Skip Navigation



PEDS Advance Access published online on March 9, 2007
Protein Engineering Design and Selection, doi:10.1093/protein/gzm006
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
20/3/143    most recent
gzm006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Pulido, M.A.
Right arrow Articles by Kanaya, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Pulido, M.A.
Right arrow Articles by Kanaya, S.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Directed evolution of Tk-subtilisin from a hyperthermophilic archaeon: identification of a single amino acid substitution responsible for low-temperature adaptation

M.A. Pulido1, Y. Koga1, K. Takano1,2 and S. Kanaya1,3

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

3 To whom correspondence should be addressed. E-mail: kanaya{at}mls.eng.osaka-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Tk-subtilisin from the hyperthermophilic archaeon Thermococcus kodakaraensis is synthesized in a prepro-form (prepro-Tk-subtilisin), secreted in a pro-form (pro-Tk-subtilisin), and matured to an active form (mat-Tk-subtilisin*; a Ca2+-bound active form of matured domain of Tk-subtilisin) upon autoprocessing and degradation of the propeptide [Tk-propeptide; propeptide of Tk-subtilisin (Gly1-Leu69)]. Pro-Tk-subtilisin exhibited halo-forming activity only at 80°C, but not at 70 and 60°C, because Tk-propeptide is not effectively degraded by mat-Tk-subtilisin* and forms an inactive complex with mat-Tk-subtilisin* at <80°C. Random mutagenesis in the entire prepro-Tk-subtilisin gene, followed by screening for mutant proteins with halo-forming activity at 70 and 60°C, allowed us to identify single Gly56 -> Ser mutation in the propeptide region responsible for low-temperature adaptation of pro-Tk-subtilisin. SDS–PAGE analyses and mat-Tk-subtilisin* activity assay of pro-G56S-subtilisin indicated more rapid maturation than pro-Tk-subtilisin. The resultant active form was indistinguishable from mat-Tk-subtilisin* in activity and stability, indicating that Gly56 -> Ser mutation does not seriously affect the folding of the mature domain. However, this mutation greatly destabilized the propeptide, making it unstructured in an isolated form. As a result, Tk-propeptide with Gly56 -> Ser mutation (G56S-propeptide) was more susceptible to proteolytic degradation and less effectively inhibited mat-Tk-subtilisin* activity than Tk-propeptide. These results suggest that pro-G56S-subtilisin is more effectively matured than pro-Tk-subtilisin at lower temperatures, because autoprocessed G56S-propeptide is unstructured upon dissociation from mat-Tk-subtilisin* and is therefore effectively degraded by mat-Tk-subtilisin*.

Keywords: directed evolution/low-temperature adaptation/propeptide/subtilisin/Thermococcus kodakaraensis


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Tk-subtilisin is a subtilisin-homolog from the hyperthermophilic archaeon Thermococcus kodakaraensis (Kannan et al., 2001Go; Pulido et al., 2006Go). It consists of a putative 24-residue signal peptide [Met(-24)-Ala(-1)], a 69-residue propeptide (Gly1-Leu69) and a 329-residue mature domain (Gly70-Gly398) (Fig. 1). The mature domain of Tk-subtilisin has an N-terminal extension and two insertion sequences (13–20 residues each) compared to bacterial subtilisins such as subtilisin E, subtilisin BPN' and subtilisin Carlsberg. Without these sequences, this domain is similar in size to bacterial subtilisins and shows the amino acid sequence identities of 45–46% to bacterial subtilisins. Similar to bacterial subtilisins, Tk-subtilisin is synthesized as prepro-Tk-subtilisin [Met(-24)-Gly398] and is presumably secreted across the cytoplasmic membrane with the aid of the signal peptide. Pro-Tk-subtilisin (Gly1-Gly398), which is secreted to the external medium, is then matured by two steps: autoprocessing and degradation of propeptide, which proceed sequentially (Pulido et al., 2006Go). Upon autoprocessing, the peptide bond between Leu69 and Gly70 is cleaved to produce Tk-propeptide [propeptide of Tk-subtilisin (Gly1-Leu69)] and mat-Tk-subtilisin* (Gly70-Gly398), which form an inactive complex. Here, mat-Tk-subtilisin* is marked by an asterisk to distinguish a Ca2+-bound active form of the mature domain of Tk-subtilisin from Ca2+-free inactive mat-Tk-subtilisin. Tk-propeptide is a potent inhibitor and, at the same time, a good substrate of mat-Tk-subtilisin*, and therefore mat-Tk-subtilisin* exhibits maximal activity only when Tk-propeptide is completely degraded. Mat-Tk-subtilisin* is a highly thermostable enzyme with half-lives of 50 min at 100°C and exhibits the highest activity at 90°C. It is also highly active and exhibits 2- to 3-fold higher activity than subtilisin E even at low temperatures (20–40°C).


Figure 1
View larger version (10K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1.. Schematic representation of the primary structure of prepro-Tk-subtilisin. Hatched box represents a putative signal peptide, gray box represents a propeptide, dark box represents a mature domain and open box represents an insertion sequence. The locations of the active site residues and the regions of the recombinant proteins analyzed in this study are also shown. It is noted that the numbering of the amino acid residue in the pro-Tk-subtilisin sequence is changed from that reported previously (Pulido et al., 2006Go), such that the positive number started from the initial glycine residue of pro-Tk-subtilisin, instead of mat-Tk-subtilisin. The GenBank accession number of this sequence is AB056701.

 
The folding pathway of bacterial subtilisins has been most extensively studied for subtilisin E (Li et al., 1995Go; Fu et al., 2000Go; Shinde and Inouye, 2000Go; Yabuta et al., 2001Go; Yabuta et al., 2003Go; Subbian et al., 2005Go). On the basis of these studies, a propeptide serves not only as an inhibitor of the mature domain, but also as an intramolecular chaperone that facilitates folding of the mature domain. In the absence of the propeptide, the mature domain is not folded into an active form, but is folded into an inactive form with molten-globular structure. Similar role of propeptide has been reported for other subtilisins as well (Eder et al., 1993aGo, bGo; Marie-Claire et al., 2001Go). In addition, pro-subtilisin E is folded and autoprocessed in the absence of Ca2+ (Yabuta et al., 2002Go). A Ca2+-independent subtilisin mutant with native-like activity has been successfully constructed by directed evolution (Gallagher et al., 1993Go; Strausberg et al., 1995Go; Bryan, 2000Go). These results indicate that bacterial subtilisins do not require Ca2+ for maturation.

In contrast, Tk-subtilisin does not require propeptide, but requires Ca2+ for maturation (Pulido et al., 2006Go), suggesting that the folding pathway of Tk-subtilisin is different from that proposed for bacterial subtilisins. Mat-Tk-subtilisin* is produced from pro-Tk-subtilisin upon autoprocessing and degradation of propeptide only in the presence of Ca2+. Likewise, mat-Tk-subtilisin is converted to mat-Tk-subtilisin* only in the presence of Ca2+, even in the absence of Tk-propeptide. These results suggest that Ca2+, instead of Tk-propeptide, is required to make the conformation of the mature domain of pro-Tk-subtilisin active. Furthermore, bacterial subtilisins are effectively matured at room temperature (Subbian et al., 2005Go), while pro-Tk-subtilisin is effectively matured only at ≥80°C (Pulido et al., 2006Go). Pro-Tk-subtilisin can be autoprocessed into mat-Tk-subtilisin* and Tk-propeptide even at 20°C, but Tk-propeptide is not fully degraded by mat-Tk-subtilisin* at ≤40°C. One of the promising strategies to understand the maturation process of pro-Tk-subtilisin and its adaptation mechanism to high temperatures is construct a mutant that adapts to low temperatures and analyze its low-temperature adaptation mechanism.

