Thermal stabilization of penicillolysin, a thermolabile 19 kDa Zn2+-protease, obtained by site-directed mutagenesis

Yuko Doi1, Hidetoshi Akiyama1, Yoshiteru Yamada1, Ch’ng Ewe Ee2, Byung Rho Lee1, Masamichi Ikeguchi1 and Eiji Ichishima1,2,3

1Laboratory of Molecular Enzymology, Graduate School of Bioengineering, Soka University, Hachioji, Tokyo 192-8577 and 2Laboratory of Molecular Enzymology, Graduate School of Agriculture Science, Tohoku University, 1–1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai 981-8555, Japan

3 To whom correspondence should be addressed. e-mail: ichisima{at}t.soka.ac.jp


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Penicillolysin is a member of the clan MX and the family of M35 proteases. The enzyme is a thermolabile Zn2+- protease from Penicillium citrinum with a unique substrate profile. We expressed recombinant penicillolysin in Aspergillus oryzae and generated several site-directed mutants, R33E/E60R, A167E and T81P, with the intention of exploring thermal stabilization of this protein. We based our choice of mutations on the structures of homologous thermally stable enzymes, deuterolysin (EC 3.4.24.39) from A.oryzae and a peptidyl-Lys metallopeptidase (GfMEP) from the edible mushroom Grifora frondsa. The resulting mutant proteins exhibited comparable catalytic efficiency to the wild-type enzyme and some showed a higher tolerance to temperature.

Keywords: aspzincin/M35 protease/mutagenesis/penicillolysin/thermal stabilization/Zn2+-protease


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Penicillolysin from Penicillium citrinum is a single-chain protein of 177 amino acid residues with a calculated molecular mass of 18 525 Da and pI of 9.6, which contains three disulfide bonds (Ichishima et al., 1991Go; Yamaguchi et al., 1993Go; Matsumoto et al., 1994Go; Ichishima, 1998Go). The specificity of penicillolysin differs from that of other metalloproteases characterized to date (Ichishima et al., 1991Go; Yamaguchi et al., 1993Go). Penicillolysin strongly hydrolyzes the nuclear proteins clupeine, salmine and histone but does not hydrolyze casein, albumin or hemoglobin. It contains 1 mol of zinc per mole of enzyme (Ichishima et al., 1991Go; Yamaguchi et al., 1993Go). Penicillolysin has the consensus sequence HExxH of deuterolysin from Aspergillus oryzae (Matsumoto et al., 1994Go). Deuterolysin is a member of a family of Zn2+-metalloendopeptidases with a new zinc-binding motif, aspzincin, defined by the HExxH + D motif and an aspartic acid as the third zinc ligand (Fushimi et al., 1999Go). Recently, X-ray crystallographic analysis of deuterolysin indicated the Zn2+ atom is liganded by two histidine residues, His128 and His132, and an aspartic acid residue, Asp143 (McAuley et al., 2001Go). Furthermore, the active site of a new aspzincin metalloendopeptidase, a peptidyl-Lys metalloendopeptidase (GfMEP), from the fungus Grifola frondosa is composed of two helices and a loop region, which indicates the HExxH and GTxxDxxTG motifs are conserved among aspzincins (Hori et al., 2001Go). His117, His121 and Asp130 coordinate with the catalytic zinc ligands of GfMEP. Thermolysin (EC 3.4.24.27), from a family of M4 enzymes, was originally identified as a thermostable enzyme (Beynon and Beaumont, 1998Go). In large part, this thermal stability is attributable to bound calcium ions. Penicillolysin (Matsumoto et al., 1994Go) has only a low degree of sequence identity (17%) with thermolysin (Beynon and Beaumont, 1998Go) and has no calcium ion, whereas it has 68% identity with deuterolysin (Tatsumi et al., 1991Go) from A.oryzae. Penicillolysin is thermolabile and its activity is almost completely lost above 66°C. In contrast, deuterolysins from A.oryzae (Tatsumi et al., 1991Go, 1994; Doi et al., 2003Go) and A.sojae (Sekine, 1972Go) are heat resistant at 100°C for 10 min. It is interesting that, despite having a highly similar identity to and the same number, three, of disulfide bonds as deuterolysin, penicillolysin is thermolabile. Of the substitutions involving residues with largely identical structural environments in penicillolysin and deuterolysin, those for which the deuterolysin situation could be favorable for thermal stability were selected. The mutations were expected to be especially important because of their rigidifying effects on partially unfolded protein (Imanaka et al., 1986Go). The M35 proteases, deuterolysin and metalloendopeptidase from G.frondosa, are {alpha}ß proteins with a zincin-like fold (Hori et al., 2001Go; McAuley et al., 2001Go). Deuterolysin comprises eight {alpha}-helices and five strands of ß-sheet. In this study, we designed and constructed two different mutants (R33E/E60R and A167E) for the connection between the two {alpha}-helices (A and C helices or E and H helices) in the folded penicillolysin. The results illustrate the principles by which the mutants stabilize penicillolysin and suggest that strategies for the selection of potential chemical bonds, probably electrostatic interaction or hydrogen bonding, are most likely to increase the thermal stability of the enzyme. Furthermore, a general type of mutation was thought to be especially important as an example because of the proline rule for increasing protein thermal stability (Suzuki et al., 1987Go; Suzuki, 1989Go; Watanabe et al., 1997Go). The chosen mutations in penicillolysin were T81P, T87P and A112P from the structure of deuterolysin. The present results show that the region around residue 81 is involved in the step of thermal stabilization according to the proline rule. Remarkably, the stabilization obtained in the T81P mutant is considerably greater than the difference in stability between the R33E/E60R mutant and the A167E mutant.

