Analysis of a conserved hydrophobic pocket important for the thermostability of Bacillus pumilus chloramphenicol acetyltransferase (CAT-86)

H. Chirakkal, G.C. Ford and A. Moir,1

Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, UK


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Site-directed mutagenesis was carried out on Bacillus pumilus chloramphenicol acetyltransferase (CAT-86) to determine the effects of substitution at a conserved hydrophobic pocket identified earlier as important for thermostability. Mutations were introduced that would substitute residues at consensus positions 33, 191 and 203 in the enzyme, both individually and in combination. Two mutants, SDM1 (CAT-86 Y33F, A203V) and SDM5 (CAT-86 A203I), were more thermostable than wild-type and two mutants, SDM4 (CAT-86 I191V) and SDM7 (CAT-86 A203G), were less stable. Reconstruction of the residues of this hydrophobic pocket to that of a more thermostable CAT-R387 enzyme pocket (as a Y33F, I191V, A203V triple mutant) increased the thermostability of the enzyme above the wild-type, but its stability was less than that of SDM1 and SDM5. The Km values of the mutant enzymes for chloramphenicol and acetyl-CoA were essentially unaltered (in the ranges 15–30 and 26–35 µM respectively) and the specific activity of purified enzyme was in the range 270–710 units/mg protein. The possible effects of the amino acid substitutions on the CAT-86 structure were determined by homology modelling. A reduction in conformational strain and optimized hydrophobic interactions are predicted to be responsible for the increased thermostability of the SDM1 and SDM5 mutants.

Keywords: CAT-86/chloramphenicol acetyltransferase/hydrophobic pocket/site-directed mutagenesis/thermostability


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Thermostable proteins are of interest for several reasons. They can be used to improve the efficiency of many industrial processes and provide insight into the general mechanisms of protein folding and stabilization (Vogt and Argos, 1997Go). The thermostability of an enzyme is a function of the enzyme's stabilizing forces such as hydrogen bonding, hydrophobic interactions, ionic bonding, metal binding and disulphide bridges. There is a delicate balance between these stabilizing interactions and destabilization due to the loss of conformational entropy of the folded protein (Matthews, 1987Go).

Among the stabilizing factors, hydrophobic interaction is considered as a dominant force in structural stability (Matthews, 1993Go). It is believed to provide the energy required for proteins to fold in aqueous solutions. As protein cores are typically hydrophobic, increased packing efficiency is often correlated with increased hydrophobicity (Sandberg and Terwilliger, 1989Go). Both site-directed (Ishikawa et al., 1993Go) and random (Turner et al., 1992Go) mutagenesis studies have demonstrated the potential to increase thermostability by filling hydrophobic cavities in the folded structure of ribonuclease HI and chloramphenicol acetyltransferase (CAT-86), respectively.

Salt bridges represent another type of common interaction that stabilizes proteins. Studies of the molecular mechanisms of irreversible thermal denaturation of {alpha}-amylase enzymes from Bacillus licheniformis, Bacillus amyloliquefaciens and Bacillus stearothermophilus by Tomazic and Klibanov (1988) suggested that the higher thermostability of the enzyme from B.licheniformis was due to salt bridges involving a few specific lysine residues. These stabilizing electrostatic interactions reduce the extent of unfolding of the enzyme molecule at high temperatures making it less prone to forming incorrect structures and thus decreasing the overall rate of irreversible thermal denaturation. Karshikoff and Ladenstein (1998) suggested that electrostatic interactions are a common factor regulating the thermal tolerance of proteins from thermostable organisms. Based on the studies on the hexameric glutamate dehydrogenases from hyperthermophiles Thermococcus litoralis and Pyrococcus furiosus, Vetriani et al. (1998) suggested that the formation of an extensive ion-pair network may provide a general strategy for manipulating enzyme thermostability for multisubunit enzymes. Vogt and Argos (1997) noticed, from their studies on 16 protein families, a consistent increase in the number of hydrogen bonds and in polar surface area fraction with increased thermostability.