Directed evolution is a method that mimics natural selection enabling evolution and adaptation of enzymes under controlled conditions with well-defined selective pressures (Arnold, 1996Go; Arnold et al., 2001Go; Farinas et al., 2001Go). Because the enzymes with only minimum amino acid substitutions responsible for functional changes can be generated, this method has been shown to be effective to modify enzyme functions (Tao and Cornish, 2002Go; Cherry and Fidantsef, 2003Go; Bloom et al., 2005Go; Eijsink et al., 2005Go). Subtilisins have so far been extensively studied by using directed evolution method. Various subtilisin derivatives with increased stability (Bryan et al., 1986Go; Zhao and Arnold, 1999Go; Yang et al., 2000Go; Srimathi et al., 2006Go), increased activity (Chen and Arnold, 1993Go; You and Arnold, 1994Go; Kano et al., 1997Go; Taguchi et al., 1998Go), altered substrate specificity (Takagi and Takahashi, 2003Go) and Ca2+-independent activity (Strausberg et al., 1995Go; Bryan, 2000Go) have been constructed by this method. The folding process of subtilisin (Fu et al., 2000Go), role of propeptide (Lerner et al., 1990Go; Kobayashi and Inouye, 1992Go; Li et al., 1995Go) and adaptation mechanisms of subtilisin to unusual environment (Taguchi et al., 2000Go; Wintrode et al., 2001Go) have also been analyzed by this method.

In this study, pro-G56S-subtilisin with single Gly56 -> Ser mutation in the propeptide region was constructed by the combination of directed evolution and site-directed mutagenesis. This mutant protein matured more rapidly than the parent protein, especially at low temperatures. This mutant protein was overproduced in Escherichia coli, purified and characterized. G56S-propeptide was also overproduced, purified, and its biochemical properties were compared with those of Tk-propeptide. On the basis of these results, we discuss the mechanism of how pro-G56S-subtilisin adapts to low temperatures.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Cells and plasmids

E. coli HB101 [F– thi-1 hsdS20 (rB–, mB–) supE44 recA13 ara-14 leuB6 proA2 lacY1 galK2 rpsL20 (strr) xyl-5 mtl-1] was from Promega. E. coli BL21-CodonPlus (DE3)-RIL [F– ompT hsdS (rB– mB–) dcm+ Tetr gal {lambda}(DE3) endA Hte (argU ileY leuW Camr)] and E. coli BL21 (DE3) [F– ompT hsdSB (rB– mB–) dcm gal {lambda} (DE3)] were from Stratagene. Plasmid pBR322 was from New England Biolabs, and pET25b and pET28a were from Novagen.

Materials

Achromobacter protease I (API), gellan gum and casein were obtained from Wako. KOD polymerase was from Toyobo. Recombinant Taq polymerase (rTaq) was from Takara. Tween 20 was from BioRad. N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (Suc-AAPF-pNA) and azocasein were from Sigma. Pro-Tk-subtilisin, mat-Tk-subtilisin and Tk-propeptide were overproduced and purified as described previously (Pulido et al., 2006Go). A Ca2+-bound active form of mat-Tk-subtilisin (mat-Tk-subtilisin*) was prepared by incubating pro-Tk-subtilisin at 80°C for 90 min in the presence of Ca2+, as described previously in Pulido et al. (2006)Go, and used without further purification.

Plasmid construction

The pBR322 derivative with the insertion of 1560-bp DNA fragment containing the entire prepro-Tk-subtilisin gene at the HindIII site was previously constructed (Kannan et al., 2001Go). This plasmid was used as a template to amplify the 1560-bp DNA fragment containing the entire prepro-Tk-subtilisin gene by polymerase chain reaction (PCR) using primers 1 and 2 (Table I). The amplified DNA fragment was digested with HindIII and BamHI, and ligated into the HindIII–BamHI sites of pBR322 to generate pBR1560. In this plasmid, the transcription and translation of the prepro-Tk-subtilisin gene are under the control of its own promoter and Shine-Dalgarno (SD) sequence, respectively. The 5'-terminus of a putative mRNA and SD sequence is located ~40 and 8 bp upstream of the initiation codon for translation, respectively.


View this table:
[in this window]
[in a new window]

  		Table I.. Nucleotide sequences of the PCR primers

 
For the construction of the pBR1560 derivative containing the prepro-G56S-subtilisin gene, the gene encoding the prepro-region of prepro-G56S/T135S-subtilisin was amplified by PCR using primers 1 and 3, and the gene encoding the mature region of prepro-Tk-subtilisin was amplified by PCR using primers 2 and 4. The prepro-G56S/T135S-subtilisin gene was obtained by directed evolution method. These two amplified DNA fragments were used as templates to amplify the prepro-G56S-subtilisin gene by overlap PCR. For the construction of the pBR1560 derivative containing the prepro-T135S-subtilisin gene, the gene encoding the prepro-region of prepro-Tk-subtilisin was amplified by PCR using primers 1 and 3, and the gene encoding the mature region of prepro-G56S/T135S-subtilisin was amplified by PCR using primers 2 and 4. These two amplified DNA fragments were used as templates to amplify the prepro-T135S-subtilisin gene by overlap PCR. Primers 1 and 2 were used for these overlap PCRs. The resultant DNA fragments were digested with HindIII and BamHI, and ligated into the HindIII–BamHI sites of pBR322.

For the construction of the pET25b derivative for the overproduction of pro-G56S-subtilisin, the gene encoding the pro-G56S-subtilisin region of prepro-G56S-subtilisin was amplified by PCR using primers 5 and 6. The resultant DNA fragment was digested with NdeI and BamHI, and ligated into the NdeI–BamHI sites of pET25b. For the construction of the pET25b and pET28a derivatives for the overproduction of G56S-propeptide without and with a His-tag at the N-terminus, respectively, the gene encoding the propeptide region of pro-G56S-subtilisin was amplified by PCR using primers 5 and 7. The resultant DNA fragment was digested with NdeI and BamHI, and ligated into the NdeI–BamHI sites of pET25b and pET28a.

PCR was carried out using GeneAmp PCR system 2400 (Perkin-Elmer) for 30 cycles using a KOD polymerase according to the procedures recommended by the supplier. All DNA oligomers for PCR were synthesized by Hokkaido System Science. The nucleotide sequence was determined with a Prism 310 DNA sequencer (Perkin Elmer).

Random mutagenesis

Random mutations were introduced into the gene encoding prepro-Tk-subtilisin by error-prone PCR using primers 1 and 2, as described previously with slight modifications (Song and Rhee, 2000Go). Plasmid pBR1560 was used as a template. The reaction mixture (100 µl) contained 10 mM Tris–HCl (pH 8.3), 50 mM KCl, 0.16 mM dNTP, 1 mM MgCl2, 50 pmol of each primer, 10 ng of pBR1560 and 5 U of rTaq polymerase. PCR was carried out using GeneAmp PCR system 2400 at 96°C for 1 min, 55°C for 1 min and 72°C for 2 min for 30 cycles. These conditions are assumed to generate an error frequency of ~2–3 substitutions per 1000 bases (Song and Rhee, 2000Go). Amplified DNA fragments were digested with HindIII and BamHI, and ligated into the HindIII–BamHI sites of pBR322 to generate a library of the pBR1560 derivatives containing the prepro-Tk-subtilisin genes with random point mutations. Single mutant proteins are designated as pro-G56S-subtilisin, pro-T135S-subtilisin and the like, in which Gly56 is replaced by Ser and Thr135 is replaced by Ser. For mutant proteins with multiple replacements, the designations for each mutation are partitioned by a slash, as in pro-G56S/T135S-subtilisin.

