Mutational Analysis of the Major Loop of Bacillus 1,3-1,4-beta -D-Glucan 4-Glucanohydrolases
EFFECTS ON PROTEIN STABILITY AND SUBSTRATE BINDING*

(Received for publication, September 17, 1996, and in revised form, January 30, 1997)

Jaume Pons Dagger §, Enrique Querol § and Antoni Planas Dagger par

From the Dagger  Laboratory of Biochemistry, Institut Químic de Sarrià, Universitat Ramon Llull, 08017 Barcelona, Spain and the § Institut de Biologia Fonamental V. Villar Palasí and the Department de Bioquímica i Biologia Molecular, Universitat Autonoma de Barcelona, 08193 Bellaterra, Barcelona, Spain

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENT
REFERENCES


ABSTRACT

The carbohydrate-binding cleft of Bacillus licheniformis 1,3-1,4-beta -D-glucan 4-glucanohydrolase is partially covered by the surface loop between residues 51 and 67, which is linked to beta -strand-(87-95) of the minor beta -sheet III of the protein core by a single disulfide bond at Cys61-Cys90. An alanine scanning mutagenesis approach has been applied to analyze the role of loop residues from Asp51 to Arg64 in substrate binding and stability by means of equilibrium urea denaturation, enzyme thermotolerance, and kinetics. The Delta Delta GU between oxidized and reduced forms is approximately constant for all mutants, with a contribution of 5.3 ± 0.2 kcal·mol-1 for the disulfide bridge to protein stability. A good correlation is observed between Delta GU values by reversible unfolding and enzyme thermotolerance. The N57A mutant, however, is more thermotolerant than the wild-type enzyme, whereas it is slightly less stable to reversible urea denaturation. Mutants with a <2-fold increase in Km correspond to mutations at residues not involved in substrate binding, for which the reduction in catalytic efficiency (kcat/Km) is proportional to the loss of stability relative to the wild-type enzyme. Y53A, N55A, F59A, and W63A, on the other hand, show a pronounced effect on catalytic efficiency, with Km > 2-fold and kcat < 5% of the wild-type values. These mutated residues are directly involved in substrate binding or in hydrophobic packing of the loop. Interestingly, the mutation M58A yields an enzyme that is more active than the wild-type enzyme (7-fold increase in kcat), but it is slightly less stable.


INTRODUCTION

1,3-1,4-beta -D-Glucan 4-glucanohydrolase (1,3-1,4-beta -glucanase1; EC 3.2.1.73) is an endo-glycosidase that hydrolyzes beta -glucans containing mixed beta -1,3- and beta -1,4-linkages as lichenin and cereal beta -glucans. The enzyme has a strict cleavage specificity for beta -1,4-glycosidic bonds in 3-O-substituted glucopyranose units (1, 2). Genes encoding bacterial 1,3-1,4-beta -glucanases have been cloned and sequenced from different Bacillus species (3-9), Fibrobacter succinogenes (10), Ruminococcus flavofaciens (11), and Clostridium thermocellum (12). Together with 1,3-beta -glucanases ("laminarinases"), all bacterial 1,3-1,4-beta -glucanases share a high degree of sequence similarity and have been classified as members of family 16 of glycosylhydrolases (13, 14).

Bacillus licheniformis 1,3-1,4-beta -glucanase is a retaining glycosidase (2), acting by general acid/base catalysis in a double displacement mechanism (15). Glu138 has been proposed as the proton donor residue and Glu134 as the catalytic nucleophile (16, 17). The three-dimensional structure, recently refined at 0.18-nm resolution by x-ray crystallography (18), is almost identical to that of the hybrid H(A16M) between Bacillus amyloliquefaciens and Bacillus macerans (19) and the B. macerans (20) enzymes. It has a jelly-roll beta -sandwich fold, with the carbohydrate-binding cleft located on the concave face of a beta -sheet formed by seven antiparallel beta -strands (see Fig. 1). The Bacillus enzymes are unrelated to the plant 1,3-1,4-beta -glucanases in both sequence similarity (family 17 of glycosylhydrolases) and three-dimensional structure (alpha /beta -barrel structure), clearly indicating that the identical substrate specificities have arisen by convergent evolution (21). On the other hand, the Bacillus enzymes show structural similarities to plant legume lectins and family 7 cellulases. Cellobiohydrolase I from Trichoderma reesei (22) has a very similar fold, with most of the beta -sandwich residues in the protein core being superimposable, but it has long loops shaping the substrate-binding tunnel that are missing in the 1,3-1,4-beta -glucanase structure.