Matthews et al. (1987) proposed that stability of a protein can be increased by selected amino acid substitutions that decrease the configurational entropy of unfolding. A glycine, which lacks a ß-carbon, has more backbone flexibility than alanine, with a ß-carbon. Therefore, more energy is required to restrict the conformation of glycine than other residues during the transfer from unfolded to folded state. It is well known that replacements with proline residues reduce the conformational degree of freedom in the main polypeptide chain and thus can increase protein stabilization (Watanabe and Suzuki, 1998Go; Zhu et al., 1999Go). {alpha}-Helix stabilization has been observed in some thermostable enzymes (Menéndez-Arias and Argos, 1989Go). Comparison of 3-phosphoglycerate kinase enzymes of B.stearothermophilus and yeast have shown that several {alpha}-helix lysine residues that are present in the yeast enzyme are replaced by glutamine, which is a better helix stabilizer (Davies et al., 1993Go). In some cases disulphide cross-links have been found to have an important role in protein stability. Most of the early efforts to increase thermostability of proteins by site-directed mutagenesis focused on the introduction of non-native disulphide cross-links (Matthews, 1987Go). This has proved useful in certain instances but not others.

Chloramphenicol acetyltransferase (CAT, EC 2.3.1.28) is an intracellular, trimeric enzyme that is responsible for chloramphenicol (Cm) resistance in bacteria (Shaw, 1983Go). Several naturally occurring CAT variants have been described in the literature; the amino acid sequence identity of these enzymes ranges from 30 to 70%. All CAT variants have a molecular mass of about 25 kDa. Most of the information available on structure, specificity and mechanism of the CAT reaction comes from a type III enzyme, CAT-R387. Crystal structures of the binary complexes of CAT-R387 with acetyl-CoA and with Cm have been determined (Leslie et al., 1988Go; Leslie, 1990Go). A six-stranded mixed parallel and antiparallel ß-pleated sheet structure forms the backbone of the monomeric structure and five {alpha}-helices are packed against one side and the end of this sheet to form an arrangement that has been described as an open-faced sandwich. In the trimer, the subunits associate so that the six-stranded ß-pleated sheet of each subunit extends across the subunit interface via the ßH strand of the adjacent subunit. A total of eight intersubunit main-chain hydrogen bonds result from this ß-sheet extension, which presumably contributes to the stability of the CAT-R387 trimer (Leslie, 1990Go). Within CAT-R387, Cm binds in a deep pocket located at the boundary between adjacent subunits of the trimer, such that the majority of residues forming the binding pocket belong to one subunit while the catalytically essential His195 belongs to the adjacent subunit. The C-terminal residues are important in production of soluble, enzymatically active CAT, suggesting the importance of the carboxy terminal {alpha}5-helix in proper folding of the enzyme (Robben et al., 1993Go, 1995Go). A second site mutation, resulting in an L145F substitution, restored function to C-terminally truncated mutants of CAT R387 (Van der Schueren et al., 1998Go).

The CAT family of enzymes show considerable variation in thermal tolerance. For example CAT-R387 retains more than 70% activity after 1 h at 70°C (Lewendon et al., 1988Go). The CAT enzymes encoded on plasmids pSCS6 and pSCS7 are also relatively thermostable (Cardoso and Schwarz, 1992Go). These enzymes retained 55–60% of their original activity after incubation at 70°C for 15 min. By contrast, CAT-86 of Bacillus pumilus is a thermolabile enzyme and it is inactivated within a few minutes upon exposure to 70°C (Laredo et al., 1988Go; Turner et al., 1992Go). CAT-86 is an inducible, type II chloramphenicol acetyltransferase encoded by a chromosomal gene (cat-86) in B.pumilus. The nucleotide sequence of cat-86 was determined by Harwood et al. (1983) and like the other CAT enzymes it is active as a homotrimer. CAT-86 has been previously used as a model enzyme to study thermostability (Turner et al., 1992Go). Eighteen mutants were isolated by in vivo random mutagenesis, all with the same mutation, a C to T transition at base 584 of the coding sequence (changing the residue 203 from A to V). The mutant enzyme (CAT-86 A203V) was found to be more thermostable than the wild-type enzyme; it retained 50% of its original activity when incubated at 60°C for 5 min, whereas the wild-type enzyme lost 50% of its activity in 5 min at 55°C. The mutation did not change the catalytic properties of the enzyme as the Km values for chloramphenicol and acetyl-CoA for both the wild-type and mutant enzyme were identical (Turner et al., 1992Go). Their modelling of the conserved hydrophobic pocket involving residue 203 suggested that additional methyl groups could be accommodated in the pocket, although there was not a large cavity in the structure.