Screening

E. coli HB101 cells were transformed with the library of pBR1560 derivatives and grown on a Luria-Bertani (LB) medium–agar plate with 50 µg/ml ampicillin at 37°C for 16 h. Colonies were transferred to LB plate with 1% casein and 2% gellan gum and allowed to grow overnight at 37°C. The casein plates were then overlaid with 2% gellan gum and 1% Tween 20 for the lysis of E. coli cells and incubated at 60, 70, and 80°C for 14–18 h. The hydrolysis of casein by Tk-subtilisin was detected by the formation of a white halo around the colony.

Overproduction and purification

An overproducing strain of pro-G56S-subtilisin was constructed by transforming E. coli BL21-CodonPlus(DE3)-RIL with the pET25b derivative. Pro-G56S-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, as previously described for pro-Tk-subtilisin (Pulido et al., 2006Go). The overproducing strains of G56S-propeptide and His-tagged G56S-propeptide were constructed by transforming E. coli BL21-CodonPlus(DE3)-RIL with the pET25b derivative and E. coli BL21(DE3) with the pET28a derivative, respectively. For overproduction, these transformants were grown at 37°C in LB medium containing 50 µg/ml ampicillin and 34 µg/ml chloramphenicol [E. coli BL21-CodonPlus(DE3)-RIL transformants] or LB medium containing 30 µg/ml kanamycin [E. coli BL21(DE3) transformants]. When the absorbance of the culture at 660 nm reached 0.5, 0.5 mM isopropyl-ß-D-thiogalactopyranoside (IPTG) was added to the medium, and the cultivation was continued for an additional 4 h. Cells were harvested and subjected to the following purification procedures.

For the purification of G56S-propeptide, cells were suspended in 20 mM Tris–HCl (pH 7.5), disrupted by French press and centrifuged at 15 000 g for 30 min. The resultant pellet was solubilized by 20 mM Tris–HCl (pH 7.5) containing 8 M urea and applied to a HiTrap SP column (5 ml, Pharmacia Biotech) equilibrated with the same buffer. The protein was eluted with a linear gradient from 0 to 0.5 M NaCl in the same buffer. G56S-propeptide was eluted at NaCl concentration of ~0.2 M and was pooled and dialyzed against 20 mM Tris–HCl (pH 9.0) containing 8 M urea. After centrifugation at 15 000 g for 30 min, the supernatant was applied to a HiTrap Q column (5 ml, Pharmacia Biotech) equilibrated with the same buffer. The protein was eluted with a linear gradient from 0 to 1.0 M NaCl in the same buffer. G56S-propeptide was eluted as a single peak at NaCl concentration of ~0.2 M. The protein was refolded by removing urea by step-wise dialysis against 20 mM Tris–HCl (pH 9.0) containing different concentrations of urea. After centrifugation at 15 000 g for 30 min to remove any precipitate, refolded G56S-propeptide was concentrated using Centricon YM-3 (Millipore).

For the purification of His-tagged G56S-propeptide, cells were suspended in 10 mM sodium phosphate (pH 7.4) containing 30 mM imidazole and 0.5 M NaCl, disrupted by French press and centrifuged at 15 000 g for 30 min. The resultant supernatant was applied to a HiTrap Chelating HP column (5 ml, Pharmacia Biotech) equilibrated with the same buffer. The protein was eluted with a linear gradient from 0 to 0.5 M imidazole in 10 mM sodium phosphate (pH 7.4) and 0.5 M NaCl. The protein was eluted as a single peak at an imidazole concentration of ~0.2 M and was pooled and dialyzed against 20 mM Tris–HCl (pH 7.5) containing 0.5 M NaCl.

The purities of the proteins were analyzed by 15% Tricine–SDS–PAGE (SDS–PAGE using the Tricine buffer) (Yabuta et al., 2001Go; http://hincklab.uthscsa.edu/html/protocols/tricine_sds.shtml), followed by staining with Coomassie Brilliant Blue G-250 (CBB).

Protein concentration

The protein concentrations were determined from the UV absorption at 280 nm using A2800.1% values (absorbance of a 1.0-mg/ml solution at 280 nm) of 1.25 for pro-Tk-subtilisin and pro-G56S-subtilisin, 1.47 for mat-Tk-subtilisin and 0.21 for Tk-propeptide, G56S-propeptide and His-tagged G56S-propeptide. These values were calculated by using absorption coefficients of 1526 M–1 cm–1 for tyrosine and 5225 M–1 cm–1 for tryptophan at 280 nm (Goodwin and Morton, 1946Go). The concentration of mat-Tk-subtilisin* produced from pro-Tk-subtilisin and pro-G56S-subtilisin upon maturation was estimated from the intensity of the band visualized with CBB staining following 15% Tricine–SDS–PAGE with the Scion Image Program using mat-Tk-subtilisin as the standard.

Enzymatic activity

The enzymatic activity of mat-Tk-subtilisin* was determined by using azocasein or Suc-AAPF-pNA as a substrate as described previously (Pulido et al., 2006Go). The reaction mixture contained 50 mM N-cyclohexyl-3'- aminopropanesulfonic acid (CAPS)–NaOH (pH 9.5), 5 mM CaCl2 and 2% azocasein or 2 mM Suc-AAPF-pNA. When azocasein was used as a substrate, the reaction mixture (300 µl) was incubated at various temperatures for 20 min, and the enzymatic reaction was terminated by adding 200 µl of 15% trichloroacetic acid (final concentration of 6%). After centrifugation at 15 000 g for 15 min, an aliquot of the supernatant (40 µl) was withdrawn, added to 10 µl of 2 M NaOH and the absorption was measured at 440 nm (A440). One unit of enzymatic activity was defined as the amount of enzyme that increases the A440 value of the assay reaction mixture by 0.1 in 1 min. When Suc-AAPF-pNA was used as a substrate, the reaction mixture (100 µl) was incubated at 20°C, and the amount of p-nitroaniline released from the substrate was determined from the absorption at 410 nm with the absorption coefficient of 8900 M–1 cm–1 by automatic UV spectrophotometer (Beckman model DU640). One unit of enzymatic activity was defined as the amount of enzyme that produced 1 µmol of p-nitroaniline per minute. The specific activity was defined as the enzymatic activity per milligram of protein.

Circular dichroism (CD) spectra

The far-UV CD spectra were obtained using a J-725 spectropolarimeter (Japan Spectroscopic Co.) at 20°C. Pro-Tk-subtilisin and pro-G56S-subtilisin were dissolved in 20 mM Tris–HCl (pH 7.5), while Tk-propeptide, G56S-propeptide and His-tagged G56S-propeptide were dissolved in 20 mM Tris–HCl (pH 7.5) containing 0.5 M NaCl. The protein concentration was 0.1 mg/ml and a cell with an optical path length of 1 mm was used. The mean residue ellipticity, {theta} (in deg cm2 dmol–1), was calculated by using an average amino acid molecular weight of 110.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Screening of low-temperature adapted mutants of pro-Tk-subtilisin

A casein–LB–agar plate was used to examine whether prepro-Tk-subtilisin is matured in the E. coli cells, because the hydrolysis of casein by mat-Tk-subtilisin* results in the formation of a white halo. When the E. coli HB101 transformants with plasmid pBR1560 containing the wild-type prepro-Tk-subtilisin gene were expressed on this plate at 37°C, followed by maturation at elevated temperatures (60–80°C) for 14–18 h, a white halo was detected only at 80°C (Fig. 2A). This result suggests that the maturation rate of pro-Tk-subtilisin is too low to produce mat-Tk-subtilisin* in an amount sufficient for halo formation at 70 and 60°C. Alternatively, the specific activity of mat-Tk-subtilisin* is too low to form a halo at 70 and 60°C. It seems unlikely that the amount of prepro-Tk-subtilisin produced in the cells is altered at these temperatures, because the E. coli enzymes involved in the transcription and the translation would be inactivated at such high temperatures.


