis of gelatin. Lane M, low-molecular-mass marker kit (Pharmacia Biotech); lanes 1 and 3, T. kodakaraensis pro-subtilisin; lanes 2 and 4, T. kodakaraensis mat-subtilisin. The arrows indicate T. kodakaraensis pro-subtilisin (top) and T. kodakaraensis mat-subtilisin (bottom). The molecular mass of each standard protein is indicated beside the gel.


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DISCUSSION
 
Enzymatic properties of T. kodakaraensis mat-subtilisin*.
T. kodakaraensis mat-subtilisin* is a highly stable enzyme with half-lives of 9 h at 90°C and 50 min at 100°C. However, the half-lives of the protein have previously been reported to be 20 min at 90°C and 7 min at 100°C (28). This discrepancy is probably caused by differences in the concentrations of the protein analyzed for thermal stability. In the previous work, T. kodakaraensis pro-subtilisin, instead of T. kodakaraensis mat-subtilisin*, was incubated at a concentration of 42 µg/ml (approximately 1 µM) in the presence of Ca2+ and analyzed for remaining activity at 80°C. In the present work, T. kodakaraensis mat-subtilisin* was incubated at a concentration of 4 µg/ml (0.1 µM). The susceptibility of T. kodakaraensis mat-subtilisin* to self-degradation may increase, and thus its stability apparently decreases, as the concentration of T. kodakaraensis mat-subtilisin* increases.

The Km and kcat values of T. kodakaraensis mat-subtilisin* were determined to be 4.0 ± 0.5 mM and 26 ± 3.0 s–1, respectively, at 20°C using Suc-AAPF-pNA as a substrate. These values were comparable to those of subtilisin E at 37°C for the same substrate (Km, 1.9 mM, and kcat, 21 s–1) (56). Both of these values increased at 80°C compared to those at 20°C. This indicates that the hydrolysis rate of the protein increases while the substrate-binding affinity decreases at high temperatures. The specific activity of T. kodakaraensis mat-subtilisin* for Suc-AAPF-pNA at 80°C was previously reported to be 3,900 units/mg (3.9 units/mg according to the definition of the present work) (28), while in the present work, it was determined to be 140 ± 18 units/mg. This discrepancy is due to a difference in the substrate concentration, which was previously 0.1 mM and is 2 mM here. The enzymatic activity decreases as the substrate concentration decreases below the Km.

Role of Ca2+.
Crystallographic studies indicated that subtilisin BPN' (36, 41) and subtilisin Carlsberg (9, 36) have two Ca2+ binding sites, site 1 (site A) and site 2 (site B), both of which are located far from the active site. Sites 1 and 2 represent high- and low-affinity binding sites, respectively. The enzyme is greatly destabilized with respect to both thermal denaturation and autodegradation and is thereby inactivated upon removal of Ca2+ from site 1 (11, 42, 59). However, pro-subtilisin E has been reported to be folded and autoprocessed in the absence of Ca2+ (66). In addition, deletion of the loop forming site 1 of subtilisin BPN', followed by directed evolution and selection for increased stability, resulted in a Ca2+-independent subtilisin mutant with native-like activity (11, 18, 53). These results suggest that Ca2+ is not required for activity of subtilisin but is required for stability.

In this report, however, it was shown that T. kodakaraensis pro-subtilisin requires Ca2+ for maturation (autoprocessing and degradation of propeptide). It was also shown that T. kodakaraensis mat-subtilisin requires Ca2+ for activity even at mild temperatures, at which this hyperthermophilic protein should be stable. These results suggest that Ca2+ is required to make the conformation of the mature domain of T. kodakaraensis subtilisin active. Various subtilases, such as sphericase from Bacillus sphaericus (1, 2), cell envelope proteinase from Lactococcus lactis (16), and psychrophilic subtilisins (14, 37), have also been reported to exhibit Ca2+-dependent activities. These enzymes may also assume an active conformation only when the Ca2+ ions bind to the enzymes. It remains to be determined, however, whether T. kodakaraensis subtilisin has two or more Ca2+ binding sites and whether its conformation is altered upon Ca2+ binding. Binding of the Ca2+ ions has been suggested to trigger sequential concerted movements that exert long-range effects on the geometry of the catalytic site for proteinase K (6), but not for thermitase (22) and subtilisin BPN' (41). The number and location of Ca2+ binding sites vary greatly for different subtilases. For example, proteinase K (8), thermitase (21), and Bacillus sp. strain Ak. 1 protease (50) contain four, three, and four Ca2+ binding sites, respectively.