In a previous paper, a new enzymatic method for production of seasoning including 5'-deoxyribonucleotides and amino acids from salmon milt with microbial enzymes was studied (Hayashi et al., 1996Go). For efficient hydrolysis of salmon milt, we found that a two-step hydrolysis procedure was necessary. Streptomyces griseus protease, actinase AS and nuclease ‘Amano’ from Penicillum ctrinum were used to obtain the product. The study dealt with the efficient production of seasoning from salmon milt DNA through the combined addition of a microbial protease and a nuclease. The thermal stability of penicillolysin is important because the conversion of milt to peptides and amino acids during the industrial production of seasoning typically takes place at temperatures of 65–68°C. The thermal stability of mutant penicillolysin results in a more favorable temperature for enzymatic hydrolysis after the milt protein is decomposed.


    Materials and methods
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 Introduction
 Materials and methods
 Results and discussion
 References
 
Bacterial strains, plasmids, media and materials

Escherichia coli DH5{alpha} was used for DNA manipulation. Aspergillus oryzae niaD 300 (niaD), which is a nitrate reductase gene (niaD)-deficient strain (Minetoki et al., 1996Go), and a high-level expression vector plasmid pNAN8142 (9.5 kb) (Minetoki et al., 1995Go; Ozeki et al., 1996Go), were used for transformation experiments. Czapek–Dox medium, consisting of 0.3% NaNO3, 0.2% KCl, 0.1% KH2PO4, 0.05% MgSO4·7H2O, 0.002% FeSO4·H2O and 2% dextrin, adjusted to pH 5.5, was used for selection of transformants. DPY medium (2% dextrin, 1% polypeptone, 0.5% yeast extract, 0.5% KH2PO4 and 0.05% MgSO4·7H2O, pH 5.5) was used for recombinant penicillolysin production. Lysing enzyme was purchased from Sigma (St Louis, MO). N-Butoxycarbonyl- L-valyl-L-arginyl-L-arginyl-4-methylcoumaryl-7-amide (Boc-Arg-Val-Arg-Arg-MCA) and other fluorogenic peptide-MCAs were purchased from the Peptide Institute (Mino-shi, Osaka, Japan).

Construction of the expression plasmid

A 1.0 kb SalI–SphI fragment of the plnC gene was inserted into the SphI–SalI site of pNAN8142 (9.5 kb) (Minetoki et al., 1995Go; Ozeki et al., 1996Go) and in this way the penicillolysin-producing plasmid pNANplnC (10.5 kb) was constructed.

Fungal transformation

The transformation of A.oryzae was performed basically according to the method of Gomi et al. (1987Go). In our experiment, the nitrate reductase gene (niaD) and the A.oryzae niaD 300 strain (niaD) were employed as a selectable marker and host, respectively.