Figure 2
View larger version (66K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2.. Halo-forming activities of the wild-type and mutant proteins of pro-Tk-subtilisin. Halo-forming activities of pro-Tk-subtilisin (A), pro-G56S/T135S-subtilisin (B), pro-G56S-subtilisin (C), pro-T135S-subtilisin (D), pro-E61G-subtilisin (E) and pro-E61K-subtilisin (F) were analyzed at 60, 70 and 80°C as described in the Materials and methods section. (G) The E. coli HB101 transformant with pBR322 (negative control).

 
In vitro random mutagenesis was introduced into the prepro-Tk-subtilisin gene by error-prone PCR. The pBR1560 derivatives containing the mutagenized prepro-Tk-subtilisin genes were used to transform E. coli HB101 to generate a mutant library. Of the 965 transformants examined, 8 transformants gave a halo at 60, 70 and 80°C, indicating that these transformants produce low-temperature adapted mutants of prepro-Tk-subtilisin. Determination of the nucleotide sequences revealed that two of them contain only single amino acid substitutions in the propeptide region, and six of them contain single amino acid substitutions in the propeptide region and single or double amino acid substitutions in the mature region (Table II). Of the eight low-temperature adapted mutants, prepro-G56S/T135S-subtilisin with the Gly56 -> Ser mutation in the propeptide region and Thr135 -> Ser mutation in the mature region was further analyzed for adaptation mechanism to low temperatures. This mutant protein was chosen as a representative, because it gave the largest halo at 60°C. The halo sizes are expected to correlate with the ability of the cells to produce active subtilisin molecules.


View this table:
[in this window]
[in a new window]

  		Table II.. Base and amino acid substitutions found from the screening

 
Identification of a single mutation responsible for low-temperature adaptation

To examine whether both mutations are required for low-temperature adaptation of prepro-Tk-subtilisin, the pBR1560 derivatives containing either the prepro-G56S-subtilisin gene or the prepro-T135S-subtilisin gene were constructed. The mutant proteins encoded in these plasmids contain only single amino acid substitutions either in the propeptide (Gly56 -> Ser) or mature (Thr135 -> Ser) region. These plasmids were used to transform E. coli HB101 and analyzed for halo formation on a casein–LB–agar plate at 60, 70 and 80°C. The results are shown in Fig. 2. The E. coli HB101 transformants with the pBR1560 derivative containing the prepro-G56S-subtilisin gene gave a halo at all temperatures examined, whereas those with the pBR1560 derivative containing the prepro-T135S-subtilisin gene gave a halo only at 80°C. None of the transformants gave a halo at 50°C. These results indicate that the single Gly56 -> Ser mutation in the propeptide region is responsible for the low-temperature adaptation of prepro-G56S/T135S-subtilisin. The Thr135 -> Ser mutation in the mature region is probably a silent mutation, which was introduced artificially.

Overproduction and purification of pro-G56S-subtilisin

To analyze a low-temperature adaptation mechanism of prepro-G56S-subtilisin, pro-G56S-subtilisin was overproduced in E. coli and purified, because it is unlikely that a presequence (signal peptide) is involved in the maturation of Tk-subtilisin. Like pro-Tk-subtilisin, pro-G56S-subtilisin was overproduced in inclusion bodies, solubilized in the presence of 8 M urea, refolded and purified to give a single band on Tricine–SDS–PAGE (Fig. 3). The amount of pro-G56S-subtilisin purified from 1 l culture was estimated to be 10 mg. The far-UV CD spectrum of pro-G56S-subtilisin was nearly identical with that of pro-Tk-subtilisin, indicating that the mutation does not seriously affect the structure of pro-Tk-subtilisin (Fig. 4).


Figure 3
View larger version (82K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3.. Autoprocessing of pro-Tk-subtilisin and pro-G56S-subtilisin at various temperatures. Pro-Tk-subtilisin and pro-G56S-subtilisin (0.3 µM) were incubated in 1 ml of 50 mM CAPS–NaOH (pH 9.5) containing 5 mM CaCl2 at the temperatures indicated. With appropriate intervals shown at the top of the gel, the protein was precipitated by adding 112 µl of trichloroacetic acid [100% (w/v)], subjected to Tricine–SDS–PAGE (15%) and stained with CBB. The sample precipitated at 0 min was not exposed to the buffer containing Ca2+. M, low-molecular-weight marker kit (Pharmacia Biotech). Arrows indicate the positions of mat-Tk-subtilisin* (top) and Tk-propeptide or G56S-propeptide (bottom). The molecular mass of each standard protein is indicated along the gel.

 

Figure 4
View larger version (18K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4.. CD spectra. The far-UV CD spectra of pro-Tk-subtilisin (thin broken line), pro-G56S-subtilisin (thin line), Tk-propeptide (gray line), His-tagged G56S-propeptide (thick broken line) and G56S-propeptide (thick line) were measured at 20°C as described in the Materials and methods section.

 
Maturation of pro-G56S-subtilisin

The maturation of pro-G56S-subtilisin was analyzed at 20, 40, 60, and 80°C by 15% Tricine–SDS–PAGE as previously described (Pulido et al., 2006Go). Upon incubation with Ca2+, this protein was rapidly autoprocessed into G56S-propeptide and mature domain within 1 min even at 20°C (Fig. 3). However, the subsequent degradation of G56S-propeptide was temperature dependent. G56S-propeptide was almost completely degraded within 5–10 min at 80°C, 15–30 min at 60°C and 3 h at 40°C, but was not completely degraded upon incubation for 3 h at 20°C. The activity and stability of the resultant mature form of pro-G56S-subtilisin after incubation at 80°C for >1 h in the presence of Ca2+ were analyzed. Its specific activity for azocasein (2800 ± 100 U/mg) and half-life at 90°C (roughly 8 h) were comparable with those of mat-Tk-subtilisin* produced from pro-Tk-subtilisin, which have been reported to be 2600 ± 200 U/mg and roughly 9 h, respectively (Pulido et al., 2006Go). These results indicate that the Gly56 -> Ser mutation in the propeptide region does not seriously affect the folding of the mature domain of pro-G56S-subtilisin. The active mature domain of pro-G56S-subtilisin will be therefore designated as mat-Tk-subtilisin* hereafter.

For comparative purposes, the maturation of pro-Tk-subtilisin was also analyzed at 20, 40, 60 and 80°C. Like pro-G56S-subtilisin, upon incubation with Ca2+, it was rapidly autoprocessed into Tk-propeptide and mat-Tk-subtilisin* within 1 min even at 20°C (Fig. 3). However, the subsequent degradation of Tk-propeptide proceeds much more slowly than that of G56S-propeptide. Tk-propeptide was almost completely degraded only when it was incubated at 80°C for 30 min. It was not completely degraded for up to 3 h at 20, 40 and 60°C. These results indicate that the Gly56 -> Ser mutation considerably increases the maturation rate of pro-Tk-subtilisin.