Fig. 1. Structure of the wild-type 1,3-1,4-beta -glucanase showing the loop between residues Asp51 and Asn67. A, overall structure; B, hydrogen bond interactions between loop residues. Gly56-Phe59 and Arg64-Asn67 have the geometry of type II beta -turns. The loop sequence is 51DGYSNGNMFNCTWRANN67. The structures were generated using MOLSCRIPT (49).
[View Larger Version of this Image (35K GIF file)]

Except for the Bacillus brevis isozyme, all Bacillus 1,3-1,4-beta -glucanases possess a single disulfide bond at Cys61-Cys90 (B. licheniformis numbering) that connects a beta -strand (residues 87-95) with a loop from residues 51 to 67 (see Fig. 1). This major loop is located on the concave side of the molecule, is solvent-exposed, and partially covers the active-site cleft. Even though no three-dimensional structure of an enzyme-inhibitor complex with a carbohydrate inhibitor filling the entire binding cleft is yet available, the three-dimensional structure of a covalent complex between the hybrid H(A16M) and epoxybutyl beta -cellobioside (19) and the molecular model of an enzyme-substrate complex made by computational methods (23) indicate that some loop residues might interact with a substrate occupying distant subsites on the nonreducing end of the binding site cleft.

Here we use the technique of alanine scanning mutagenesis (24) to analyze the role of loop residues (from Asp51 to Arg64) in B. licheniformis 1,3-1,4-beta -glucanase in substrate binding and stability by means of equilibrium urea denaturation, enzyme thermotolerance, and kinetics. Previous studies of the disulfide bond at Cys61-Cys90 have shown the deleterious effect of cysteine-to-alanine mutations on protein stability and activity, but no effect of disulfide bond reduction on activity (25). These results suggested that the loop has little flexibility and that the disulfide bond is not required to keep the structural integrity of the loop. Other hydrophobic interactions may position the loop to shape the active-site cleft.


MATERIALS AND METHODS

Bacterial Strains and Culture Media

Escherichia coli TG1 (supE hsdDelta 5 thiDelta (lac-proAB) F' [traD36 proAB+ lacIq lacZDelta M15]) was used for plasmid propagation, transformation with the mutagenic polymerase chain reaction (PCR), and protein expression. For plasmid isolation, bacteria were grown in 2YT medium (26), and 2SB medium (27) was used for protein expression. Ampicillin at 100 µg/ml was added when appropriate.

Chemicals and Enzymes

Urea (molecular biology-grade) was purchased from Sigma; dithiothreitol, 3,5-dinitrosalicilic acid, and 5,5'-dithiobis(2-nitrobenzoic acid) were from Fluka. Restriction endonucleases and T4 DNA ligase were from Boehringer Mannheim, and DeepVent® polymerase was from New England Biolabs Inc. alpha -35S-ATP was purchased from Amersham Corp. DNA sequencing was performed with the T7 sequencing kit from Pharmacia Biotech Inc. Oligonucleotides were synthesized by Boehringer Mannheim. Barley beta -glucan was from Megazyme (Sydney, Australia). All buffers and solutions for kinetic and urea denaturation experiments were degassed prior to use.

Site-directed Mutagenesis by PCR

The gene coding for B. licheniformis beta -glucanase previously cloned from the genomic DNA (8) and subcloned in pUC119 as a 1.21-kilobase SacI/SphI fragment (16) was used as the template for mutagenic PCR following the method previously reported for other 1,3-1,4-beta -glucanase mutants (17). The first PCR used the mutagenic primers and the reverse universal primer flanking the 5'-end of the 1,3-1,4-beta -glucanase gene. The primers were as follows (mismatches are in boldface): D51A, 5'-CCATTCGAGTACCCAGCTGCTTTTTGCCAT-3'; G52A, 5'-GTTTCCATTCGAGTACGCATCTGCTTTTTGCC-3'; Y53A, 5'-AAACATGTTTCCATTCGAGGCCCCATCTGCTTTTTGCC-3'; S54A, 5'-AAACATGTTTCCATTCGCGTACCCATCTGCTTTTTGCC-3'; N55A, 5'-TAAACATGTTTCCAGCCGAGTACCCATCTGCTTTTTGCC-3'; G56A, 5'-ACAGTTAAACATGTTTGCATTCGAGTACCCATC-3'; N57A, 5'-ACAGTTAAACATGGCTCCATTCGAGTACCC-3'; M58A, 5'-CGCCACGTACAGTTAAACGCGTTTCCATTCGAGT-3'; F59A, 5'-GCACGCCACGTACAGTTAGCCATGTTTCCATTCG-3'; N60A, 5'-GCACGCCACGTACAGGCAAACATGTTTCCATTCG-3'; T62A, 5'-TGTTTGCACGCCACGCACAGTTAAACATGTTTCC-3'; W63A, 5'-GGAGACATTGTTTGCACGCGCCGTACAGTTAAACATGTTTCC-3'; and R64A, 5'-GGAGACATTGTTTGCAGCCCACGTACAGTTAAAC-3'. The second PCR used the product of the first PCR as a primer and the forward universal primer to yield the whole 1,3-1,4-beta -glucanase gene with the desired mutation. The mutated gene was cut with EcoRI/HindIII and ligated again to a pUC119 vector. After transformation of E. coli TG1 cells, transformants were screened by DNA sequencing using appropriate primers located ~100 bases from the mutation point. Positive clones were confirmed by complete sequencing of the entire gene.