Production and purification of penicillolysin and mutant enzymes expressed in A.oryzae

The conidiospores from a selected transformant were inoculated into 50 ml of DPY medium in a 500 ml Sakaguchi flask and cultured at 30°C for 3 days. The filtrate was salted out by adding ammonium sulfate up to a 70%-saturated concentration and centrifuged for 15 min at 10 000 g. The following operations were carried out at below 4°C unless noted otherwise. The precipitate was dissolved in 20 ml of 10 mM sodium acetate buffer, pH 5.0. The solution was dialyzed against 10 mM sodium acetate buffer, pH 5.0 and then applied to a cation-exchange CM Sepharose Fast Flow column (1x 30 cm) (Amersham Pharmacia Biotech, Uppsala, Sweden) equilibrated in 10 mM sodium acetate buffer, pH 5.0. Elution was effected with a linear gradient of 30–75 mM NaCl in 10 mM sodium acetate buffer, pH 5.0. The active enzyme fraction eluted from the CM Sepharose Fast Flow column was dialyzed against Milli-Q water at 4°C. The purified penicillolysin migrated with homogeneity on SDS–PAGE, which was performed by the method of Laemmli (1970Go). Purification procedures of the mutant enzymes were performed using the same method as for wild-type penicillolysin.

Site-directed mutagenesis

Site-directed mutagenesis of the plnC gene was performed by the Kunkel (1985Go) method. Escherichia coli strain CJ236 was used to generate dU-substituted DNA and single-stranded DNA was isolated using the helper phage M13K07 (Bio-Rad, Hercules, CA). The oligonucleotides used for generation of the mutants were as follows: 5'-TACGGCAGCCgaGTCTGGATCcGCCTCCAAGT-3' for R33E; 5'-AGCACGTCTAcgaGCTGTGGCT-3' for E60R; 5'-GCAGTGACcCCcTaGGATA CTGCGA-3' for T81P; 5'-GATACTGCGAGcCtAACGTGCTaGCTTATACCCT-3' for T87P; 5'-TCTTATCTGCCAcCcTTGGCcGGcACCTGCCATCA-3' for A112P; 5'-CAC GACAACTCTtCgcGAGTTCACCC-3' for H128R; 5'-CAACTCTCCATcAGTTCACCCAT-3' for E129Q; 5'-CCATGAGTTCACtCgaGCACCTGGTG-3' for H132R; 5'-GTGTAT AGCCCcGGgACCGATaACCTGGGATA-3' for D143N; 5'-GTATTGAATGCgaAtTCGTATGCCC-3' for D164N; and 5'-CTGACTCGTATGagCTCTACGCCAACG-3' for A167E. Substituted nucleotides are indicated by lower-case letters. Italic letters show the sites recognized by the restriction endonucleases used. Mutations were verified based on restrictive differences or DNA sequencing before construction of the expression plasmid pNANplnC.

Enzyme assay

The enzyme activity of penicillolysin was routinely assayed with Boc-Arg-Val-Arg-Arg-MCA as a substrate at 30°C in 50 mM Tris–HCl buffer, pH 8.0. One katal (kat) of enzyme activity in SI units (Price and Stevens, 1999Go) is defined as the amount of enzyme that liberates 1 mol of 4-methylcoumaryl-7-amide (MCA) from the fluorogenic peptide substrate per second at 30°C and pH 8.0 (Doi et al., 2003Go). The specific activity of the enzyme was expressed as katal/kg protein. The purified enzyme was pre-incubated in 895 µl of 50 mM Tris–HCl buffer, pH 8.0, at 30°C for 10 min, to which 5 µl of 10 mM peptide-MCA substrate had been added. The initial rate of increase in the 7-amino-4-methylcoumarin of enzymatic hydrolysates was monitored fluorometrically using a Shimadzu RF-5000 spectrophotometer with excitation at {lambda}ex = 360 nm and emission at {lambda}em = 440 nm. The kinetic parameters Km and Vmax were determined directly from Hanes–Woolf plots. Values for kcat were derived from Vmax = kcat[E]0, where [E]0 is the enzyme concentration.