To confirm the above results, pro-G56S-subtilisin and pro-Tk-subtilisin were incubated at 20, 40, 60 and 80°C in the presence of Ca2+. The activity of the aliquot taken at appropriate intervals was determined at 20°C using Suc-AAPF-pNA as a substrate. This temperature was chosen for assay, because the maturation of this protein is expected to be too slow to proceed during assay. If the maturation of these proteins did not seriously proceed during assay, the activity of pro-G56S-subtilisin or pro-Tk-subtilisin relative to that determined after incubation at 80°C for >1 h, which represents the maturation yield, would increase until the maturation is completed. As shown in Fig. 5, the maturation of pro-Tk-subtilisin was almost fully completed within 90 min at 80°C, but was not completed within 3 h at 60°C. It was very slow at 20 and 40°C, and <10% of pro-Tk-subtilisin was matured upon incubation at these temperatures for 3 h. These maturation rates are lower than, but comparable, those reported previously (Pulido et al., 2006Go).


Figure 5
View larger version (16K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5.. Temperature dependence of maturation. Pro-Tk-subtilisin (A) and pro-G56S-subtilisin (B) were incubated at the concentration of 0.3 µM in 50 mM CAPS–NaOH (pH 9.5) containing 5 mM CaCl2 at 20°C (cross), 40°C (open circle), 60°C (solid circle) and 80°C (open square). With appropriate intervals, an aliquot (10 µl) was withdrawn and enzymatic activity was determined at 20°C using Suc-AAPF-pNA as a substrate as described in the Materials and methods section. The absorbance at 410 nm, which increases as the amount of mat-Tk-subtilisin* increases, was plotted as a function of time.

 
In contrast, the maturation of pro-G56S-subtilisin was almost fully completed within 30 min at 80°C and 1 h at 60°C. It was not completed within 3 h at 20 and 40°C, but the amounts of mat-Tk-subtilisin* produced from pro-G56S-subtilisin upon incubation at 20 and 40°C for 3 h were higher than those produced from pro-Tk-subtilisin by three to four times, respectively. These results are consistent with those obtained by Tricine–SDS–PAGE analysis. It is noted that the maturation rate of pro-G56S-subtilisin analyzed by the activity of mat-Tk-subtilisin* was lower than that analyzed by Tricine–SDS–PAGE. For example, Tricine–SDS–PAGE analysis indicates that pro-G56S-subtilisin is almost fully matured within 5–10 min at 80°C (Fig. 3). However, the resultant mat-Tk-subtilisin* protein is not fully active (Fig. 5). The reason behind this remains to be understood. A trace amount of the undegraded propeptide may inhibit the mat-Tk-subtilisin* activity.

Overproduction and purification of G56S-propeptide

To analyze the mechanism by which G56S-propeptide is degraded by mat-Tk-subtilisin* more rapidly than Tk-propeptide, G56S-propeptide was overproduced in E. coli, using the same overproduction system for Tk-propeptide (Pulido et al., 2006Go). Using this system, Tk-propeptide has been shown to be overproduced in a soluble form in the E. coli cells. However, G56S-propeptide was overproduced in the E. coli cells in an insoluble form. Attempts to overproduce this protein in a soluble form, such as overproduction at low temperatures, induction with low concentrations of IPTG and addition of glucose and ethanol to the culture medium have so far been unsuccessful. These results suggest that G56S-propeptide is structurally less stable than Tk-propeptide. G56S-propeptide accumulated in the cells in an insoluble form was solubilized by 8 M urea, purified in the presence of 8 M urea and refolded by the removal of urea. The amount of G56S-propeptide purified from 1 l culture was roughly 12 mg. The far-UV CD spectrum of G56S-propeptide was considerably different from that of Tk-propeptide (Fig. 4), suggesting that G56S-propeptide is almost fully unstructured.

In contrast to G56S-propeptide, however, His-tagged G56S-propeptide was overproduced in the E. coli cells in a soluble form and purified to give a single band on Tricine–SDS–PAGE (data not shown). The amount of the protein purified from 1-l culture was roughly 14 mg. The far-UV CD spectrum of this protein was nearly identical to that of the refolded G56S-propeptide (Fig. 4), indicating that the structure of G56S-propeptide is not seriously changed by the refolding process. This was confirmed by the urea-induced unfolding and refolding of Tk-propeptide. The far-UV CD spectrum of Tk-propeptide was not seriously changed by this refolding process (data not shown). Because G56S-propeptide is almost fully unstructured, regardless of whether it is overproduced in an insoluble form and refolded, or purified in a soluble form in a His-tagged form, and because a His-tag may affect the susceptibility of the propeptide to proteolytic degradation and interaction between the propeptide and mature domain, we used refolded G56S-propeptide for further biochemical analyses.

Susceptibility to proteolytic degradation

To compare the susceptibility of G56S-propeptide to proteolytic degradation with that of Tk-propeptide, the proteins were incubated with API in 100 mM Tris–HCl (pH 9.0) at 37°C at an enzyme to substrate ratio of 1:20 (w/w) and subjected to 15% Tricine–SDS–PAGE with appropriate intervals. As shown in Fig. 6, G56S-propeptide was rapidly degraded to produce peptide(s) with smaller molecular masses. It was almost fully converted to these peptides within 1 min. In contrast, Tk-propeptide was highly resistant to API digestion and most of the propeptide were kept intact up to 90 min. These results indicate that G56S-propeptide is more susceptible to API digestion than Tk-propeptide. Similar results were obtained when these proteins were digested with chymotrypsin (data not shown). In both cases, however, G56S-propeptide was not cleaved at all possible sites, but was apparently cleaved at a limited site. G56S-propeptide consists of 69 amino acid residues and contains 7 lysine residues at the positions 15, 17, 35, 50, 52, 53 and 59. The cleavage sites of this protein by API remain to be determined.


Figure 6
View larger version (88K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 6.. Susceptibilities to proteolytic degradation. Tk-propeptide (A) and G56S-propeptide (B) were incubated with API for 1 (lane 3), 5 (lane 4), 10 (lane 5), 15 (lane 6), 20 (lane 7), 30 (lane 8), 60 (lane 9), and 90 (lane 10) min in 100 mM Tris–HCl (pH 9.0) at 37°C at an enzyme to substrate ratio of 1:20 (w/w) and subjected to 15% Tricine–SDS–PAGE. The protein was stained with CBB. M, low-molecular-weight marker kit (Pharmacia Biotech). Lane 1, API alone. Lane 2, Tk-propeptide or G56S-propeptide alone. The thin and thick arrows indicate the positions of API and Tk-propeptide or G56S-propeptide, respectively. The molecular mass of each standard protein is indicated along the gel.

 
Inhibition of mat-Tk-subtilisin* activity by G56S-propeptide

Inhibition of the enzymatic activity of mat-Tk-subtilisin* (2 nM) with G56S-propeptide was analyzed at 20°C using Suc-AAPF-pNA as a substrate. At this temperature, the concentration of G56S-propeptide is not significantly changed during the assay, because G56S-propeptide is not effectively cleaved by mat-Tk-subtilisin* (Fig. 3). Inhibition of mat-Tk-subtilisin* activity with Tk-propeptide was also analyzed similarly for comparative purpose. As shown in Fig. 7, the enzymatic activity of mat-Tk-subtilisin* was inhibited by G56S-propeptide and Tk-propeptide in a concentration-dependent manner. Like the interactions between bacterial subtilisins and propeptides (Li et al., 1995Go; Huang et al., 1997Go), these interactions appear to be slow binding. However, ~500 nM of G56S-propeptide is required to obtain an inhibition of mat-Tk-subtilisin* to an extent to that by 20 nM of Tk-propeptide. These results suggest that G56S-propeptide is ~25-fold weaker inhibitor of mat-Tk-subtilisin* than Tk-propeptide. This weaker inhibition of mat-Tk-subtilisin* activity by G56S-propeptide may occur as a consequence of a disordered structure.


