Stabilization of a chitinase from Serratia marcescens by Gly->Ala and Xxx->Pro mutations

Sigrid Gåseidnes1, Bjørnar Synstad1, Xiaohong Jia1, Hege Kjellesvik1, Gert Vriend2 and Vincent G.H. Eijsink1,3

1Department of Chemistry and Biotechnology, Agricultural University of Norway, PO Box 5040, 1432 Ås, Norway and 2Center for Molecular and Biomolecular Informatics, University of Nijmegen, PO Box 9010, 6500 GL Nijmegen, The Netherlands

3 To whom correspondence should be addressed. e-mail: vincent.eijsink{at}ikb.nlh.no


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This paper describes attempts to increase the kinetic stability of chitinase B from Serratia marcescens (ChiB) by the introduction of semi-automatically designed rigidifying mutations of the Gly->Ala and Xxx->Pro type. Of 15 single mutants, several displayed significant increases in thermal stability, whereas most mutants showed minor effects. All mutations with non-marginal effects on stability clustered in a limited, surface-exposed region of the enzyme, indicating that this region is involved in a partial unfolding process that triggers irreversible thermal inactivation (aggregation). A double mutant containing two stabilizing mutations in this region (G188A, A234P) displayed a 10-fold increase in half-life at 57°C and a 4.2°C increase in apparent Tm. These results show that entropic stabilization works well for ChiB and they pinpoint a region whose unfolding may be crucial for the kinetic stability of this enzyme.

Keywords: chitinase/proline/thermal stability/unfolding


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chitin, a 1,4-ß-linked polymer of N-acetyl-ß-D-glucosamine (GlcNAc), is the second most abundant polymer in nature (after cellulose). It is a major structural component of the exoskeleton of insects and crustaceans and it occurs in the cell walls of a variety of fungi. Chitinases are of biotechnological interest for several reasons. One of them is that they may be used to convert chitin-containing biomass into chito-oligosaccharides, which have various potentially interesting applications (Tharanathan and Kittur, 2003Go). The Gram-negative bacterium Serratia marcescens is one of the most effective degraders of chitin and it secretes several chitinases (Watanabe et al., 1997Go). One of the best characterized chitinases from S.marcescens is an exochitinase called chitinase B (ChiB). ChiB is a modular enzyme with a catalytic domain and a domain involved in substrate binding (van Aalten et al., 2000Go) (Figure 1).



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Fig. 1. Structure of ChiB. The C{alpha} atoms of mutated residues are indicated by balls, labelled with the residue name. The side chain of the catalytic acid, Glu144, is shown as sticks. The part of the protein marked ChBD is the chitin-binding domain.

 
Efficient industrial use of chitinases requires these enzymes to be sufficiently stable. Stability is a beneficial parameter for biocatalysis (for instance, stable enzymes allow higher process temperatures) and the prolonged lifetime of the catalyst reduces process costs. This makes stability an important parameter to improve during the development of industrial enzymes.

Studies of the thermodynamic stability of small proteins and their mutated variants have provided some fundamental insights into factors that determine protein stability (Fersht and Serrano, 1993Go; Matthews, 1995Go; Vieille and Zeikus, 2001Go). From these studies, several important strategies for protein stabilization have arisen. Successful stabilization strategies include the introduction of disulphide bridges (Matsumura et al., 1989Go; Mansfeld et al., 1997Go) and the optimization of helices and helix caps (Serrano et al., 1992Go; Blaber et al., 1993Go). Another strategy, termed ‘entropic stabilization’ (Matthews et al., 1987Go), involves the introduction of mutations that reduce the conformational freedom of the unfolded state. This may be achieved by the introduction of disulphide bridges, but also by simpler types of mutations that reduce the conformational freedom of the residue in question. These simpler mutations concern removal of glycine residues (thus restricting the {phi} and {psi} dihedral angles) and introduction of proline residues (thus strongly restricting the {phi} angle) (Matthews et al., 1987Go; Hardy et al., 1993Go; Van den Burg et al., 1998Go; Bryan, 2000Go; Watanabe et al., 2000Go).