Effect of temperature

The measurements were performed at a low concentrations in 10 mM sodium acetate buffer at pH 5.0. Heating took place in a thermostatically regulated PCR Thermal Cycler 480 thermocycler (Takara, Kyoto, Japan). As the control, unheated samples were kept at 20°C until assayed. The enzyme was incubated at a given temperature for 10 min before being cooled on ice for 10 min and was then assayed for remaining enzymatic activity at pH 8.0 to determine the degree of irreversible thermal denaturation. Thermal stability was quantified as the half-life of activity, T50, being the temperature (°C) of incubation at which 50% of the initial activity of a solution of enzyme is retained in 10 min. The stability of the mutant enzyme is expressed as {delta}T50, being the difference in T50 between the wild-type (T50 = 62°C) and mutant enzymes. The error margins in the {delta}T50 values are estimated to be 0.2–0.4°C.

Protein determination

Protein concentrations were usually measured by the method of Bradford (1976Go) with bovine serum albumin (BSA) as a standard protein.

CD measurements

Penicillolysin was dialyzed against 10 mM sodium acetate buffer, pH 5.0, then diluted in the same buffer to 0.2 mg protein/ml. Far-UV CD spectra were measured with a Jasco J-720 spectrophotometer at 25°C in a cuvette with a 1 mm pathlength (Yang et al., 1986Go). Unfolding curves were measured at 222 nm in the temperature scan mode with a gradient of 1°C/min until a temperature of 80°C was reached.

Atomic absorption spectrometric (AAS) analysis

Zinc analysis of the recombinant penicillolysin was carried out byAAS using a Shimadzu Model AA-660 (P/N206-1000-02) instrument.


    Results and discussion
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 Abstract
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 Materials and methods
 Results and discussion
 References
 
Recombinant penicillolysin expressed in A.oryzae

A recombinant penicillolysin was expressed in A.oryzae. The system is capable of making as much as 15 mg of enzyme per liter of culture. The recombinant penicillolysin was secreted as an active form in culture broth and purified to a homogeneous state by one-step column chromatography. The molecular mass was determined as 23.7 kDa by SDS–PAGE. The N-terminal amino acid sequence of the enzyme was found to be TKET. These results suggest that the recombinant enzyme was accurately processed and the number of amino acid residues was assumed to be 177 from the predicted amino acid sequence described previously (Matsumoto et al., 1994Go). The recombinant penicillolysin contained 1 mol of zinc per mol of enzyme with a molecular mass of 18 525 Da.

Penicillolysin does not hydrolyze small synthetic peptides (Yamaguchi et al., 1993Go). To obtain a sensitive small substrate of penicillolysin, 45 kinds of fluorogenic peptide-MCAs were tested. The recombinant penicillolysin displayed hydrolytic activity towards Boc-Arg-Val-Arg-Arg-MCA (Boc-RVRR-MCA), with optimal activity at pH 8.0. These findings indicate that penicillolysin exhibits a similar specificity to that of deuterolysin (Doi et al., 2003Go). The properties of the recombinant penicillolysin expressed in A.oryzae are summarized in Table I.


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Table I. Summarized molecular and enzymatic properties of the recombinant penicillolysin from P.citrinum overexpressed in A.oryzae
 