Figure 7
View larger version (16K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 7.. Inhibition of the mat-Tk-subtilisin* activity by Tk-propeptide and G56S-propeptide. The indicated concentration of Tk-propeptide (A) or G56S-propeptide (B) was premixed with the substrate (Suc-AAPF-pNA) before mat-Tk-subtilisin* was added. Hydrolysis of Suc-AAPF-pNA (2 mM) by mat-Tk-subtilisin* (2 nM) was analyzed at 20°C by monitoring an increase in absorbance at 410 nm, which results from a release of p-nitroaniline.

 
Folding of mat-Tk-subtilisin using G56S-propeptide

We previously showed that mat-Tk-subtilisin refolded in the absence of Ca2+ is converted to an active form (mat-Tk-subtilisin*) upon Ca2+ binding in the absence of Tk-propeptide, but with a very low yield (Pulido et al., 2006Go). This yield greatly increases when Tk-propeptide is added in trans, suggesting that Tk-propeptide is required to assist an effective folding of the mature domain. Because G56S-propeptide can inhibit mat-Tk-subtilisin* ~25-fold weaker than Tk-propeptide, we examined its ability to assist folding of mat-Tk-subtilisin. When mat-Tk-subtilisin was incubated at 80°C for 30 min in the presence of Ca2+ and in the absence of Tk-propeptide, very small amount of the protein remained intact (Fig. 8, lane 2). The amount of this protein was not seriously changed upon further incubation (data not shown). The specific activity of this protein was comparable with that of the mat-Tk-subtilisin*, indicating that mat-Tk-subtilisin was converted to mat-Tk-subtilisin* with a very low yield upon incubation with Ca2+ at 80°C, and pre-activated mat-Tk-subtilisin was completely degraded by mat-Tk-subtilisin* within 30 min. This yield greatly increased when Tk-propeptide was added in trans (Fig. 8, lane 3), whereas it only slightly increased when G56S-propeptide was added in trans (Fig. 8, lane 4). These results suggest that G56S-propeptide assists folding of mat-Tk-subtilisin with a lower efficiency than Tk-propeptide. Thus, the efficiency of the propeptide to assist protein folding correlates with its ability to bind to the folded mature domain.


Figure 8
View larger version (98K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 8.. Comparison of the yields of mat-Tk-subtilisin* in the presence and absence of G56S-propeptide. Refolded mat-Tk-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 of 0.6 µM of Tk-propeptide (lane 3) or G56S-propeptide (lane 4), and subjected to 15% Tricine–SDS–PAGE. The protein was stained with CBB. Lane 1, refolded mat-Tk-subtilisin without exposure to the buffer containing Ca2+. M, low-molecular-weight marker kit (Pharmacia Biotech). The arrow indicates the position of mat-Tk-subtilisin or mat-Tk-subtilisin*. The molecular mass of each standard protein is indicated along the gel.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
Localization of the mutation sites

By the directed evolution method, eight pro-Tk-subtilisin variants that adapt to low temperatures were isolated. Two of them contain only single mutations at Glu61 within the propeptide region, while six of them contain single mutations at Lys16, Gly56, Val60 or His64 within the propeptide region and single or double mutations at Ser107, Trp131, Thr135, Ile357 or Asp379 within the mature region (Table II). If the positive numbering of the amino acid residue in the pro-subtilisin E sequence started from the initial alanine residue of the propeptide region, instead of that of the mature region, the amino acid residues corresponding to those which mutated in the low-temperature adapted variants of pro-Tk-subtilisin are Thr17 for Lys16, Ser64 for Gly56, Val68 for Val60, Glu69 for Glu61, His72 for His64, Asn102 for Ser107, Ala125 for Trp131, Pro129 for Thr135, Trp318 for Ile357 and Ser337 for Asp379. According to the crystal structure of the complex between the propeptide and mature domain of subtilisin E (Jain et al., 1998Go), either one of these residues in the propeptide region is not located too close to directly interact with either one of these residues in the mature region (Fig. 9). These results suggest that the mutations at the propeptide and mature regions do not cooperatively contribute to low-temperature adaptation of the pro-Tk-subtilisin variants with multiple mutations both at propeptide and mature regions.


Figure 9
View larger version (80K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 9.. Three-dimensional structure of an autoprocessed Ser221Cys–subtilisin E-propeptide complex. The backbone structure (Jain et al., 1998Go) was drawn with the program RasMol. N and C represent the N- and C-termini of this structure. The side chains of Thr17 (Lys16), Ser64 (Glu56), Val68 (Val60), Glu69 (Glu61), His72 (His64), Asn102 (Ser107), Ala125 (Trp131), Pro129 (Thr135), Trp318 (Ile357) and Ser337 (Asp379), as well as the position of the active site are indicated (the amino acid residues in the parentheses represent the corresponding residues in the pro-Tk-subtilisin sequence). The dark and light gray regions correspond to the propeptide and mature domain, respectively. This structure is stored at the Protein Data Bank under the accession number 1SCJ.

 
Possible low-temperature adaptation mechanism

In this study, the single Gly56 -> Ser mutation in the propeptide region was shown to be responsible for low-temperature adaptation of pro-G56S/T135S-subtilisin. The Thr135 -> Ser mutation in the mature region alone did not seriously affect either the maturation temperature of pro-Tk-subtilisin or the enzymatic properties of mat-Tk-subtilisin*. In contrast to Tk-propeptide, G56S-propeptide is mostly unstructured in an isolated form even at 20°C. This unstructurality of G56S-propeptide can account for its increased susceptibility to proteolytic degradation and decreased binding affinity to mat-Tk-subtilisin*. Pro-G56S-subtilisin is effectively matured even at 60°C, probably because G56S-propeptide autoprocessed from mat-Tk-subtilisin* is unstructured when it is dissociated from mat-Tk-subtilisin* and therefore it is rapidly degraded by mat-Tk-subtilisin* at this temperature. However, the reason why the mutation of Gly56 -> Ser is detrimental to propeptide folding remains to be understood. The corresponding residue of subtilisin E (Ser64) is located far from the interface between the propeptide and mature domain (Fig. 9), and the amino acid residues located around this residue are not well conserved in the pro-Tk-subtilisin sequence. Pro-E61G-subtilisin and pro-E61K-subtilisin also exhibited a halo-forming activity at 80, 70 and 60°C, indicating that the single Glu61 -> Gly or Glu61 -> Lys mutation in the propeptide region is responsible for low-temperature adaptation of these mutant proteins (Fig. 2E and F). Glu61 is conserved in the subtilisin E (as Glu69) and subtilisin BPN' sequences and is located at the interface between the propeptide and mature domain (Fig. 9). The mutation of this residue probably reduces the binding affinity of the propeptide to mat-Tk-subtilisin* and thereby shifts the temperature for maturation to lower ones. It remains to be analyzed for other protein variants whether the single mutations are responsible for their low-temperature adaptations.

