Engineering a substrate-specific cold-adapted subtilisin

Nikolaj Tindbaek1, Allan Svendsen, Peter Rahbek Oestergaard and Henriette Draborg

Molecular Biotechnology, Novozymes A/S, Krogshoejvej 36, DK-2880 Bagsvaerd, Denmark

1 To whom correspondence should be addressed. e-mail: nikolaj_tindbaek{at}webspeed.dk


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
One region predicted to be highly flexible for a psychrophilic enzyme, TA39 subtilisin (S39), was transferred in silico to the mesophilic subtilisin, savinase (EC 3.4.21.62), from Bacillus lentus (clausii). The engineered hybrid and savinase were initially investigated by molecular dynamic simulations at 300 K to show binding region and global flexibility. The predicted S39 region consists of 12 residues, which due to homology between the subtilisins, results in a total change of eight residues. By site-directed modifications, the region was transferred to the binding region of savinase, thus a savinase-S39 hybrid, named H5, was constructed. The designed hybrid showed the same temperature optimum and pH profile as savinase, but H5 had higher specific activity on the synthetic substrate N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (AAPF) at all temperatures measured and, at the same time, H5 showed a decrease in thermostability. The H5 hybrid showed broader substrate specificity, measured at room temperature, due to an increase in catalytic efficiency on AAPF, AAPA and FAAF compared with savinase (N-succinyl-XXXX-pNA; XXXX = AAPF, AAPA and FAAF). The H5 hybrid showed increased activity at low temperature, increased binding region and global flexibility, as investigated by molecular dynamic simulations, and global destabilization from differential scanning calorimetry measurements. These psychrophilic characteristics indicated an increase in binding site flexibility, probably due to the modifications P129S, S130G, P131E, and thus we show that it is possible to increase low temperature activity and global flexibility by engineered flexibility in the binding region.

Keywords: cold adaptation/molecular dynamics/protein engineering/protein stability/psychrophilic enzymes


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Subtilisins are endoproteases which constitute an important class of enzymes in the laundry detergent industry, where they help remove protein stains from textiles. It is of great interest from an industrial and environmental point of view to design an enzyme which functions with higher specific activity at low temperatures. Cold-adapted enzymes will enhance wash performance in low temperature laundry, where also reduction in energy consumption and reduction in wear of textiles are obvious advantages, but possible drawbacks of using cold-adapted proteases are their instability at higher temperatures and under storage conditions. Therefore, many attempts have been made to confer a broad temperature spectrum on detergent proteases.

Subtilisins isolated from bacilli native to cold environments generally exhibit higher catalytic efficiency at low temperatures and relative instability at high temperatures (Miyazaki et al., 2000Go). Local or global flexibility seems to be the main adaptive character of psychrophilic enzymes responsible for the decrease in activation enthalpy and increase in entropy that efficiently increases the catalytic constant, kcat, at low temperatures (Lonhienne et al., 2000Go).

The psychrophilic enzymes S39 and S41, subtilisins from the Antarctic bacteria Bacillus TA39 and TA41, respectively, show, by modelling, a significant decrease in the number and strength of intramolecular weak bonds such as salt bridges and aromatic interactions (Miyazaki et al., 2000Go). Furthermore, they lack the P129 (BPN' numbering; Perona and Craik, 1995Go), which severely restricts the mobility of a substrate binding loop in mesophilic and thermophilic subtilisins (Davail et al., 1994Go). Also, the affinity for calcium is almost three orders of magnitude lower than that of mesophilic subtilisins and the interaction with the solvent is significantly higher due to an increase in the number of Asp residues in the loops connecting secondary structures (Narinx et al., 1997Go).

Extensive attempts to engineer cold-adapted proteases from subtilisin BPN', a mesophilic and industrially useful serine protease, have previously been made (Kano et al., 1997Go; Taguchi et al., 1998Go, 2000Go). Another strategy to evolve a serine protease with enhanced thermostability and activity has been to use the psychrophilic subtilisin S41 (Miyazaki et al., 2000Go; Wintrode et al., 2000Go). In these studies, several enzyme variants, as starting points for random mutagenesis, exhibited a catalytic efficiency higher than the wild-type at low temperatures, when N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (AAPF) was used as substrate.

Here we report an attempt to engineer a site-directed serine protease hybrid designed on the basis of molecular dynamic simulations. Based on homology modelling of the psychrophilic S39 subtilisin from the Antarctic bacteria Bacillus TA39 (Narinx et al., 1992Go), a 12 amino acid region of the binding cleft, residues MSLGSSGESSLI (positions 138–149), was predicted to be highly flexible and transferred in silico to the mesophilic Bacillus lentus subtilisin, savinase, replacing residues LSLGSPSPSATL (positions 124–135, BPN' numbering) in the binding site. In a 1.2 ns molecular dynamic simulation, the savinase hybrid (H5) showed increased global flexibility together with increased mobility in the transferred region, which now comprised eight amino acid changes compared with savinase wild-type, including P129S, S130G and P131E.