ChiB unfolds irreversibly at higher temperatures, meaning that only the kinetic (and not thermodynamic) stability of this enzyme may be addressed. It has been suggested that kinetic stability is determined by the rate of a partial (as opposed to global) unfolding event that triggers an irreversible process, e.g. aggregation or, in the case of proteases, autolysis (Vriend and Eijsink, 1993Go; Vriend et al., 1998Go; Zhao and Arnold, 1999Go; Bryan, 2000Go; Machius et al., 2003Go). Stabilizing mutations are most likely to be effective if they affect these stability-determining parts (‘weak spots’) of the protein (Mansfeld et al., 1997Go; Vriend et al., 1998Go; Machius et al., 2003Go). Thus, it is useful to start a mutagenesis strategy aimed at improvement of kinetic stability by a search for crucial unfolding regions in a protein (Eijsink et al., 1995Go; Machius et al., 2003Go). It has been suggested that limited proteolysis may be used for the identification of such regions, since this technique may reveal areas that are particularly flexible and/or prone to local unfolding (Fontana, 1988Go; Arnold et al., 1996Go, 1999; Fontana et al., 1997Go).

Here we describe attempts to stabilize ChiB by introducing prolines and removing glycines. Since limited proteolysis experiments did not reveal any potential ‘weak spot’ (see below), mutations were introduced all over the protein. Mutations were designed using semi-automated design methods implemented in the molecular modelling package of WHAT IF (Vriend, 1990Go). The results show that this strategy worked well in the sense that more stable variants of ChiB were obtained. On the other hand, many of the mutations had no or hardly any effect on ChiB stability. Possible explanations for these observations are discussed.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Mutant design

Mutations were designed with the WHAT IF automatic mutation prediction options. These options predict how well each of the 20 possible amino acids fit at specific positions in the protein and calculate a quality parameter for each fit. Special options exist for scanning proteins to find good positions for prolines, cysteines or alanines. These options are all based on the position-specific rotamer technology as originally described by Jones and Thirup (Jones and Thirup, 1986Go) and Filippis et al. (Filippis et al., 1994Go). For each putative rotamer, the fitness in the local environment is calculated using the same scoring functions as used in the WHAT IF homology modelling procedure (Chinea et al., 1995Go).

Note that the WHAT IF options used in this study do not explicitly predict thermostabilizing mutations, but merely mutations that are likely to be compatible with the structure. Other knowledge is needed to select those mutations that are likely to stabilize the protein among all predicted mutations. In this case, for reasons explained above, we focused on mutations of the Gly->Ala and Xxx->Pro type.

All automatically detected mutations were visually inspected and a further selection of mutations was made on the basis of the following criteria:

1. The residue is surface located. This criterion was based on the notion that irreversible thermal inactivation is likely to be governed by unfolding processes that primarily involve surface-located regions of the protein. Also, surface-located residues make fewer contacts, reducing the chance for mutation design errors and ‘side effects’ (see next criterion).

2. The mutation does not lead to removal of favourable interactions or to introduction of unfavourable (steric hindrance) interactions.

3. The mutation does not affect the catalytic centre of the enzyme.

This manual process yielded 15 mutations, which were all made. Some details of the structural context of the mutated residues are provided in Table I. The pictures presented in this report were made with PyMOL (DeLano Scientific, San Carlos, CA, USA).


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Table I. Structural context of mutated residues
 
Limited proteolysis

Thermolysin (Roche Molecular Biochemicals, Basel, Switzerland) was dissolved to 10 mg/ml in 50 mM Tris–HCl, pH 8.0, 10 mM CaCl2 and stored in aliquots at –20°C. For proteolysis, 20 µg of purified ChiB (see below) were incubated with various amounts of thermolysin in 50 mM Tris–HCl, pH 8 at various temperatures (reaction volume 100 µl). Reactions were stopped by adding 50 mM EDTA to a final concentration of 5.5 mM, followed by addition of one volume of an SDS–PAGE sample buffer containing 62.5 mM Tris–HCl, pH 6.8, 10% (v/v) glycerol, 2% (w/v) SDS, 5% (v/v) ß-mercaptoethanol and 0.025% (w/v) bromophenol blue. After incubation of the samples at 95°C for 4 min, SDS–PAGE was conducted essentially according to Laemmli (Laemmli, 1970Go), using a 12% seperating gel, in a Bio-Rad Mini Protean II system.