Role of T. kodakaraensis propeptide.
Unlike propeptides of bacterial subtilisins, T. kodakaraensis propeptide is not required for folding of the mature domain of T. kodakaraensis subtilisin, because the mature domain alone is refolded and exhibits Ca2+-dependent activity in the absence of T. kodakaraensis propeptide. Refolding of the mature domain without the assistance of propeptide has been reported for subtilisin BPN' covalently attached to agarose beads (23), a Ca2+-independent subtilisin mutant (11), and a thermolysin-like neutral protease (TLP-ste) from Bacillus stearothermophilus (32). In the case of TLP-ste, the refolding yield of the active mature TLP-ste is not significantly changed in the presence or absence of its cognate propeptide. However, the amount of T. kodakaraensis mat-subtilisin* produced from T. kodakaraensis pro-subtilisin was approximately six times higher than that produced from refolded T. kodakaraensis mat-subtilisin when 0.3 µM of the refolded proteins was incubated at 80°C in the presence of Ca2+. The latter amount increased by five times when T. kodakaraensis propeptide was added in an isolated form (Fig. 9). In addition, Ca2+-free T. kodakaraensis mat-subtilisin is rapidly degraded to small fragments by T. kodakaraensis mat-subtilisin* under conditions in which T. kodakaraensis mat-subtilisin* is not self-degraded (S. Tanaka, unpublished data). These results suggest that T. kodakaraensis propeptide is required to allow the mature domain of T. kodakaraensis pro-subtilisin or T. kodakaraensis mat-subtilisin to complete its Ca2+-induced folding. In the absence of T. kodakaraensis propeptide, the T. kodakaraensis mat-subtilisin molecules, which are converted to active form earlier, may rapidly degrade those that are converted to active form later. However, the possibility that T. kodakaraensis propeptide is not required for folding of the mature domain but assists its effective folding cannot be ruled out.

Inhibition mode of T. kodakaraensis propeptide.
Like the propeptides of bacterial subtilisins, T. kodakaraensis propeptide inhibits the activity of T. kodakaraensis mat-subtilisin* when added in trans. However, its inhibition mode is different from those of bacterial propeptides. The propeptides of bacterial subtilisins have been reported to be slow and tight binding inhibitors of the mature cognate, with inhibition constants (Ki) of 1.0 to 6.1 nM (24, 31). The progress curves for the inhibition of these subtilisins by their cognate propeptides reveal a hyperbolic pattern. In contrast, T. kodakaraensis propeptide did not exhibit slow binding inhibition at 20°C (Fig. 7). The enzymatic reaction of T. kodakaraensis mat-subtilisin* followed Michaelis-Menten kinetics in the presence of T. kodakaraensis propeptide. A Lineweaver-Burk plot revealed noncompetitive inhibition, suggesting that the major binding site of T. kodakaraensis propeptide in T. kodakaraensis mat-subtilisin* is different from the substrate-binding site. The Ki value of T. kodakaraensis propeptide (25 ± 3.0 nM) is comparable to that of the propeptide of thermolysin. The propeptide of thermolysin has been reported to act as a mixed, noncompetitive inhibitor of the mature cognate with a 50% inhibitory concentration of 14 nM and a Ki value of 6 to 20 nM (39). It has been noted that propeptides of bacterial subtilisins bind to the mature cognates more tightly than T. kodakaraensis propeptide, but the concentrations of these propeptides sufficient for complete inhibition of the mature cognates are 0.5 to 5 µM, which are at least 10 times higher than that of T. kodakaraensis propeptide, because of their slow binding properties. It remains to be determined whether the enzymatic activity of T. kodakaraensis mat-subtilisin* is inhibited by T. kodakaraensis propeptide in a slow-binding mode at higher temperatures close to the optimum, because T. kodakaraensis propeptide is rapidly degraded by T. kodakaraensis mat-subtilisin* at temperatures higher than 80°C. Construction of the active-site mutants may facilitate quantitative analysis of the interaction between T. kodakaraensis propeptide and T. kodakaraensis mat-subtilisin* at higher temperatures.

Adaptation to high temperature.
The maturation process of T. kodakaraensis pro-subtilisin is bound at an intermediate stage of autoprocessing and degradation of propeptide at temperatures below 60°C. At high temperatures close to the optimum growth temperature of the source organism (90°C), however, T. kodakaraensis propeptide* is rapidly degraded by T. kodakaraensis mat-subtilisin*. Therefore the maturation process of T. kodakaraensis pro-subtilisin is not bound at this intermediate stage. At high temperatures, the activity of T. kodakaraensis mat-subtilisin* increases, the stability of T. kodakaraensis propeptide* decreases, and the binding affinity of T. kodakaraensis propeptide* to T. kodakaraensis mat-subtilisin* probably also decreases. The combination of these effects may facilitate rapid degradation of T. kodakaraensis propeptide* and thereby facilitate rapid maturation of T. kodakaraensis pro-subtilisin at high temperatures. In this regard, T. kodakaraensis subtilisin is well adapted to a hyperthermic environment. Phylogenetic analyses have suggested that hyperthermophilic archaea and bacteria retain a trace of early life forms and produce enzymes which may represent prototypes in the same protein family (63). Therefore, T. kodakaraensis subtilisin may represent a prototype of subtilisin in which a propeptide functions as a potent inhibitor but does not exhibit a chaperoning property. A similar function of propeptide has been reported for a thermostable subtilisin homologue, aqualysin I from Thermus aquaticus (34).


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ACKNOWLEDGMENTS
 
We thank Nao Maeda for technical assistance.

This work was supported in part by a Grant-in-Aid for the National Project on Protein Structure and Functional Analyses and by a Grant-in-Aid for Scientific Research (no. 16041229) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Material and Life Science, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan. Phone and fax: 81-6-6879-7938. E-mail: kanaya{at}mls.eng.osaka-u.ac.jp. Back

{dagger} M. Pulido and K. Saito contributed equally to this work. Back

{ddagger} Present address: Laboratory of Environmental Molecular Biology, Graduate School of Environmental Earth Science, Hokkaido University, Sapporo 060-0810, Japan. Back


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Applied and Environmental Microbiology, June 2006, p. 4154-4162, Vol. 72, No. 6
0099-2240/06/$08.00+0     doi:10.1128/AEM.02696-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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