The hybrid was created by site-directed mutagenesis and showed psychrophilic properties like enhanced activity at low temperature, a flexible binding region and global weak stability due to increased overall flexibility.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Homology modelling and molecular dynamics

Public databases were searched with BLASTP 2.1.2 (Altschul et al., 1997Go) and due to its high sequence homology the Bacillus sphaericus subtilisin, SSII (Wati et al., 1997Go), Protein Data Bank accession no. 1EA7, was chosen as a template structure for modelling of S39. The modules HOMOLOGY and BIOPOLYMER, INSIGHTII version 2000 from Accelrys, were used to align the SSII and S39 sequences, assign coordinates, insert calcium atoms, generate and search loops, and finally build the complete model. Savinase coordinates were downloaded from the Protein Data Bank (accession no. 1SVN). Before each simulation, structures were evaluated and corrected for errors with WHATCHECK (WHATIF; Vriend, 1990Go) and crystal water was only deleted from the binding site when collision with AAPF was possible. Docking of AAPF was done by superimposing AAPF on the chymotrypsin inhibitor 2 (CI-2). In silico construction of hybrid H5 was performed using a rotamer library, choosing only side chain conformations with lowest energies; residue substitutions are described in the section ‘Construction of hybrid’. Water was added to each structure using PBC with a TIP3 water box of dimension 60 Å3, enclosing the enzyme with at least 12 Å of water on each side.

The molecular dynamic simulations of savinase and the H5 hybrid with AAPF were done at 300 K using NAMD version 2.1 (Kalé et al., 1999Go) and a CHARMm force field (Brooks et al., 1983Go). C{alpha} isotropic fluctuations were obtained by identifying atom positions to various time intervals (trajectories) for 1.2 ns and fluctuations were compared with a minimized average structure of the enzymes.

At every simulation, structures were minimized using a 100 step conjugated gradient (step size 0.1 fs), and subsequently equilibrated at 10 000 steps (step size 1 fs). The dynamics were run for 1.2 ns and the first 0.3 ns were excluded because simulations were not stable before that time. Each dynamic simulation was performed with the following parameters: cut off, 10; pairlistdist, 11; switchdist, 8.

Materials

Synthetic substrates, N-succinyl-XXXX-pNA (XXXX = AAPF, AAVA, AAPA, FAAF and YVAD) were purchased from Bachem AG (Weil am Rhein, Germany) and dissolved in dimethyl sulfoxide before being diluted in buffer. Bacitracin was obtained from Lundbeck A/S (Copenhagen, Denmark). PCR primers were purchased from DNA Technology (Aarhus, Denmark).

Expression systems

Bacillus subtilis DN1885, with disrupted apr and npr genes (Diderichsen et al., 1990Go), was used as the host strain for the screening of the subtilisin hybrid on LB-agar skim milk plates containing 1% skim milk and 6 µg/ml chloramphenicol (CAM). One litre of LB-agar contained 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl and 15 g of agar. Bacillus subtilis DN1885 was also used as the host strain for production of wild-type savinase and hybrid in liquid Bacillus medium containing 100 g of pearl sugar, 40 g of soy, 10 g of Na2HPO4, 0.1 ml of Pluronic PE 6100 (from BASF), 10 µg/ml CAM and water added to 1 l.

Construction of hybrid

Site-directed mutagenesis was performed using standard primers and PCR methods (Horton et al., 1990Go). Construction of the H5 hybrid was done with deletion of residues LSLGSPSPSATL (positions 124–135; BPN' numbering) in savinase and subsequent insertion of residues MSLGSSGESSLI (positions 138–149) from TA39 subtilase (Siezen and Leunissen, 1997Go) between N123 and E136 in savinase. Another modification, V104S, was added due to steric limitation between V104 and L135I (BPN' numbering is used throughout the paper).

The PCR mixture was incubated in an MJ Research PTC-200 thermal cycler and the amplified product was isolated by agarose gel electrophoresis and gel purification with a GFX purification kit. The hybrid gene was chromosomally integrated in the B.subtilis genome (Declerck et al., 1988Go). Transformation in B.subtilis was done as described by Yasbin et al. (1975)Go. The hybrid protein sequence compared with savinase, with silent modifications at S125, L126, G127, S128 and S132 became H5 (V104S, L124M, S125, L126, G127, S128, P129S, S130G, P131E, S132, A133S, T134L, L135I).