Metalloproteases are a well-known class of proteolytic enzymes and have been classified into more than 30 different families, many of which contain an HExxH motif that forms part of the metal-binding site (Hooper, 1994Go; Rawlings and Barrett, 1995Go; Turner, 1998Go; Beynon and Bond, 2001Go). Several candidates for zinc ligands of penicillolysin were mutated. Substitutions of His128 and His132 with Arg, of Glu129 with Gln, of Asp143 with Asn and of Asp164 with Asn resulted in a complete loss of both the enzyme activity and zinc binding ability of all mutant enzymes except D164N (Table II). As for the mutant D164N, the zinc content was determined as 1.0 mol/mol of the enzyme and the specific activity was found to be 53%. This finding indicates that the Zn2+ atom is bound by two histidine residues (His128 and His132) and an aspartic acid residue (Asp143) as shown in Table II. Substitution of Glu129, a catalytically crucial residue in penicillolysin, with glutamine resulted in a mutant enzyme (E129Q) which exhibited no activity. In a previous study in our laboratory, based on site-directed mutagenesis experiments, it was demonstrated that the three binding amino acid residues, His128, His132 and Asp164, provided the Zn2+ ligands of deuterolysin (Fushimi et al., 1999Go). McAuley et al. (2001Go) showed that two histidine residues, His128 and His132, an aspartic acid residue (Asp143) and two water molecules formed a distorted octahedral geometry. Hori et al. (2001Go) reported that Fushimi et al. (1999Go) proposed a new family name ‘aspzincin’ based on a site-directed mutagenesis experiment with deuterolysin, although their assignment of the third zinc ligand of the Zn atom had been an Asp residue corresponding to Asp164 (numbering for deuterolysin) rather than Asp130 (Asp143 in deuterolysin) of GfMEP from G.frondosa. In the present study, we confirmed the third Zn binding site, Asp143, in the penicillolysin by site-directed mutagenesis (Table II). Deuterolysin is an {alpha}ß protein with a zincin-like fold (McAuley et al., 2001Go), which comprises eight {alpha}-helices and five ß-sheets (Figure 1). There are three disulfide bridges between residues Cys6–Cys78, Cys85–Cys103 and Cys117–Cys177; the last of these involves the C-terminal residue (McAuley et al., 2001Go).


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Table II. Specific activity and zinc content of the recombinant penicillolysin and mutants
 


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Fig. 1. Secondary structure of deuterolysin reported by McAuley et al. (2001Go) (A) and sequence alignment of penicillolysin (PLN) and deuterolysin (DLN) (B). The locations of Thr124 in {alpha}E and Ala167 in {alpha}H helices, Glu33 in {alpha}A and Arg60 in {alpha}C helices and Pro81 in the loop between ß3 and ß4 sheets in deuterolysin are shown in (A). A closed circle gives the position of the catalytic residue in penicillolysin. Asterisks mark the His and Asp residues corresponding to the zinc ligands in the enzyme. The positions of the disulfide bonds in deuterolysin were reported by Tatsumi et al. (1994Go).

 
Structural determination of thermal instability of penicillolysin by site-directed mutagenesis

Protein engineering is a promising tool for obtaining stable proteins. Comparisons between homologous penicillolysin and deuterolysin can reveal the features responsible for the enhanced stability of penicillolysin. Thermally unstable penicillolysin has 68% sequence identity with the thermally stable deuterolysin from A.oryzae; however, in the N-terminal region from amino acid residues 1 to 60, only 60% identity was observed (Figure 1B). This shows that the region was a target for mutation. Thermally unstable penicillolysin had Arg33 and Glu60, whereas thermally stable deuterolysin had Glu33 and Arg60. From the crystal structure of deuterolysin from A.oryzae (Figure 1A), the N-terminal region of the enzyme is composed of three helices, {alpha}A, {alpha}B and {alpha}C, and a single ß-structure, ß1, as shown in Figure 1B (McAuley et al., 2001Go). Glu33 in deuterolysin is located in the {alpha}A long helix region and Arg60 in the {alpha}C long helix. In this study, our choice of mutation was R33E/E60R in the N-terminal structure of penicillolysin. For thermal stabilization of penicillolysin, we expected that on the polar surface of the helix, electrostatic interactions between acidic and basic residues at Glu33 and Arg60 would copy the N-terminal sequence of deuterolysin. We generated the mutant, R33E/E60R.

Generally, proline residues have less conformational freedom in unfolded structures than any other residues since the proline side chain is fixed by a covalent bond to the main chain. From the structure of deuterolysin, it is evident that three proline residues, Pro81, Pro87 and Pro112, are present in the molecule, but not in penicillolysin (Figure 1B). Pro81 and Pro87 are located in the loop region between ß3 and ß4 and Pro112 is located in the loop region between {alpha}D and {alpha}E. We decided on the position and species of amino acids to be replaced to give enhanced thermal stability. We generated the mutants T81P, T87P and A112P.

In {alpha}/ß proteins, {alpha}-helices and ß-sheets alternate along the peptide chain. For example, the greater the fraction of the non-polar surface buried, the lower is the stability of the protein, suggesting that the alternative polar surface buried, in hydrogen-bonded form, contributes more to stability (Darby and Creighton, 1993Go).