Structurality of propeptide

It has been reported that the 77-mer propeptide of subtilisin BPN' is mostly unstructured (Shinde et al., 1993Go; Wang et al., 1995Go; Huang et al., 1997Go) and is 97% unfolded even under optimal folding conditions (Wang et al., 1998Go). Similarly, the propeptides of subtilisin E (Hu et al., 1996Go) and subtilisin Carlsberg (Huang et al., 1997Go) are almost fully disordered in the aqueous solution. Subbian et al. (2005)Go have reported that the propeptide of subtilisin E exhibits properties similar to an emerging family of intrinsically unstructured proteins (IUPs). Tompa (2002)Go describes IUPs as those proteins that lack a folded structure but display a highly flexible, random, coil conformation and/or little secondary structure under physiological conditions. Dyson and Wright (2005)Go further described these IUPs as disordered segments that fold upon binding to their biological targets (coupled folding and binding). Nevertheless, these propeptides act as a potent inhibitor of subtilisins, suggesting that they are structured when they bind to subtilisins. In fact, the crystal structures of propeptide–subtilisin BPN' complex (Gallagher et al., 1995Go) and autoprocessed Ser221Cys–subtilisin E-propeptide complex (Jain et al., 1998Go) have shown that the propeptides form a compact structure, which binds to subtilisins through an interface mediated by the two parallel {alpha}-helices of subtilisins (Fig. 9).

G56S-propeptide is mostly unstructured in an isolated form. In contrast, Tk-propeptide retains a folded structure even in an isolated form at 20°C. Pro-Tk-subtilisin is not effectively matured at lower temperatures, <80°C, probably because Tk-propeptide is not unstructured upon dissociation from mat-Tk-subtilisin* and is therefore highly resistant to degradation by mat-Tk-subtilisin* and retains high binding affinity to mat-Tk-subtilisin*. It has been reported that the folding of subtilisin BPN' is accelerated by the propeptide mutant that retains a folded structure in an isolated form (Kojima et al., 2001Go). It has also been reported that the propeptide of aqualysin I, which is a subtilisin homolog from Thermus aquaticus, is structured in an isolated form and more strongly inhibits subtilisin BPN' than the propeptide of subtilisin BPN' (Marie-Claire et al., 2001Go). These results may suggest that stabilization of the propeptide structure is one of the strategies employed by hyperthermophilic proteases for their adaptation to high temperatures. It remains to be determined whether Tk-propeptide is folded at 80°C upon dissociation from the mature domain. However, analyses for the thermal denaturation using CD suggests that Tk-propeptide in an isolated form, which is not unfolded up to 90°C (M. Pulido, unpublished results).

Role of propeptide

The role of Tk-propeptide is not fully understood yet. Unlike bacterial subtilisins, the mature domain of Tk-subtilisin alone (mat-Tk-subtilisin) is refolded and converted to an active Ca2+-bound form (mat-Tk-subtilisin*) even in the absence of Tk-propeptide (Pulido et al., 2006Go). This result indicates that Tk-propeptide is not required for folding of the mature domain of Tk-subtilisin. However, the yield of mat-Tk-subtilisin* greatly increases when Tk-propeptide is added in trans, or pro-Tk-subtilisin is matured, suggesting that Tk-propeptide exhibits a chaperon function and assists the effective folding of the mature domain. Tk-propeptide is probably also required to prevent degradation of pre-activated mat-Tk-subtilisin by mat-Tk-subtilisin*, because Tk-propeptide is a potent inhibitor of mat-Tk-subtilisin* and Ca2+-free mat-Tk-subtilisin is rapidly degraded by mat-Tk-subtilisin* (S. Tanaka, unpublished data). Two conflicting results have been reported for the relationships between the inhibitory and chaperon functions of the propeptides. One indicates that they are correlated (Li et al., 1995Go; Wang et al., 1995Go), whereas the other indicates that they are not necessarily correlated (Marie-Claire et al., 2001Go; Yabuta et al., 2003Go). Our results that G56S-propeptide exhibits lower inhibitory and chaperon activities than Tk-propeptide suggests that they are correlated with each other.

Both the inhibitory and chaperon functions of G56S-propeptide were greatly decreased compared with those of Tk-propeptide. Nevertheless, no significant difference was detected in the yield of mat-Tk-subtilisin* between pro-Tk-subtilisin and pro-G56S-subtilisin at 80°C (Figs. 3 and 5). The propeptide region of pro-G56S-subtilisin probably assumes a folded structure, which is similar to that of the propeptide region of pro-Tk-subtilisin, because the far-UV CD spectrum of pro-G56S-subtilisin is nearly identical to that of pro-Tk-subtilisin (Fig. 4). In the absence of Ca2+, the mature domain of pro-G56S-subtilisin is folded into an inactive form. However, this structure may be sufficient to stabilize a folded structure of the propeptide region of pro-G56S-subtilisin. If the propeptide region of pro-G56S-propeptide was folded into a functional structure regardless of whether its mature domain assumes an active or inactive conformation, this mutant propeptide region would interact with the mature domain equally as with the wild-type propeptide region. This may be the reason why the maturation yield of pro-G56S-subtilisin is similar to that of pro-Tk-subtilisin at 80°C.

It is noted that the inhibition mode of Tk-propeptide is revised in this study. We previously showed that Tk-propeptide does not exhibit slow binding inhibition, but exhibits non-competitive inhibition (Pulido et al., 2006Go). However, the progress curves for the inhibition of mat-Tk-subtilisin* by Tk-propeptide and G56S-propeptide reveal a hyperbolic pattern as shown in Fig. 7. This discrepancy is probably caused by the difference in the concentration of mat-Tk-subtilisin* used for inhibition assays, which is previously 0.1 nM and currently 2 nM. The mat-Tk-subtilisin* concentration of 0.1 nM is probably too low to clearly detect the slow binding inhibition. However, the amount of Tk-propeptide required to inhibit mat-Tk-subtilisin* was not seriously changed when the mat-Tk-subtilisin* concentration increased to 2 nM.

It has been reported for subtilisin E that the mutations within the propeptide region seriously affect the folding of the mature domain due to a protein memory function of propeptide and an inactive enzyme production (Lerner et al., 1990Go; Kobayashi and Inouye, 1992Go) or an active one with an altered conformation which differs from that of the wild-type enzyme (Shinde et al., 1997Go). The effect of this mutation can be suppressed by the second-site mutation within the mature domain (Li et al., 1995Go). This protein memory function was not observed in this study, because the activity and stability of mat-Tk-subtilisin* produced from pro-G56S-subtilisin were identical with those produced from pro-Tk-subtilisin. Further mutational and structural studies will be required to analyze the role of Tk-propeptide for folding of the mature domain.


   Footnotes
 
Abbreviations: Tk-subtilisin, subtilisin from Thermococcus kodakaraensis; prepro-Tk-subtilisin, Tk-subtilisin in a prepro-form [Met(-24)-Gly398]; pro-Tk-subtilisin, Tk-subtilisin in a pro-form (Gly1-Gly398); pro-G56S/T135S-subtilisin, pro-Tk-subtilisin with the Gly56 -> Ser and Thr135 -> Ser mutations; pro-G56S-subtilisin, pro-Tk-subtilisin with the Gly56 -> Ser mutation; pro-T135S-subtilisin, pro-Tk-subtilisin with the Thr135 -> Ser mutation; mat-Tk-subtilisin, mature domain of Tk-subtilisin (Gly70-Gly398) in a Ca2+-free inactive form; mat-Tk-subtilisin*, a Ca2+-bound active form of mat-Tk-subtilisin; Tk-propeptide, propeptide of Tk-subtilisin (Gly1-Leu69); G56S-propeptide, Tk-propeptide with the Gly56 -> Ser mutation; Suc-AAPF-pNA, N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide; bp, base pair(s); API, Achromobacter protease I.

Edited by Taiji Imoto


   Acknowledgments
 
This work was supported in part by a Grant-in-Aid for National Project on Protein Structural and Functional Analyses and by a Grant-in-Aid for Scientific Research on Priority Areas "Systems Genomics" from the Ministry of Education, Culture, Sports, Science and Technology of Japan.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Arnold F.H. (1996) Chem. Eng. Sci. 51:5091–5102.