DNA sequencing

The DNA analysis of the hybrid gene was carried out by the dideoxynucleotide chain-termination method (Morinaga et al., 1984Go) using BigDye v. 3.1 and an ABI 3700 Capillary Sequencer.

Fermentation and purification

Fermentation was done in 300 ml of Bacillus medium (as described earlier) at 37°C, 250 r.p.m.; thereafter, the cultures were fermented for 5 days at 22°C and 250 r.p.m. The H5 fermentation culture was centrifuged and the supernatant was filtered through a SEITZ depth filter plate, followed by a buffer exchange to 50 mM boric acid, 5 mM succinic acid, 1 mM CaCl2, pH 6.0, on a G25-Sephadex column. After the G25-Sephadex step, H5 was purified on an SP-Sepharose high-performance column (Amersham Biosciences) and eluted in the buffer system described above by a 0.5 M NaCl linear gradient, followed by a bacitracin affinity chromatography step (Mortensen et al., 1989Go), where the protease was eluted in 75 mM boric acid, 7.5 mM succinic acid, 1.5 mM CaCl2, 25% isopropanol, pH 7.0, using a 1 M NaCl linear gradient. The isopropanol and NaCl were removed immediately after the elution by another G25-Sephadex exchange because of the denaturating properties of isopropanol.

Savinase was purified by a bacitracin affinity chromatography column under the same conditions as H5. The purity of the recovered fractions was verified by SDS–PAGE, using Cambrex PAGEr Gold Precast 4–20% tris-(hydroxymethyl)-aminomethane (Tris)-glycine gels.

Mass spectrometry

The protein mass was confirmed using electrospray ionization mass spectrometry on a VG-platform from Micromass, connected to a Waters HPLC, model 2690. The sample (0.1–1.0 mg) was desalted on a reverse-phase C4 column before the spectrometry analysis. The software Maximum Entropy was used for data analysis to determine the mass.

Measurements of enzyme activity

Wild-type and hybrid activities were assayed on a SPECTRAmax Plus 384 spectrophotometer by monitoring the release of pNA at 405 nm following enzymatic hydrolysis of the synthetic tetrapeptides (0–5.4 mM) in 100 mM Tris buffer, 2 mM CaCl2, 0.025% Triton X-100 at pH 9.0 and an extinction coefficient of 10 500 M–1 cm–1. These conditions were used unless stated otherwise. Km and kcat were determined by fitting the data to the Michaelis–Menten equation using the least squares method (Cornish-Bowden, 1995Go) and weighting each data point according to the standard deviation.

The absolute enzyme concentration was estimated by active-site titration (AST) using the tight-binding inhibitor CI-2 and AAPF as substrate (Knight, 1995Go).

Measurements of heat inactivation

A 1 ml aliquot of 0.3 µM purified enzyme was incubated at 68°C in the activity buffer together with 1 mg/ml bovine serum albumin (BSA) to measure autocatalysis (1:50 000, enzyme to BSA) and 20 µl samples of each enzyme pool were taken out at various time intervals and immediately cooled on ice. The residual subtilisin activity was measured with AAPF (1.58 mM) in the buffer described above.

Thermal denaturation

Differential scanning calorimetry (DSC) was performed on Microcal VP-DSC and Origin software was used for data acquisition and analysis. A protein concentration of 38.5 mM in 100 mM boric acid, 2 mM CaCl2 at pH 9 was used. The scan rate was 90°C/h spanning from 10 to 100°C at 20 p.s.i. pressure.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Homology modelling and molecular dynamics

Homology modelling of the cold-adapted subtilisin S39 was based on the structure of the mesophilic subtilisin SSII from B.sphaericus, which shared an amino acid sequence identity of 74% with S39.

The savinase-hybrid H5 was designed in silico with a region including an eight amino acid change in the active site (Figure 1). To investigate whether the S39 region increased the flexibility in H5 compared with savinase, a molecular dynamic simulation at 300 K was performed with the AAPF substrate docked in H5 and savinase binding clefts (Figure 2). The transferred S39 region in H5 showed an increase of 18% in C{alpha} isotropic fluctuations compared with the corresponding savinase region 124–135. In particular, the transferred residues 127–131, including P129S, S130G and P131E, were highly flexible with an increase of 45% in C{alpha} isotropic fluctuations. This showed that the region had a more flexible backbone structure when AAPF was bound and this could also be sufficient to conduct an increase in low temperature activity and catalytic efficiency to the thermostable backbone of savinase. The other side of the savinase binding cleft, residues 98–104, showed higher flexibility than the corresponding H5 region, but in overall flexibility, H5 showed a 20% increase in C{alpha} isotropic fluctuations compared with savinase; in particular, residues 190–210 were highly mobile. From the structure of the H5 hybrid it was predicted that V104 and L135I could give steric hindrance in the entrance to the active site and V104 was therefore modified to the spatially smaller residue side chain of serine.