Mutagenesis

ChiB mutants were made with the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) essentially as described by the manufacturer. DNA sequences of mutated gene fragments were sequenced with an ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit and an ABI PRISM 377 DNA Sequencer (Perkin-Elmer Applied Biosystems, Foster City, CA). Mutated gene fragments were used to construct variants of the wild-type production plasmid pMAY2-10 (Brurberg et al., 1996Go). Wild-type and mutant chib genes were expressed in Escherichia coli DH5{alpha} (Life Technologies, Rockville, MD, USA) and ChiB variants were purified as described earlier (Brurberg et al., 1996Go).

Catalytic activities of ChiB variants were determined using the substrate 4-methylumbelliferyl-ß-D-N,N'-diacetylchitobioside [4MU-NAG2 (Sigma-Aldrich, St. Louis, MO), an analogue of the natural substrate (NAG)3], as described previously (Brurberg et al., 1996Go). Protein concentrations were determined with the Protein Assay solution from Bio-Rad, based on staining with Coomassie blue.

Stability (t1/2)

Purified ChiB variants (20 nM) were incubated in 50 mM citrate-phosphate buffer, pH 6.2 (Stoll and Blanchard, 1990Go) and 0.1 mg/ml BSA at 57°C (BSA was added to prevent unspecific binding of proteins to the test tube). At regular intervals (depending on the enzyme tested), 25 µl of the enzyme solution was transferred to ice. After the last sample had been removed from the water bath, residual catalytic activity was tested. Five microlitres of the 20 nM enzyme reaction was transferred to a test tube containing 95 µl of a solution of 68 µM substrate (4MU-NAG2) and 0.1 mg/ml BSA in 50 mM citrate-phosphate buffer, pH 6.2. After 10 min, the reaction was stopped with 1.9 ml of 0.2 M Na2CO3 and the fluorescence measured with a Hoefer DyNA Quant 200 fluorimeter. Thermal inactivation followed first-order kinetics and plots of relative remaining activity versus t were fitted to a first-order exponential curve. The fit normally gave curves with a correlation higher than 0.99. Half-lives were calculated using the equation t1/2 = 0.6931/kapp, where kapp is the apparent rate constant of the reaction. The t1/2 values presented are average values derived from three independent measurements; the error margins reported correspond to one standard deviation.

Circular dichroism (CD) measurements

CD measurements were performed using a Jasco J-810 spectropolarimeter (Jasco) calibrated with ammonium d-camphor-10-sulfonate (Icatayama Chemicals, Tokyo, Japan). The experimental conditions were: protein concentration, 0.1 mg/ml; buffer, 20 mM sodium phosphate, pH 8.0; pathlength, 1 mm; wavelength, 222 nm; scan rate, 1°C/min. The apparent fraction of unfolded protein (Fapp) was calculated using the equation Fapp = (YYN)/(YUYN), where Y is the observed signal and YU and YN are the formulae of the linear baselines of unfolded and native protein, respectively [see Pace (Pace, 1990)Go for details on the calculations]. Apparent Tm values were calculated from a plot of the first derivative of the two-state unfolding curve.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Limited proteolysis

In an attempt to identify unstable regions (‘weak spots’) of ChiB, we conducted a series of limited proteolysis experiments with thermolysin. The rate of proteolysis increased upon increasing the incubation temperature from 50 to 60°C (Figure 2). At 50°C, no proteolysis was detected, indicating that ChiB is stable. At 60°C, however, most of the enzyme was degraded during the 20 min incubation period. Thus, ChiB seems to unfold, and become more prone to proteolysis, in the 50–60°C interval (see also the stability measurements described below).



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Fig. 2. SDS–PAGE analysis of limited proteolysis of ChiB with thermolysin. The pictures show three series of six samples each. Each series represents a gradient in incubation temperature from 50 (left) to 60°C (right) with a 2°C interval. The series are separated by lanes with marker proteins (molecular masses are indicated in kDa). The three series represent different incubation times: 0 (series A), 10 (series B) or 20 min (series C). The arrows indicate ChiB bands that clearly show a loss of intensity, demonstrating the occurrence of proteolysis. Note that the loss of intensity in the ChiB band is accompanied by the appearance of low molecular mass material at approximately the same position as the 14.4 kDa marker

 
The limited proteolysis experiments with thermolysin (and other broad-specificity proteases; data not shown) did not reveal intermediate products and thus did not reveal any information as to where on the protein the proteolytic process could start. Apparently, a first cleavage of ChiB leads to destabilization and rapid complete hydrolysis of the molecule.