Protein Expression and Purification of Wild-type and Mutant Enzymes

Proteins were purified from the supernatant of E. coli TG1 cultures harboring the mutagenized plasmids basically as described before (28) with an additional purification step of fast protein liquid chromatography on an ion-exchange TSK CM-3SW column in 5 mM acetate buffer, pH 5.6, and elution with a linear gradient of 0-0.4 M NaCl in the same buffer. The proteins were analyzed by SDS-polyacrylamide gel electrophoresis as described (29) and by fast protein liquid chromatography on a TSK CM-3SW column at pH 5.6. Enzyme concentrations were determined by absorbance at 280 nm using A1 mg/ml = 14.5 absorbance units for the wild-type enzyme and by the Bradford protein assay (30) for the mutants using the wild-type enzyme as a standard. Spectrophotometric and kinetic measurements were performed on a Varian Cary 4 spectrophotometer with a Peltier temperature control system.

Enzyme Assay and Kinetics

1,3-1,4-beta -Glucanase activity on plates was detected by the Congo red assay after growing the E. coli TG1 cells containing the mutagenized plasmids on LB plates supplemented with 0.04% (w/v) barley beta -glucan (31). Activity in the supernatant from liquid cultures and the enzyme activity of purified enzymes were determined as previously reported (28) by measuring the net release of reducing sugars from barley beta -glucan in citrate/phosphate buffer (6.5 mM citric acid, 87 mM Na2HPO4), pH 7.2, 0.1 mM CaCl2 using the 3,5-dinitrosalicilic acid reagent (32). Kinetic measurements were performed using the synthetic substrate 4-methylumbelliferyl 3-O-beta -cellobiosyl-beta -D-glucopyranoside in citrate/phosphate buffer, pH 7.2, 0.1 mM CaCl2 by measuring the release of 4-methylumbelliferone at 365 nm (33, 34). Kinetic parameters were derived by fitting the data to a substrate inhibition model (v = kcat·[E]0/([S] + Km + [I]2/KI)) by means of nonlinear regression analysis (50).

Equilibrium Urea Denaturation

Unfolding was monitored by fluorescence spectroscopy in a Perkin-Elmer LS50 spectrofluorometer, with excitation at 282 nm (3-nm slit) and the emission spectra being recorded from 270 to 440 nm (8-nm slit) and measured at 340 nm, in thermostatted cuvette holders at 37 °C. For each data point collected, wild-type or mutant 1,3-1,4-beta -glucanases in citrate/phosphate buffer, pH 7.2, were diluted to 1 µg/ml in degassed urea solution in the same buffer and incubated overnight at 37 °C. To obtain reduced enzymes, the protein stock solutions (25 µg/ml) were made 200 mM dithiothreitol and incubated for 30 min at 25 °C before being added to the denaturant solution. The final dithiothreitol concentration was 10 mM, enough to avoid reoxidation for at least 24 h as shown by free sulfhydryl titration with 5,5'-dithiobis(2-nitrobenzoic acid) (after removal of the excess dithiothreitol by means of a pD10 G25 desalting column from Pharmacia).

Enzyme Inactivation and Thermotolerance Measurements

For determination of enzyme thermotolerance, samples of 50 µg/ml enzyme in 50 mM sodium acetate buffer, pH 6.0, 20 mM CaCl2 were incubated at 65 or 70 °C. Aliquots of 80 µl were withdrawn at various time intervals (until complete inactivation) and immediately diluted 5-fold in ice-cold water. The residual activity was determined at 45 °C using 4-methylumbelliferyl 3-O-beta -cellobiosyl-beta -D-glucopyranoside (3 mM assay concentration) and barley beta -glucan (5 mg/ml assay concentration) in citrate/phosphate buffer, pH 7.2, 0.1 mM CaCl2. The enzymatic half-life (t50) was calculated by fitting the first phase of the plot residual activity versus incubation time to a single exponential decay.