View larger version (52K):
[in this window]
[in a new window]
 
Fig. 1. Savinase is shown in cartoon style using PyMOL (DeLano, 2003Go). Red represents the region where H5 is different from savinase, corresponding to V104S, L124M (front left and deep in the binding site, respectively), P129S, S130G, P131E (the binding loop), A133S, T134L and L135I (red region at the end of the {alpha}-helix). The binding region is seen to the left of the red loop. The catalytic triad is indicated with yellow sticks (D32, H64 and S221) and is placed right over the binding region and on the lower left side of the central {alpha}-helix.

 


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 2. R.m.s.d. subtraction plot shows C{alpha} isotropic fluctuations of H5 compared with savinase at 300 K. R.m.s.d. of H5 was subtracted from savinase values (H5 – savinase) as a function of residue numbers, with positive values for H5. The tetrapeptide AAPF was docked in the enzyme binding site under the simulations, which in both enzymes are residues 98–104 and 124–135.

 
Purification of hybrid H5 and savinase

Exchange of an eight amino acid savinase region with the corresponding region of S39 was made by a site-directed approach, transferred into B.lentus and clones expressing the active protease hybrid were identified for halo formation around the colony on LB-agar containing 1% skim milk.

A 300 ml fermentation of Bacillus-expressed H5 was centrifuged, filtered and buffer exchanged on a G25-Sephadex column, followed by purification on an SP-Sepharose high-performance column and a bacitracin affinity chromatography column. Coomassie Brilliant Blue stained SDS–PAGE of the purified samples of savinase and H5 showed only single bands, indicating pure samples (data not shown). Mass spectrometry confirmed that the purified enzymes were of the correct size and H5 was determined to be 26 676 ± 3 Da, with a theoretical value of 26 678.5 Da.

Temperature and pH dependence in savinase and hybrid

To study whether the hybrid had changed pH properties compared with savinase, a pH profile study of H5 and savinase was performed in a multi-pH stable buffer measuring the cleavage of AAPF (Figure 3). The pH spectra spanned the range 2–12, taking into account the alkaline autolysis of the phenylalanine–pNA peptide bond at pH >11. The hybrid showed no difference in pH profile compared with savinase and both had an optimum at pH 9.0 (24°C). The protein concentration of the H5 hybrid and savinase was determined by AST with the reversible CI-2 so as to get an absolute enzyme concentration. AST inhibition curves of H5, altered in the CI-2 binding site, were linear, and differences in the enzyme concentration of H5 and savinase determined by AST and OD280 were 17 and 10%, respectively. Therefore, concentration determination by AST was taken as reliable and the purified enzyme samples were of high quality.



View larger version (12K):
[in this window]
[in a new window]
 
Fig. 3. The pH–activity profile of wild-type and hybrid was tested in a pH activity assay measuring on AAPF (1.58 mM) at 24°C in 100 mM succinic acid, 100 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 100 mM 2-(cyclohexylamino)-ethanesulfonic acid (CHES), 100 mM 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS), 1 mM CaCl2, 150 mM KCl, 0.01% Triton X-100. Initial velocities were followed spectrophotometrically over 1 min and standard deviations for all data sets were under 10%.

 
Increased specific activities of H5 compared with savinase on AAPF were observed at every temperature spanning the interval from 5 to 85°C at pH 9 (Figure 4A). The temperature profiles showed a similar trend and at 10 and 20°C, H5 had an increased activity of 121 and 73%, respectively, compared with savinase (Figure 4B). H5 and savinase showed the same temperature optimum at 65°C, although H5 had 37% higher specific activity at that temperature.




View larger version (29K):
[in this window]
[in a new window]
 
Fig. 4. (A) Temperature dependence of savinase and H5 measured on AAPF (1.58 mM) in 100 mM succinic acid, 100 mM HEPES, 100 mM CHES, 100 mM CABS, 1 mM CaCl2, 150 mM KCl, 0.01% Triton X-100, pH 9.0. Protease activity was stopped after 1 min by adding 600 µl of 0.5 M succinic acid to the reaction mixture, cooled on ice and the endpoint measured at OD405. Standard deviations for all data sets were under 10%. (B) Percentage increase of H5 compared with savinase calculated as specific activity [(H5/savinase] – 1)x100%, and plotted as a function of the temperature.