In this study we attempted to elucidate further the role of this conserved hydrophobic pocket in the thermostability of the enzyme. The amino acid residues interacting with residue 203 were identified and substituted. Changes in thermostability are discussed with respect to the predicted 3D structure of the protein.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial strains and plasmids

Construction of vectors and expression experiments were carried out in Escherichia coli JM109 (DE3). E.coli expression vector pTB361 was obtained from Dr Peter Barth (Zeneca Pharmaceuticals, UK). Plasmid pPL608 (Williams et al., 1981Go), carrying cat-86, was donated by P.S.Lovett (University of Maryland, Catonsville, MD).

Site-directed mutagenesis

Site-directed mutagenesis was carried out according to Higuchi (1990). First, two overlapping halves of the gene were synthesized separately using one mutagenic primer and one flanking primer. These primary PCR products were then used as template DNA for the secondary PCR to synthesize the complete gene. The template DNA and oligonucleotide primers used for the construction of each site-directed mutant are listed in Table IGo. The full-length DNA was synthesized by a second PCR using the `outside' primers and a mixture of the primary PCR products as template DNA. All PCR products were purified by gel excision.


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Table I. Primers used for site-directed mutagenesisa
 
Cloning of mutant cat-86 DNA in pTB361 and transformation of E.coli JM109 (DE3)

All routine molecular biology experiments were carried out according to Sambrook et al. (1989). The purified secondary PCR products were digested with NdeI and BglII and ligated downstream of the T7 promoter in the DNA of pTB361, which had been linearized by the same enzymes. The ligation mixtures were used for transforming E.coli JM109 (DE3) by electroporation. The transformants were selected directly on LB agar containing chloramphenicol (20 µg/ml). Plasmids were extracted from the transformants and their restriction pattern checked by agarose gel electrophoresis. The complete cat-86 gene in each plasmid was sequenced to confirm that the expected mutations were the only base changes present.

Overexpression and purification of the mutant enzymes

Enzymes were prepared from E.coli JM109 (DE3) carrying appropriate plasmids. Cultures (50 ml) induced with 0.4 mM IPTG were used for enzyme purification, using affinity chromatography on chloramphenicol caproate–agarose (Turner et al., 1992Go). The fractions containing enzyme activity were pooled and dialysed against 50 mM Tris–HCl (pH 7.8), 1 mM EDTA to remove salt and chloramphenicol. The purified enzymes were stored on ice and used within 24 h.

Enzyme assays

The spectrophotometric assay of Shaw (1975) was used to measure CAT-86 activity. The assay buffer was 50 mM Tris–HCl (pH 7.8), 1 mM EDTA. Protein was estimated by the method of Bradford (1976). A unit of CAT activity is defined as the number of micromoles of chloramphenicol acetylated per minute per milligram of protein at 30°C. Thermal inactivation studies were performed as described by Turner et al. (1992). Samples (120 µl, at 10 µg/ml in assay buffer) of enzyme were heated in small (70x8 mm) Pyrex tubes and then cooled quickly on ice. The remaining activity was assayed immediately at 30°C.