RESULTS

Enzyme Expression and Purification

Point mutations to alanine in the loop residues from Asp51 to Arg64 (Fig. 1) were prepared by site-directed mutagenesis by PCR. The mutant proteins were purified up to 95% as judged by SDS-polyacrylamide gel electrophoresis following the procedure described for the wild-type enzyme (28). Expression and purification yields were similar for all the mutant and wild-type enzymes. Proteins were stored in their oxidized form. Reduction of the disulfide bond at Cys61-Cys90 (reduced enzymes) was done just before their use as described under "Materials and Methods."

Analysis of Stability by Equilibrium Urea Denaturation

The stability of the enzymes reported in this study was examined by urea denaturation assuming a two-state transition using the model of Clarke and Fersht (35). Unfolding was monitored by measuring the dependence of fluorescence intensity on urea concentration. The data were analyzed as described previously for other beta -glucanase mutants (25) using Equation 1 (for its derivation, see Ref. 35),
F=(<UP>a</UP><SUB><UP>F</UP></SUB>+<UP>b</UP><SUB><UP>F</UP></SUB>[D]+(<UP>a</UP><SUB><UP>U</UP></SUB>+<UP>b</UP><SUB><UP>U</UP></SUB>[D])<UP>exp</UP>(m([D]−[D]<SUB>50%</SUB>)/RT))/(1+<UP>exp</UP>(m([D]−[D]<SUB>50%</SUB>)RT)) (Eq. 1)
where F is the measured fluorescence, aF and aU are the intercepts and bF and bU are the slopes of the base lines at low (F) and high (U) denaturant concentrations, [D] is the denaturant (urea) concentration, [D]50% is the concentration of denaturant at which 50% of the protein is unfolded, and m (=delta Delta GU/delta [D]) is the slope of the transition. The free energy of unfolding in the absence of denaturant (Delta GUH2O) is then calculated as shown in Equation 2.
&Dgr;G<SUB><UP>U</UP></SUB><SUP><UP>H</UP><SUB>2</SUB><UP>O</UP></SUP>=m[D]<SUB>50%</SUB> (Eq. 2)
Previous experiments with the wild-type enzyme (25) have shown the urea-induced denaturation to be reversible and independent of protein concentration.

The calculated values for m and [D]50% are given in Table I, and Fig. 2 plots the transition curves of the wild-type enzyme and one mutant as an example. Inspection of the m values for the oxidized and reduced enzymes shows that they are grouped in two clusters, one for the oxidized proteins with an average m value (mav) of 2.3 ± 0.4 kcal·mol-1·M-1 and the other for the reduced forms with an mav value of 1.2 ± 0.2 kcal·mol-1·M-1. This difference has proved to be significant by a Student's t test (epsilon  = 0.05), indicating that the presence or absence of the disulfide bond has a significant and constant effect on the unfolding behavior.

Table I. Equilibrium urea denaturation data for reduced and oxidized wild-type and mutant 1,3-1,4-beta -glucanases and thermotolerance of the oxidized enzymes


Mutant Urea denaturationa
Thermotolerance,b t50
mred [D]50%(red) mox [D]50%(ox)

kcal·mol-1·M-1 M kcal·mol-1·M-1 M min
Wild-type 1.12  ± 0.12 4.13  ± 0.01 1.58  ± 0.28 4.61  ± 0.07 70
D51A 1.08  ± 0.06 2.84  ± 0.02 2.33  ± 0.36 3.63  ± 0.04 3
G52A 1.39  ± 0.28 3.01  ± 0.01 2.39  ± 0.23 3.85  ± 0.07 10
Y53A 1.35  ± 0.18 2.75  ± 0.07 2.84  ± 0.08 3.83  ± 0.04 NDc
S54A 1.32  ± 0.16 3.31  ± 0.14 2.25  ± 0.12 4.02  ± 0.01 26
N55A 0.86  ± 0.03 3.57  ± 0.03 2.03  ± 0.04 4.15  ± 0.02 ND c
G56A 1.45  ± 0.04 3.01  ± 0.06 2.36  ± 0.05 3.91  ± 0.02 9
N57A 0.99  ± 0.03 3.96  ± 0.08 1.94  ± 0.26 4.42  ± 0.01 105
M58A 0.99  ± 0.09 3.23  ± 0.07 2.07  ± 0.02 4.20  ± 0.03 30
F59A 1.11  ± 0.09 3.27  ± 0.12 2.37  ± 0.20 4.13  ± 0.01 ND c
N60A 0.91  ± 0.06 3.23  ± 0.07 2.17  ± 0.06 4.06  ± 0.03 31
T62A 1.33  ± 0.04 2.78  ± 0.11 3.13  ± 0.29 3.84  ± 0.09 7
W63A 1.56  ± 0.12 2.49  ± 0.03 2.17  ± 0.08 3.65  ± 0.01 ND c
R64A 1.41  ± 0.02 2.92  ± 0.03 2.46  ± 0.05 3.89  ± 0.04 13
mavd 1.2  ± 0.2 2.3  ± 0.4