 
Kinetics of savinase and hybrid

To characterize the kinetic properties of the H5 hybrid and savinase, Km and kcat were determined using five different synthetic tetrapeptides: AAPF, AAVA, AAPA, FAAF and YVAD (Table I). H5 showed a 66% higher Km and 117% higher kcat on AAPF compared with savinase. Both enzymes showed a very low Km on AAVA. The activity relative to the wild-type was increased for H5 on AAPF, AAPA and FAAF, and decreased on AAVA and YVAD. FAAF substrate showed a kcat/Km (specificity constant) of 767 x 103 M–1 s–1 and 418 x 103 M–1 s–1 for H5 and savinase, respectively.


View this table:
[in this window]
[in a new window]
 
Table I. Kinetic parameters of H5 hybrid and savinase for hydrolysis of various tetrapeptides (0–4.8 mM) at 24°C
 
Heat inactivation of savinase and H5

Heat inactivation was determined as the rate of irreversible inactivation caused by autocatalysis or thermal unfolding at 68°C for up to 134 min (Narinx et al., 1997Go). The half-lives of enzyme inactivation were 78 min for savinase and 49 min for H5, as presented in Figure 5. Clearly, H5 was sensitive to heat treatment at 68°C, but no autocatalysis could be detected using BSA controls and, furthermore, H5 ± BSA and savinase ± BSA curves were found to be similar, proving heat inactivation was due to thermal unfolding and not due to autocatalysis. This indicated that the S39 region transferred temperature instability to the enzyme due to global weak stability.



View larger version (13K):
[in this window]
[in a new window]
 
Fig. 5. Heat inactivation of savinase and H5. The residual activity after exposure to 68°C in various time intervals was assayed at 24°C with 1.58 mM AAPF as substrate. BSA (1 mg/ml) was included to measure the autocatalysis. Standard deviations for all data sets were under 10%.

 
Thermal denaturation of savinase and H5

Thermal denaturation was measured with DSC (Figure 6) and temperature instability and weak global stability from the heat inactivation assay were substantiated here. The DSC thermogram of H5 was broader compared with savinase and unfolding was accomplished over a larger temperature range. The unfolding temperature (Tm) was 72.7°C for H5 and 75.5°C for savinase, and this DSC profile indicated a less cooperative unfolding compared with savinase. Furthermore, the thermograms showed a one transition state unfolding of both enzymes according to only one unfolding peak and this property corresponded to a one domain denaturation process for both enzymes.



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 6. Thermal denaturation as monitored by DSC. Baseline subtracted DSC data have been normalized for protein concentration. Samples were dialysed overnight against the appropriate buffer, the latter being used in the reference cell and for baseline determination. Due to the minimal difference in autocatalysis of H5 and savinase, measurements could be performed at pH optima, e.g. pH 9.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The psychrophilic engineering concept described is to transfer a binding region, with different substrate specificities and loop flexibility, from a psychrophilic subtilisin, S39, to the more stable backbone of savinase. Transfer of the S39 binding region to savinase was based on previous speculation on local flexibility as a mechanism for cold adaptation (Lonhienne et al., 2001Go); while P129 severely restricts the mobility of the substrate binding loop in mesophilic and thermophilic subtilisins, its lack in S39 might explain its cold adaptation. Furthermore, removal of P131 had been involved in destabilization of savinase (Betzel et al., 1992Go).

The overall amino acid identity between savinase and S39 is 44% and the S39 region 124–135 shares 50% sequence identity with the corresponding region in savinase. Transfer of the 12 residue long region from the subtilisin S39 binding site to the savinase binding site introduces four changes in the S4 binding site (V104S, P129S, S130G and L135I; Siezen, 1996Go). V104S and L135I are at the rim of S4, and by these four modifications S4 becomes more hydrophilic and able to bind more voluminous and more hydrophilic residues in the P4 site of the substrate.