Because we had no clue as to which parts of ChiB could be involved in the partial unfolding processes that lead to thermal inactivation, mutations were deliberately scattered over the complete ChiB molecule. Thus, we optimized the chance of hitting a ‘weak spot’ with the mutations.

Production and characterization of mutants

All mutants made in this study could be produced in normal amounts under standard conditions and purified using the standard procedure for ChiB. The half-lives and specific activities of the purified ChiB variants are presented in Table II.


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Table II. Half-lives and specific activities of mutant enzymes
 
Of the 15 initial mutants, only four displayed changes in t1/2 of >50%: W252P, G188A and A234P raised t1/2 ~1.5-, ~2- and ~5-fold, respectively, whereas E253P reduced t1/2 by ~3-fold. The mutations that had these larger effects on stability are clustered in a limited part of the ChiB molecule, both in structure (Figure 1) and in sequence (Figure 3). A double mutant containing the two most stabilizing single mutations, G188A and A234P, showed a 10-fold increase in half-life. Interestingly, the G188A mutation had a beneficial effect on specific activity, meaning that the G188A mutant and the stable G188A_A234P mutant are not only more stable, but also more active than wild-type ChiB.



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Fig. 3. Graphical representation of the stability of ChiB variants. Each spot represents the half-life of a mutant (in min), positioned in the graph according to the sequence number of the mutated residue. Spots representing mutants displaying the most prominent mutational effects are labelled. The half-life of the wild-type enzyme was 26 ± 4 min.

 
In addition to the 15 initial single mutants, two other ChiB variants were made. The A234G variant was constructed to check for the importance of main chain rigidity at this position where the introduction of rigidity had been found to be clearly beneficial (see the A234P mutant). As expected, the introduction of glycine led to reduced stability, but the effect was modest (reduction in t1/2 at 57°C from 26 to 16 min).

Highly solvent-exposed hydrophobic residues such as Trp252 are often observed in glycoside hydrolases because they contribute to the binding of longer sugar chains (van Aalten et al., 2000Go; Uchiyama et al., 2001Go). Such highly exposed hydrophobic residues do not seem favourable for stability and, thus, we wondered whether the stabilizing effect of the W252P mutation could be a result of ‘non-entropic’ effects (see below for further discussion). Therefore, as a control, we replaced Trp252 with a polar, charged residue, glutamate, which we assumed to be beneficial for stability. Indeed, the W252E mutant showed a considerable increase in stability, surpassing that of the W252P mutant (Table II).

Melting curves of ChiB and its G188A_A234P mutant (Figure 4) showed an irreversible transition that led to almost complete loss of the CD signal at 222 nm and to aggregation. The melting curves permitted the determination of apparent Tm values of 59.9 and 64.1°C, respectively.



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Fig. 4. Thermal unfolding monitored by CD. The apparent fraction of unfolded protein Fapp was derived from measurements of the CD signal at 222 nm at varying temperatures, as described in Materials and methods. The curves shown represent the wild-type enzyme (solid line) and the G188A_A234P double mutant (dotted line), at pH 8.0.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The present study shows that the semi-automated procedures for mutant design implemented in WHAT IF worked well in the sense that all mutant proteins were well expressed and reasonably stable. Apparently, we did not make major design errors leading to proteins that cannot fold or that are highly unstable. The application of these procedures to design Gly->Ala and Xxx->Pro mutations in ChiB indeed resulted in stabilization of the enzyme by up to a factor of 10.

In the design of mutants, we aimed at selecting mutants with relatively ‘clean’ effects. That is, we discarded mutations which we expected to have major effects in terms of removal or introduction of interactions. We cannot exclude though that seemingly minor changes in beneficial or unfavourable interactions contribute to some of the observed mutational effects, for example, in the case of the A234P and G188A mutations.

The Cß of Ala234 is highly solvent exposed and has few contacts. Its closest contact is with the C{delta} of the solvent-accessible hydrophobic residue Leu438 (distance 3.84 Å). A model of the A234P mutant shows that the introduction of proline leaves the hydrophobic contact between residues 234 and 438 intact, and that a new contact (Pro234C{delta}–Lys362C{epsilon}; 3.5 Å) is created. Since such hydrophobic contacts at the surface may be beneficial for stability (Van den Burg et al., 1994Go; Machius et al., 2003Go) they may contribute to the stabilizing effect of the A234P mutation. Likewise, the destabilizing effect of the A234G mutation may in part be a result of the loss of the contact between Ala234Cß and Leu438C{delta}.