Molecular modelling

Molecular modelling of CAT-86 was carried out using the molecular graphics program MidasPlus (Ferrin et al., 1988Go; Huang et al., 1991Go) running on an SGI VGX Indigo workstation and FORTRAN 77 programs compiled under Salford FORTRAN 77 and run on a personal computer.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Mutant enzymes and their properties are listed in Table IIGo. Amino acids in the CAT sequence were numbered according to the standard CAT alignment (Murray et al., 1988Go). The amino acid changes made in this study fall into two categories. The first category of mutants (A203I, A203L and A203G) was created to study whether the thermostability of the enzyme can be enhanced further by introducing an amino acid with a larger hydrophobic side chain at position 203. The second category of amino acid substitutions was intended to recreate elements of the hydrophobic pocket of CAT-R387 in CAT-86. Amino acid residues of CAT-86 at all three positions 33, 191 and 203 were substituted by the amino acids in the corresponding sites of CAT-R387, both individually and in combination. All mutants were made by site-directed mutagenesis, expressed in E.coli and purified by affinity chromatography. The purity of the proteins was checked by SDS–PAGE and was >95%. Specific activity and Km were measured (Table IIGo). None of the changes appeared to have dramatically altered the enzyme activity. Whether the twofold increase in the specific activity of SDM1 and SDM8 is significant is not clear; it may reflect the increased stability of these proteins during purification. The rate of irreversible thermal inactivation of various CAT proteins was measured at 55°C (Table IIGo) and 60°C (Figure 1Go). Comparison of the half-lives of the proteins at 60°C provided an estimate of the stabilizing energy difference between the wild-type and mutant proteins ({Delta}G{ddagger}, as proposed by Perutz and Raidt, 1975).


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Table II. Comparison of enzyme propertiesa
 


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Fig. 1. Rate of thermal inactivation of the purified WT and mutant CAT-86 enzymes at 60°C. All values are averages of three independent experiments.

 
Effect of substitution at position 203 on thermostability

Of the three mutants (A203I, A203L and A203G) the enzyme with the A203I substitution (SDM5) proved to be the most thermostable at both 55 (Table IIGo) and 60°C (Figure 1bGo; {Delta}G{ddagger} = –4.6 kJ/mol). In contrast, the enzyme with an A203G substitution (SDM7) appeared to be very thermolabile. Incubation for 15 min at 55°C resulted in the loss of ~95% of its activity. At 60°C, 98% of enzyme activity was lost in 5 min and the activity was undetectable after 10 min. The A203L substitution (SDM6) gave unusual properties; the protein showed a higher thermostability at 55°C than the wild-type enzyme, but this substitution significantly decreased the stability of the enzyme at 60°C.

Effect of substitution at positions 33 and 191 on thermostability

Mutants SDM2 and SDM4 carry the Y33F and I191V substitutions, respectively, in CAT-86. Mutants SDM1 and SDM3 also contain these changes, but in a protein that already contains the A203V alteration. The stability of these four mutants is compared with the controls in Figure 1Go and Table IIGo. SDM1 (F, I and V at positions 33, 191 and 203, respectively) showed higher stability than all the other mutants. After incubation for 30 min at 55°C this mutant retained about 80% of its original activity (Table IIGo) and it retained about 50% activity after heating at 60°C for 30 min (Figure 1aGo). SDM4 (YVA) appeared to be the least thermostable mutant; it retained only 4% of its activity after incubation for 15 min at 55°C. No detectable activity remained after 10 min at 60°C. SDM2 and SDM3 showed intermediate levels of stability.

Altering the hydrophobic pocket in CAT-86 towards that of R387

The CAT-86 enzyme with the CAT-R387 hydrophobic pocket (Y33F I191V A203V; SDM8) showed higher thermostability at 55°C than the WT CAT-86 enzyme or the A203V mutant. At 60°C, the increase in stability was much less pronounced (Figure 1Go). This mutant could also be compared with those differing by a single change at one of the three positions. The Y33F substitution would have helped stabilize the enzyme as it did for other mutants. The I191V substitution did not stabilize the SDM3 mutant (I191V, A203V) mentioned earlier.