V104S modification was introduced as a consequence of a steric hindrance between L135I and V104. Furthermore, V104 is believed to have an important function at the entrance to the active site and is necessary to maintain activity at high pH (Betzel et al., 1992Go). In our case, changing V104S had no altering effect on the pH profile and it is most likely that the substitution of V104S is not the reason for the increased low temperature activity in H5 (Davail et al., 1994Go). L124M is located in the S1 binding site and this change would make the site become more hydrophilic, but not affect the spatial size of S1. Due to higher isotropic fluctuations [high root-mean-square deviation (r.m.s.d.)], as seen in Figure 2, P129S, S130G and P131E are shown by the molecular dynamic simulation to introduce flexibility in the binding loop and could also change the electrostatics towards binding positively charged residues with higher affinities. Modifications of P129 and P131 remove rigid constraints on rotation about the N–C{alpha} bond of the backbone and also lower the likelihood of the preceding peptide bond to adopt the cis configuration. Changing S130G would also introduce greater conformational flexibility to the binding loop due to the absence of the larger –CH2OH serine side chain and the H atom symmetry in the glycine (Creighton, 1993Go). Furthermore, the three modifications increase the negative charge and may result in a poorer binding constant on AAPF and increase the mobility of the region, and may explain why an increased activity of H5 relative to savinase was observed (Table I). A133S and T134L are located in the {alpha}-helix N-terminal part and do not form any of the substrate (S) sites; changing them would probably not affect the substrate binding directly, but it cannot be excluded that they can influence binding site mobility or conformation with long range charge interaction.

Specific activity of H5 is improved on AAPF compared with savinase at all tested temperatures (Figure 4A and B) and from Table I, improvements of kcat/Km are observed in three out of five substrates. Regarding AAPF, the increase in Km is probably due to the altered S4 binding site, making AAPF bind with less affinity, but at the same time increasing the kcat and overall catalytic efficiency by a factor of 1.3, in agreement with S1 and S4 pockets determining the substrate specificity (Mulder et al., 1999Go).

Cold-adapted enzymes optimize their catalytic efficiency by increasing kcat, decreasing Km or by changes in both parameters (Feller et al., 1994Go; Narinx et al., 1997Go; D’Amico et al., 2002Go). The increased low temperature activity could be explained kinetically by a combined kcat and Km effect; in this study these parameters increased 118 and 66%, respectively, under steady state, when the enzyme was saturated with AAPF. This kcat and Km effect is in agreement with the temperature-adaptive hypothesis, which states that an increase in kcat will occur concomitantly with an increase in Km, due to the higher conformational mobility of the binding region required for rapid catalysis (high kcat) in the cold-adapted form. This will lead to a higher number of conformational states available to the enzyme and, as a result, to a larger proportion of enzyme conformations that bind substrates poorly or not at all. This will result in higher Km and kcat in the cold-adapted enzyme (Fields and Somero, 1998Go).

Thermal inhibition of enzyme reaction rates is clearly seen in Figure 4B, which compares the thermal dependence of enzyme activity. Hybrid H5 demonstrates adaptation to catalysis at low temperatures that must be due to the low activation energy associated with the unusually increased kcat. These optimized thermodynamic properties are probably due to a change of weak interactions in the catalytic centre, involved in substrate binding at low temperatures, which is also shown for the Antarctic Arthrobacter chitobiase (Lonhienne et al., 2001Go).

The high thermostability of savinase is verified in the heat inactivation and DSC assay. These show the stable structure of savinase with high Tm and the acquired heat sensitivity of H5 with reduced melting temperature. The heat inactivation assay gives evidence of infinitesimal autocatalysis of both enzymes, even when measured at their pH optima and when the enzyme to BSA ratio was 1:50 000. Therefore, DSC could be measured at pH 9 without protease inhibitors, which could severely change enzyme unfolding profiles, which could also be changed at low pH values. Protein stability increases from H5 to savinase analogues, going from psychrophilic to mesophilic and thermophilic enzymes. The mechanism of unfolding in subtilisins is primarily due to loss of a calcium atom from the strong cation binding site at residue 73–83, an unfolding which is irreversible. In addition, protein instability of H5 might be conducted by the P131E modification, which was previously shown to be involved in savinase stability (Betzel et al., 1992Go).

Evidence of protein instability was established by DSC measurements, showing that the DSC profile of H5 is broader compared with savinase, although with a lower Tm. The broadening of the DSC profile is a normal result of the fact that there is a difference between the Cp values of the native and denatured forms of the protein, as a result of the exposure of the hydrophobic core on denaturation.

Microcalorimetric studies of psychrophilic enzymes have revealed two main types of stability patterns. One group of cold-active enzymes displays a global decrease of stability with highly cooperative unfolding (D’Amico et al., 2001Go), whereas the other group displays only local increases in flexibility (Bentahir et al., 2000Go; Lonhienne et al., 2001Go). Categorizing H5 into one of these groups is not straightforward, due to both the highly mobile binding region and the global instability and flexibility. In comparison, the psychrophilic {alpha}-amylase (D’Amico et al., 2001Go) and the psychrophilic xylanase (Collins et al., 2003Go) show global weak stability and improve the kcat values at low temperatures at the expense of the Km values, probably as a result of a less efficient substrate binding into the mobile active site. These kinetic and stability characteristics are also symptomatic for H5, and the higher flexibility of the entire enzyme molecule is accompanied by a broader distribution of conformational states, leading to poor ligand binding and high Km. This trend is also seen from the kinetics measured on the five tetrapeptides, where Km on four out of five substrates is increased. The differences in kinetic constants may also be related to the size of the substrate (Collins et al., 2002Go), where macromolecular substrates, hydrolysed by subtilisins, probably require large conformational changes of the entire protein compared with hydrolysis of small substrates.