In GfMEP from G.frondosa, the active-site helix, helix {alpha}D, contains two zinc ligands, His117 and His121, and the catalytic residue Glu118 as components of the HExxH motif (Hori et al., 2001Go). The loop {alpha}D–{alpha}E includes the GTxDxxYG segment, which is strictly conserved among aspzincins and contains Asp130 as the third zinc ligand. Asp130 makes a unique bifurcated interaction with the zinc ion. Such a bifurcated interaction has not observed in other metalloendopeptidases whose third zinc ligand is Asp or Glu. Thr125 in penicillolysin, Thr125 in the {alpha}E helix of deuterolysin and Thr114 in the {alpha}D helix of GfMEP from G.frondosa were conserved in the common TxxHExxH motif at the C-terminal end of aspzincin family enzymes and the {alpha}E of deuterolysin and {alpha}D of GfMEP from G.frondosa are called the active-site helix.

The Ala167 residue of the C-terminal of penicillolysin was located in the corresponding region of {alpha}H in deuterolysin. On the other hand, the Glu157 residue in the {alpha}F helix in the C-terminal region of aspzincin from G.frondosa (GfMEP), a thermally stable fungal metalloendopeptidase, corresponded to Ala167 in penicillolysin. We expected penicillolysin to have a hydrogen bond between the acidic residue Glu at position 167 and Thr at position 125. We therefore generated the mutant A167E.

The polypeptide backbone of L-amino acid residues is chiral and gives an intense CD signal in the far-UV region (180– 240 nm) that is dependent on the conformation of the backbone. In particular, {alpha}-helices and ß-structures each give characteristically different spectra in the wavelength range. The approximate average content of such structures in a protein can be estimated from the CD spectrum, by comparison to a database of spectra of proteins of known structure (Figure 2). Although the CD spectra of the wild-type and mutants have slight differences (Figure 2), the enzyme activity of the mutants was not largely affected. Kinetic parameters of the mutant R33E/E60R are similar to those of the wild-type enzyme (Table III). Similar kcat/Km parameters were obtained for the two mutants R33E/E60R and T81P. The kcat/Km value of the A167E mutant was 1.6-fold higher than that of the wild-type enzyme.



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Fig. 2. Far-UV spectra of wild-type penicillolysin (open circles) and site-directed mutants R33E/E60R (upright filled triangles), A167E (inverted filled triangles) and T81P (filled squares). The spectra were measured with a Jasco J-700 spectrophotometer at room temperature and at an enzyme concentration of 0.2 mg/ml of 100 mM sodium acetate buffer, pH 5.0.

 

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Table III. Kinetic parameters of site-directed mutants of penicillolysin
 
The stability of both the wild-type and mutant proteins is expressed as the melting temperature, Tm, which is the temperature at which 50% of the enzyme is denatured during irreversible heat denaturation. To prevent the self-digestion of penicillolysin, heat treatment was carried out at pH 5.0 because the proteolytic activity is substantially reduced at this pH and the active form of the enzyme is stable. For the wild-type enzyme and the three mutants, the thermal stabilities and the Tm changes were assessed at pH 5.0 by measuring the far-UV CD spectrum at 222 nm as a function of temperature (Figure 3). For the wild-type penicillolysin, the Tm value was determined as 68°C at pH 5.0 (Table IV). The chosen mutation in penicillolysin was T81P, which caused an increase in the melting temperature to 71°C at pH 5.0. Unfortunately, we could not obtain thermally stable forms for the two mutants, T87P and A112P, of penicillolysin. The two single mutants, T87P and A112P, were found to be fairly unstable enzymes. The destabilizing effect of T87P is probably due to the location of Thr87, which is near the disulfide bridge, Cys85 and Cys103. Ala112 is located in a loop between {alpha}D and {alpha}E. The {alpha}E helix includes a catalytic residue, Glu129, and Zn2+-binding residue, His128. When Ala112 is substituted with a more rigid residue, Pro, a minor structural change to the loop and the {alpha}E helix regions might occur. The chosen mutations in penicillolysin, R33E/E60R and A167E, caused an increase in the Tm to 70°C at pH 5.0 (Table IV).