Modelling of CAT-86 structure

The molecular modelling of CAT-86 was carried out based on the coordinates of CAT-R387, which shares 79 out of 220 identical residues. Alignment of the R387 and the CAT-86 sequence (Steffen and Matzura, 1989Go) suggests that many of the substitutions that occur in the CAT-86 protein sequence with respect to the R387 sequence are relatively conservative. The conserved quaternary structure, reaction mechanism and active site residues suggested that the R387 structure could serve as a basis on which the CAT-86 structure could be modelled. Figure 2aGo shows this structure in the region of residue A203. Amino acid residues different in CAT-86 and CAT-R387 sequence that lie within a 10 Å distance from the 33, 191 or 203 residues in the CAT-R387 structure were identified by a FORTRAN program. Within the pocket, residue 203 interacts with five side chain groups all from the same subunit. These residues are F33, L35, V191, V193 and C198. The residues that comprise this pocket are conserved as hydrophobic residues in all CAT sequences determined to date with the exception of residue 35 (Turner et al., 1992Go). Both CAT-86 and CAT-R387 share the same residues at positions 35, 193 and 198. Therefore, in our studies we substituted the residues F33, V191 and V203 of the CAT-R387 with Y, I and A, respectively, to mimic the hydrophobic pocket of CAT-86.



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Fig. 2. Computer graphic representation of the modelled structure of CAT-86 proteins around the hydrophobic region involving the 203 residue. The green and red strands represent regions from two different monomers of the trimeric protein. The residues to be altered (33, 191 and 203) are coloured white. (a) CAT-R387 enzyme (YIA), showing the region of the hydrophobic pocket. Four additional residues are identified (V193, H195, F206 and I207) to orient the image. (b) Model illustrating the contacts made by the O{eta} atom of Y33 in the CAT-86 A203V mutant. These are with the main chain oxygen and carbonyl carbon residue 199, C{alpha} and main chain nitrogen of residue 200 and C{gamma} of residue 203. (c) A close-up view of the potential interactions between residues in SDM6 (YIL). The close distances between L203 and C or O atoms in Y33, I191 and V193 are indicated by yellow dotted lines. Additional residues labelled for orientation are H202, R205 and I207. The molecular graphics images were produced using the MidasPlus program (Huang et al., 1991Go).

 
Substitution at position 33 by phenylalanine

One of the important observations made from the analysis of thermostability of these site-directed mutants was the increase in thermostability of CAT-86 when residue 33 is F rather than Y (Figure 1aGo). The increase in thermostability was very dramatic when the substitution was made in conjunction with the A203V mutation (SDM1), so that the three residues in the hydrophobic pocket are F33, I191 and V203. A less dramatic increase was observed when the combination of residues was F, I and A. On the basis of these comparisons we speculate that in CAT-86, the hydroxyl group of Y33 causes steric hindrance resulting in a decreased conformational stability of the hydrophobic pocket. Molecular modelling revealed that the O{eta} atom of Y33 made close contacts with residue 199, 200 and 203 as shown in Figure 2bGo. The distance of these interactions are 2.5 Å with the main chain O and 2.7 Å with the carbonyl carbon of residue 199, 3.3 Å with the C{alpha} and 3.1 Å with the main chain N of residue 200 and 3.1 Å with the C{gamma} of residue V203. The O{eta} atom of Y33 lies below the peptide plane of residue 199–200 and makes an out-of-plane angle of about –90° with the C=O bond. This angle is unfavourable for a hydrogen bond between O{eta} (Y33) and O (199) (reviewed by Baker and Hubbard, 1984). The close contacts of O{eta} (Y33) are within van der Waals separation and are likely to cause steric hindrance in the pocket. It is likely that these contacts are not favourable to the stability of the protein, but they may have been compensated by additional favourable van der Waals interactions. The relative thermostability data for the proteins A203V and SDM1 (which combines Y33F and A203V substitutions) in Table IIGo and Figure 1Go suggest that although the extra hydroxyl group is not completely destabilizing the enzyme, F33 gives greater stability than does Y33, especially as additional methyl groups are added to the pocket.