The domain structure of savinase was not expected to be changed due to transfer of the S39 region and, in agreement with this, thermograms show a one transition state unfolding of both enzymes, meaning that only one domain is unfolding.

Additionally, shown by molecular dynamics, flexibility engineering of the binding site not only changed the flexibility profile of the binding site, but also affected the entire enzyme. It is then possible that increased kcat is due not only to the flexible binding region, but also to the highly flexible enzyme edifice, which makes fast hydrolysis possible.

As seen in Table I, FAAF is the preferred substrate to both savinase and H5, and compared with AAPF, activity on FAAF increases by a factor of 2.6 and 3.7, respectively, and may be considered as a more sensitive substrate in screening assays for savinase hybrids. In general, substrates such as FAAF and similar, with hydrophobic residues in positions P1 and P4 and succinyl at the N-terminus to increase water solubility, are better substrates to savinase and variants thereof than suc-AAPF. FAAF seems to be water soluble at least up to 5 mM concentration, while substrates with more hydrophobic residues are insoluble in water at the concentrations required for kinetic studies.

The pH optimum of H5 is not affected by these regional changes when using AAPF, but it cannot be excluded that H5 has a different pH profile compared with savinase when using other synthetic tetrapeptides. When using synthetic tetrapeptides in measuring enzyme activity it generally should be considered that they are not natural substrates and this substrate bias could be why low temperature optima of cold-adapted subtilisins remain to be demonstrated in the literature using tetrapeptides as substrates (D’Amico et al., 2002Go).

In conclusion, H5 shows psychrophilic properties, such as increased low temperature activity, binding site flexibility and global destabilization as compared with savinase (Davail et al., 1994Go).

Our results illustrate that the stable backbone of savinase is able to adopt eight new residues in the binding region and at the same time become heat sensitive. The results also demonstrate that it is possible to increase low temperature activity on small substrates by engineering an increase in binding site flexibility, which affects the stability of the whole enzyme edifice and conducts a weak global structure with increased overall flexibility. Therefore, global flexibility conducted by flexibility changes in the binding region is one of perhaps several mechanisms used to increase low temperature activity, but not necessarily to a low temperature optimum.


    Acknowledgements
 
We thank Dr Darren Dafydd Jones, Dr Steffen Ernst and Dr Leonardo di Maria for advice and fruitful discussions and Dr Anders Dybdal Nielsen for assistance with DSC. We also thank Dr Esben Friis, Dr Thomas Agersten Poulsen and Dr Olivier Taboreau for help and advice with the molecular dynamic simulations and Dr Marianne Vind for help with the mass spectrometry.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Altschul,S.F., Thomas,L.M., Schäffer,A.A., Zhang,J., Zhang,Z., Miller,W. and Lipman,D.J. (1997) Nucleic Acids Res., 25, 3389–3402.[Abstract/Free Full Text]

Bentahir,M., Feller,G., Aittaleb,M., Lamotte-Brasseur,J., Himri,T., Chessa,J.P. and Gerday,C. (2000) J. Biol. Chem., 275, 11147–11153.[Abstract/Free Full Text]

Betzel,C., Klupsch,S., Papendorf,G., Hastrup,S., Branner,S. and Wilson,K.S. (1992) J. Mol. Biol., 223, 427–445.[ISI][Medline]

Brooks,B.R., Bruccoleri,R.E., Olafson,B.D., States,D.J., Swaminathan,S. and Karplus,M. (1983) J. Comput. Chem., 4, 187–217.[ISI]

Collins,T. et al. (2002) In Pandalai,S.G. (ed.), Recent Research Developments in Proteins, Vol. 1. Transworld Research Network, Trivandrum, pp. 13–26.

Collins,T., Meuwis,M., Gerday,C. and Feller,G. (2003) J. Mol. Biol., 328, 419–428.[CrossRef][ISI][Medline]

Cornish-Bowden,A. (1995) Analysis of Enzyme Kinetic Data. Oxford University Press, Oxford.

Creighton,T.E. (1993) Proteins: Structures and Molecular Properties, 2nd edn. W.H. Freeman and Company, New York.