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Fig. 3. Determination of melting temperature (Tm) for site-directed mutants measured by far-UV CD spectroscopy at 222 nm. Curve a, wild-type penicillolysin (open circles); curve b, R33E/E60R (upright filled triangles); curve c, A167E (inverted filled triangles); curve d, T81P (filled squares) at pH 5.0. The curves were recorded at 222 nm in the temperature scan mode with a gradient of 1°C/min until a temperature of 80°C was recorded.

 

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Table IV. Melting temperature (Tm) and half-life of activity (T50) of site-directed mutants of penicillolysin
 
Thermal stabilization of penicillolysin by site-directed mutagenesis

When the rate of degradation of enzyme activity is measured, it is more common to express the result in terms of the half-life of enzyme activity, T50, rather than in terms of the rate constant, kd. For a first-order reaction the relationship is t1/2 = ln2/kd = 0.69/kd (Price and Stevens, 1999Go). In this study, thermal stability was quantified as T50, being the temperature at which 50% of the initial activity was retained for 10 min. The thermal stability of the mutants also depended on temperature, as shown in Figure 4A and B. The double mutant R33E/E60R was found to be fairly thermally stable, as shown in Figure 4A and B. The T50 value, 66°C, of R33E/E60R was higher than that of the wild-type penicillolysin (Table IV). It is interesting that two mutations probably due to favorable interactions between the two {alpha}-helices (A and C helices) were generated and these interactions are important in the thermal stability of the mutant enzyme R33E/E60R. The A167E mutant showed the same thermal stability as R33E/E60R. The A167E mutation was also sufficient to increase the T50 value from 62 to 65°C (Table IV). The single A167E mutation was useful for the thermal stabilization of penicillolysin, indicating that the connection between the {alpha}E and {alpha}H helices may be important. Among the three proline mutants, the T81P mutant significantly increased the T50 value from 62 to 68°C, whereas T87P and A112P had no clear stabilizing effects. For the 50% residual activity, the thermal stability at 65°C was 4, 12, 12 and 21 min for the wild-type, R33E/E60R, A167E and T81P, respectively (Figure 4B). When the T81P mutation was introduced, the enzyme was found to be more thermally stable than the two mutants, R33E/E60R and A167E. It is interesting that T81P similarly enhanced the thermal stability of penicillolysin; however, the other two mutants, T87P and A112P, showed decreased thermal stability. The T81P mutant of penicillolysin may have a structure essentially identical to that of the wild-type of deuterolysin. There are indications that the proline residues contribute to enzyme thermal stability (Matthews et al., 1987Go; Suzuki, 1989Go). The contribution can be explained in terms of entropic effects (or chain flexibility) since proline is more restricted conformationally in the unfolded state than any other amino acid (Matthews et al., 1987Go). Since proline residues are restricted with respect to their {phi}{psi} angles, their introduction can easily cause conformational strain in the backbone. Suzuki et al. (1987Go) demonstrated that an increase in the frequency of proline in ß-turns and in the total number of hydrophobic residues can enhance protein thermal stability.



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Fig. 4. (A) Determination of the half-life of activity, T50, for wild-type penicillolysin (open circles) and mutants R33E/E60R (upright filled triangles), A167E (inverted filled triangles) and T81P (filled squares). (B) Stability at 65°C of wild-type penicillolysin (open circles), R33E/E60R (upright filled triangles), A167E (inverted filled triangles) and T81P (filled squares).

 
For further investigation, the triple mutant R33E/E60R/T81P was constructed. Surprisingly, the triple mutation was found to produce a fairly thermally unstable enzyme. The T50 value of the triple mutant was determined as 58°C (data not shown).

The thermal stability of an enzyme is influenced by many factors, including amino acid sequence, three-dimensional structure, cofactors and pH. However, at present we cannot explain the results obtained. The present findings show that the thermal stability of penicillolysin is increased dramatically by just a few mutations in one particular region of the protein (Table IV).


    References
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 Abstract
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 Materials and methods
 Results and discussion
 References
 
Beynon,R.J. and Beaumont,A. (1998) In Barrett,A.J., Rawlings,N.D. and Woessner,J.F. (eds), Handbook of Proteolytic Enzymes. Academic Press, San Diego, pp. 1037–1046.

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Received December 18, 2003; revised March 26, 2004; accepted April 1, 2004 Edited by Taiji Imoto





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