Arnold F.H., Wintrode P.L., Miyazaki K., Gershenson A. (2001) Trends Biochem. Sci. 26:100–106.[CrossRef][Web of Science][Medline]

Bloom J.D., Meyer M.M., Meinhold P., Otey C.R., MacMillan D., Arnold F.H. (2005) Curr. Opin. Struct. Biol. 15:447–452.[CrossRef][Web of Science][Medline]

Bryan P.N. (2000) Biochim. Biophys. Acta 1543:203–222.[CrossRef][Medline]

Bryan P.N., Rollence M., Pantoliano M., Wood J., Finzel B., Gilliland G.L., Howard A., Poulos T. (1986) Proteins 1:326–334.[CrossRef][Medline]

Chen K. and Arnold F.H. (1993) Proc. Natl Acad. Sci. USA 90:5618–5622.[Abstract/Free Full Text]

Cherry J.R. and Fidantsef A.L. (2003) Curr. Opin. Biotechnol. 14:438–443.[CrossRef][Web of Science][Medline]

Dyson H.J. and Wright P.E. (2005) Nat. Rev. Mol. Cell. Biol. 6:197–208.[CrossRef][Web of Science][Medline]

Eder J., Rheinnecker M., Fersht A.R. (1993a) Biochemistry 32:18–26.[CrossRef][Medline]

Eder J., Rheinnecker M., Fersht A.R. (1993b) J. Mol. Biol. 233:293–304.[CrossRef][Web of Science][Medline]

Eijsink V.G., Gaseidnes S., Borchert T.V., van den Burg B. (2005) Biomol. Eng. 22:21–30.[CrossRef][Web of Science][Medline]

Farinas E., Butler T., Arnold F.H. (2001) Curr. Opin. Biotechnol. 12:545–551.[CrossRef][Web of Science][Medline]

Fu X., Inouye M., Shinde U. (2000) J. Biol. Chem. 275:16871–16878.[Abstract/Free Full Text]

Gallagher T., Bryan P.N., Gilliland G.L. (1993) Proteins 16:205–213.[CrossRef][Web of Science][Medline]

Gallagher T., Gilliland G.L., Wang L., Bryan P.N. (1995) Structure 3:907–914.[Medline]

Goodwin T.W. and Morton R.A. (1946) Biochem. J. 40:628–632.[Medline]

Huang H.W., Chen W.C., Wu C.Y., Yu H.C., Lin W.Y., Chen S.T., Wang K.T. (1997) Protein Eng. 10:1227–1233.[Abstract/Free Full Text]

Hu Z., Haghjoo K., Jordan F. (1996) J. Biol. Chem. 271:3375–3384.[Abstract/Free Full Text]

Jain S.C., Shinde U., Li Y., Inouye M., Berman H.M. (1998) J. Mol. Biol. 284:137–144.[CrossRef][Web of Science][Medline]

Kannan Y., Koga Y., Inoue Y., Haruki M., Takagi M., Imanaka T., Morikawa M., Kanaya S. (2001) Appl. Environ. Microbiol. 67:2445–2452.[Abstract/Free Full Text]

Kano H., Taguchi S., Momose H. (1997) Appl. Microbiol. Biotechnol. 47:46–51.[CrossRef][Web of Science][Medline]

Kobayashi T. and Inouye M. (1992) J. Mol. Biol. 226:931–933.[CrossRef][Web of Science][Medline]

Kojima S., Yanai H., Miura K. (2001) J. Biochem. (Tokyo) 130:471–474.[Abstract/Free Full Text]

Lerner C., Kobayashi T., Inouye M. (1990) J. Biol. Chem. 265:20085–20086.[Abstract/Free Full Text]

Li Y., Hu Z., Jordan F., Inouye M. (1995) J. Biol. Chem. 270:25127–25132.[Abstract/Free Full Text]

Marie-Claire Y., Yabuta Y., Suefuji K., Matsuzawa H., Shinde U. (2001) J. Mol. Biol. 305:151–165.[CrossRef][Web of Science][Medline]

Pulido M., Saito K., Tanaka S., Koga Y., Takano K., Kanaya S. (2006) Appl. Environ. Microbiol. 72:4154–4162.[Abstract/Free Full Text]

Shinde U. and Inouye M. (2000) Semin. Cell Dev. Biol. 11:35–44.[CrossRef][Web of Science][Medline]

Shinde U., Liu J., Inouye M. (1997) Nature 389:520–522.[CrossRef][Medline]

Shinde U., Li Y., Chatterjee S., Inouye M. (1993) Proc. Natl Acad. Sci. USA 90:6924–6928.[Abstract/Free Full Text]

Song J.K. and Rhee J.S. (2000) Appl. Environ. Microbiol. 66:890–894.[Abstract/Free Full Text]

Srimathi S., Jayaraman G., Narayanan P.R. (2006) Enzyme Microb. Tech. 39:301–307.

Strausberg S.L., Alexander P.A., Gallagher D.T., Gilliland G.L., Barnett B.L., Bryan P.N. (1995) Biotechnology 13:669–673.

Subbian E., Yabuta Y., Shinde U. (2005) J. Mol. Biol. 347:367–383.[CrossRef][Web of Science][Medline]

Taguchi S., Komada S., Momose H. (2000) Appl. Environ. Microbiol. 66:1410–1415.[Abstract/Free Full Text]

Taguchi S., Ozaki A., Momose H. (1998) Appl. Environ. Microbiol. 64:492–495.[Abstract/Free Full Text]

Takagi H. and Takahashi M. (2003) Appl. Microbiol. Biotechnol. 63:1–9.[CrossRef][Web of Science][Medline]

Tao H. and Cornish V. (2002) Curr. Opin. Chem. Biol. 6:858–864.[CrossRef][Web of Science][Medline]

Tompa P. (2002) Trends Biochem. Sci. 27:527–533.[CrossRef][Web of Science][Medline]

Wang L., Ruan B., Ruvinov S., Bryan P.N. (1998) Biochemistry 37:3165–3171.[CrossRef][Medline]

Wang L., Ruvinov S., Strausberg S., Gallagher D.T., Gilliland G.L., Bryan P.N. (1995) Biochemistry 34:15415–15420.[CrossRef][Medline]

Wintrode P., Miyazaki K., Arnold F.H. (2001) Biochim. Biophys. Acta 1549:1–8.[CrossRef][Medline]

Yabuta Y., Subbian E., Oiry C., Shinde U. (2003) J. Biol. Chem. 278:15246–15251.[Abstract/Free Full Text]

Yabuta Y., Subbian E., Takagi H., Shinde U., Inouye M. (2002) J. Biochem. (Tokyo) 131:31–37.[Abstract/Free Full Text]

Yabuta Y., Takagi H., Inouye M., Shinde U. (2001) J. Biol. Chem. 276:44427–44434.[Abstract/Free Full Text]

Yang Y., Jiang L., Zhu L., Wu Y., Yang S. (2000) J. Biotechnol. 81:113–118.[CrossRef][Web of Science][Medline]

You L. and Arnold F.H. (1994) Protein Eng. 9:77–83.

Zhao H. and Arnold F.H. (1999) Protein Eng. 12:47–53.[Abstract/Free Full Text]

Received November 9, 2006; revised December 27, 2006; accepted January 10, 2007.


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?



This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
20/3/143    most recent
gzm006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Pulido, M.A.
Right arrow Articles by Kanaya, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Pulido, M.A.
Right arrow Articles by Kanaya, S.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?