D’Amico,S., Gerday,C. and Geller,G. (2001) J. Biol. Chem., 276, 25791–25796.[Abstract/Free Full Text]

D’Amico,S., Claverie,P., Collins,T., Georlette,D., Gratia,E., Hoyoux,A., Meuwis,M., Feller,G. and Gerday,C. (2002) Philos. Trans. R. Soc. Lond. B, 357, 917–925.[CrossRef][ISI][Medline]

Davail,S., Feller,G., Narinx,E. and Gerday,C. (1994) J. Biol. Chem., 269, 17448–17453.[Abstract/Free Full Text]

Declerck,N., Joyet,P., Lecoq,D. and Heslot,H. (1988) J. Biotechnol., 8, 23–38.[CrossRef][ISI]

DeLano,W.L. (2003) The PyMOL Molecular Graphics System. DeLano Scientics LLC, San Carlos, CA. http://www.pymol.org.

Diderichsen,B., Wedsted,U., Hedegaard,L., Jensen,B.R. and Sjoeholm,C. (1990) J. Bacteriol., 172, 4315–4321.[ISI][Medline]

Feller,G., Payan,F., Theys,F., Qian,M., Haser,R. and Gerday,C. (1994) Eur. J. Biochem., 222, 441–447.[Abstract]

Fields,P. and Somero,G. (1998) Proc. Natl Acad. Sci. USA, 95, 11476–11481.[Abstract/Free Full Text]

Horton,R.M., Cai,Z.L., Ho,S.N. and Pease,L.R. (1990) Biotechniques, 8, 528–535.[ISI][Medline]

Knight,C.G. (1995) Methods Enzymol., 248, 85–101.[ISI][Medline]

Kalé,L., Skeel,R., Bhandarkar,M., Brunner,R., Gursoy,A., Krawetz,N., Phillips,J., Shinozaki,A., Varadarajan,K. and Schulten,K. (1999) J. Comput. Phys., 151, 283–312.[CrossRef][ISI]

Kano,H., Taguchi,S. and Momose,H. (1997) Appl. Microbiol. Biotechnol., 47, 46–51.[CrossRef][ISI][Medline]

Lonhienne,T., Gerday,C. and Feller,G. (2000) Biochim. Biophys. Acta, 1543, 1–10.[ISI][Medline]

Lonhienne,T., Zoidakis,J., Vorgias,C.E., Feller,G., Gerday,C. and Bouriotis,V. (2001) J. Mol. Biol., 310, 291–297.[CrossRef][ISI][Medline]

Miyazaki,K., Wintrode,P., Grayling,R.A., Rubingh,D.N. and Arnold,F.H. (2000) J. Mol. Biol., 297, 1015–1026.[CrossRef][ISI][Medline]

Morinaga,Y., Franceschini,T., Inouye,S. and Inouye,M. (1984) BioTechnology, 2, 636–639.[ISI]

Mortensen,S.B., Thim,L., Christensen,T., Woeldike,H., Boel,E., Hjortshoej,K. and Hansen,M.T. (1989) J. Chromatogr., 476, 227–233.[CrossRef]

Mulder,F.A.A., Schipper,D., Bott,R. and Boelens,R. (1999) J. Mol. Biol., 292, 111–123.[CrossRef][ISI][Medline]

Narinx,E., Davail,S., Feller ,G. and Gerday,C. (1992) Biochim. Biophys. Acta, 1131, 111–113.[ISI][Medline]

Narinx,E., Baise,E. and Gerday,C. (1997) Protein Eng., 10, 1271–1279.[CrossRef][ISI][Medline]

Perona,J.J. and Craik,C.S. (1995) Protein Sci., 4, 337–360.[Abstract/Free Full Text]

Siezen,R.J. (1996) In Bott,R. and Betzel,C. (eds), Subtilisin Enzymes: Practical Protein Engineering. Plenum Press, New York, pp. 63–74.

Siezen,R.J. and Leunissen,J.A.M. (1997) Protein Sci., 6, 501–523.[Abstract/Free Full Text]

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

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

Vriend,G. (1990) J. Mol. Biol., 8, 29, 52–56.

Wati,M.R., Thanabalu,T. and Porter,A.G. (1997) Biochim. Biophys. Acta, 1352, 56–62.[ISI][Medline]

Wintrode,P.L., Miyazaki,K. and Arnold,F.H. (2000) J. Biol. Chem., 275, 31635–31640.[Abstract/Free Full Text]

Yasbin,R.E., Wilson,G.A. and Young,F.E. (1975) J. Bacteriol., 121, 296–304.[ISI][Medline]

Received September 25, 2003; revised December 15, 2003; accepted January 5, 2004 Edited by Jacques Fastrez