Substitutions at positions 191 and 203

The C{delta}1 of I191 in SDM5 would make contact with C{delta}1 of I203 (centre to centre distance 4.0 Å); this is the only additional interaction made by introducing I in place of V at position 203. Although the A203L substitution in the wild-type enzyme contributes the same number of methyl groups as the A203I substitution, the stability of the enzyme was much lower (Figure 1Go). Modelling of the A203L mutant (Figure 2cGo) showed that the C{delta}1 atom of the L203 side chain could make many additional contacts compared to I203, namely with C{delta}1 of I191 (separation: 3.1 Å), with C{varepsilon}1 of Y33 (separation: 2.3 Å), with C{gamma}1 of V193 (3.0 Å) and with the OH group of Y33 (2.5 Å). As modelled in the CAT-R387 structure, some of these interactions are too close and likely to cause steric problems. It is therefore not surprising that the thermostability of this mutant at 60°C was lower than that of the wild-type enzyme.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This study confirms the findings of Turner et al. (1992) that the hydrophobic pocket involving residue 203 is important in the thermostability of CAT-86. Furthermore, analysis of the thermostability of mutants with substitutions at position 203 highlights the importance of methyl groups in hydrophobic interactions and their contribution to protein thermostability. Kauzmann (1959) was the first to point out the importance of the contribution of hydrophobic effects to the stability of globular proteins. Now it is generally agreed that such hydrophobic effects represent the major factor stabilizing folded structures of globular proteins (Dill, 1990Go; Kim and Baldwin, 1990Go).

There are several reports implicating the role of buried hydrophobic pockets in thermal stability of proteins. Ishikawa et al. (1993) achieved stabilization of E.coli ribonuclease H1 by a cavity filling mutation within a hydrophobic core that does not change the enzymatic activity. Their results demonstrated that by the introduction of a methyl group into the cavity without causing steric clash, thermostability can be achieved.

Table IIGo summarizes the consequences of increasing the number of methyl groups in the 203 pocket of CAT-86: in general, the higher the number of methyl groups in the 203 pocket, the greater is the thermal stability of the CAT-86 enzyme. For example, the addition of three methyl groups in an A to I change provides a stabilization energy of –4.8 kJ/mol in SDM5. The Y33F substitution increased the potential stabilizing effect of two methyl groups from –1.9 to –5.4 kJ/mol, comparing A203V and SDM1.

Pace (1992) calculated that on average each buried CH2 group contributes 3.3–7.5 kJ/mol to the conformational stability of a globular protein. Our experiments suggest that the average contribution from adding methyl groups to the 203 pocket in CAT-86 is rather less, with an increase of two methyl groups contributing 2–5 kJ/mol. The lowering of free energy of the folded protein by favourable enthalpic interactions within the structure could also contribute to stabilization. The structure model of SDM5 suggests a stabilizing interaction between I191 and I203. This is one of the most stable CAT-86 mutants constructed in this study. Mutants with one fewer methyl group in the hydrophobic pocket compared with wild-type (SDM4 and SDM7) were destabilized by nearly 4 kJ/mol.

Although the A203L mutant showed a slight increase in thermostability over wild-type enzyme at 55°C (Table IIGo), it was less stable at 60°C. Structural analysis (Figure 2cGo) showed that L203 would make a number of close contacts that may be destabilizing. Similarly, Zhang et al. (1995) reported an increase in thermostability of T4 lysozyme when an I3L change was introduced. This suggests that not only the extent of hydrophobicity, but also the configuration of the atoms is important in achieving thermal stability.

The CAT-86 enzyme with a reconstituted CAT-R387 pocket was not as thermostable as R387 and the thermostability was intermediate compared with that of the other mutants constructed. This observation suggests that, although the 203 hydrophobic pocket has an important role in thermostability, there are other factors responsible for the higher thermostability of CAT-R387. In contrast, the hydrophobic interactions in the 203 pocket are crucial to the thermostability of CAT-86 and they can be optimized considerably beyond the native configuration.


    Notes
 
1 To whom correspondence should be addressed. E-mail: a.moir{at}sheffield.ac.uk Back


    Acknowledgments
 
We thank Dr M.J.Horsburgh for advice on site-directed mutagenesis and Drs I.Murray, A.Lewendon and W.V.Shaw for useful discussions during the course of this work. This work was supported by a postgraduate studentship to H.C. by the Government of India. The Sgi VGX workstation used in this study was provided by the Wellcome Trust. The MidasPlus program from the Graphics Laboratory, UCSF, was supported by NIH grant RR-01081.


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Received May 18, 2000; revised December 5, 2000; accepted December 20, 2000.





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