The Cß of Ala188 is expected to have a hydrophobic contact with the C{delta}2 of Leu216 (3.69 Å), which may contribute to the stability of the G188A mutant. Interestingly, and unexpectedly, the stabilizing G188A mutation also yielded an ~2-fold increase in activity. Leu216 is next to Asp215, which is known to be important in catalysis (van Aalten et al., 2001Go). One might thus speculate that small structural adjustments of and around Asp215 account for the beneficial effect of the G188A mutation on activity.

In general, one could expect that at least some of the rigidifying mutations would reduce catalytic efficiency, based on the general notion that enzyme flexibility is important for catalysis. In some cases, small reductions in activity were indeed observed (Table I), but the general picture is that the mutations had only minor effects on catalytic activity. This was expected since mutations located close to the catalytic centre were discarded during the mutant design process.

The two mutations present in the most stable ChiB variant (G188A, A234P) as well as all other mutations with larger effects on stability (A234G, W252P, W252E, E253P) are clustered on one side of the ChiB molecule. Apart from Gly188, all these mutated residues are located in an exposed surface meander running from residue 230 to 265 (Figure 5). Gly188 is located just in front of an {alpha}-helix on which the surface meander packs (there are many contacts between residues 189–190 and residues 239–243; Ala189Cß also has a contact with Phe263C{zeta}). Thus, it is conceivable that mutation of residue 188 somehow affects the 230–265 surface meander. The presence of a region in which mutations have relatively strong effects on stability can be explained by assuming that the unfolding process that leads to thermal inactivation involves only part of the protein. Only mutations involved in these stability-determining parts will have noticeable stability effects, as observed in protein engineering work on proteases and amylases (Vriend and Eijsink, 1993Go; Vriend et al., 1998Go; Zhao and Arnold, 1999Go; Machius et al., 2003Go). The present results suggest that the partial unfolding process that precedes irreversible thermal inactivation (aggregation) of ChiB involves the 230–265 meander, which thus may be a ‘weak spot’. In this respect it is interesting to note the large stabilizing effect of the W252E mutation. One might speculate that the reduction of surface hydrophobicity in an area that supposedly unfolds relatively easily will lead to a decreased tendency to aggregate.



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Fig. 5. The 230–265 surface meander in ChiB. The picture shows a C{alpha} trace of ChiB where the thicker line indicates the region 230–265. The modelled side chains of Ala188 and Pro234 as well as the side chain of Trp252 are also shown.

 
Obviously, the fact that so many mutations have little effect on stability should not only be explained by assuming that these mutations are located in parts of the protein that are not involved in the thermal inactivation process. The clearest example in this study is the A189P mutation, which by all criteria (located in the N-terminal turn of a helix; located next to Gly188) should be stabilizing, but which slightly destabilized the enzyme. In this case, the design process indicated two steric clashes involving Phe263 and Leu265 (Pro189C{gamma}–Phe 263C{zeta}, 3.03 Å; Pro189C{delta}–Leu265C{delta}1, 3.04 Å). These problems were judged to be surmountable by minor local structural adjustments, but they may in fact outweigh the beneficial effect of introducing a proline.

In general, it cannot be excluded that the designed mutations have small negative side effects that outweigh the beneficial effects of the mutation and that have been overlooked in the design process. It seems unlikely though that this explains the fact that all mutations outside the 188–252 region had such small effects on the stability of ChiB. We thus conclude that ChiB has a ‘weak spot’ (Vriend and Eijsink, 1993Go) or ‘nucleation site for unfolding’ [a term derived from helix–coil theory as explained by Machius et al. (Machius et al., 2003Go)]. As shown previously for proteases (Eijsink et al., 1995Go) and amylases (Machius et al., 2003Go) and as shown in this study, the identification and mutation of such a nucleation site for unfolding is a prerequisite for successful attempts to increase the kinetic stability of a protein.


    Acknowledgements
 
We thank Dimitrios Mantzilas, Department of Biochemistry at the University of Oslo, for help with the CD measurements and Alexei V.Finkelstein for helpful discussions. This work was supported by grants from the Norwegian Research Council (122004/130 and 140440/130) and by EU grant BIO4-CT-960670.


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 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received June 18, 2003; revised September 3, 2003; accepted September 12, 2003.