a Values are averages of two experiments. Conditions were as follows: citrate/phosphate buffer, pH 7.3; 37 °C; and [urea] = 0-8 M and [enzymes] = 1 µg·ml-1. m and [D]50% values were calculated by fitting the fluorescence data to Equation 4 as described under "Results."
b Thermotolerance at 65 °C is expressed as enzymatic half-life (t50). Conditions were as follows: 50 mM acetate buffer, pH 6.0, 20 mM CaCl2; 65 °C; and [enzymes] = 50 µg·ml-1 (oxidized forms).
c ND, not determined.
d mav is the average m values for reduced and oxidized enzymes.


Fig. 2. Equilibrium urea denaturation curves for the wild-type enzyme (left panel) and the N55A mutant (right panel). Fluorescence intensity values were fitted to Equation 1. Normalized curves are plotted as fraction native (fN) = (F - (aU + bU[D]))/(aF - aU + [D](bF - bU)), where aU, aF, bU, and bF are the adjusted parameters from Equation 1. Left panel: bullet , oxidized; black-square, reduced; right panel: bullet , oxidized; black-square, reduced. wt, wild-type enzyme.
[View Larger Version of this Image (11K GIF file)]

Since individual m values for each mutant are subjected to large standard errors, we used the corresponding mav value to calculate the free energies of unfolding in the absence of denaturant for the oxidized and reduced enzymes, respectively. Then, Equation 2 becomes Equation 3.
&Dgr;G<SUB><UP>U</UP></SUB><SUP><UP>H</UP><SUB>2</SUB><UP>O</UP></SUP>=m<SUB><UP>av</UP></SUB>[D]<SUB>50%</SUB> (Eq. 3)
The difference in stability between two enzymes is evaluated as shown in Equation 4,
&Dgr;&Dgr;G<SUB><UP>U</UP></SUB>=&Dgr;G<SUB><UP>U</UP></SUB><SUP><UP>H</UP><SUB>2</SUB><UP>O</UP></SUP>(a)=&Dgr;G<SUB><UP>U</UP></SUB><SUP><UP>H</UP><SUB>2</SUB><UP>O</UP></SUP>(b) (Eq. 4)
where a and b are the mutant and wild-type enzymes, respectively, in their oxidized or reduced forms (Delta Delta GU(ox) or Delta Delta GU(red)). The calculated Delta GUH2O and Delta Delta GU values are listed in Table II.

Table II. Free energies of unfolding for wild-type and mutant 1,3-1,4-beta -glucanases determined by equilibrium urea denaturation at 37 °C


Mutant  Delta GUH2O(red)a  Delta GUH2O(ox)a  Delta Delta GU(red)b  Delta Delta GU(ox)b  Delta Delta GS-Sc

kcal·mol-1 kcal·mol-1 kcal·mol-1 kcal·mol-1 kcal·mol-1
Wild-type 4.98 10.50 5.6
D51A 3.42 8.30 1.6 2.2 4.9
G52A 3.63 8.79 1.4 1.7 5.2
Y53A 3.32 8.76 1.7 1.7 5.4
S54A 4.00 9.19 1.0 1.3 5.2
N55A 4.30 9.48 0.7 1.1 5.2
G56A 3.63 8.94 1.4 1.6 5.3
N57A 4.78 10.10 0.2 0.4 5.3
M58A 3.89 9.59 1.1 0.9 5.7
F59A 3.94 9.44 1.1 1.1 5.5
N60A 3.90 9.28 1.1 1.2 5.4
T62A 3.35 8.78 1.6 1.8 5.4
W63A 3.00 8.34 2.0 2.2 5.3
R64A 3.52 8.88 1.5 1.6 5.4
Mean 5.3 ± 0.2

a Delta GUH2O = mav[D]50% using mav = 1.2 kcal·mol-1·M-1 for reduced enzymes and mav = 2.3 kcal·mol-1·M-1 for oxidized enzymes (see "Results"). Values are averages of two experiments. The estimated average error for Delta GU values is ±0.05.
b Delta Delta GU = Delta GUH2O(mut) - Delta GUH2O(wt) for reduced and oxidized enzymes, respectively.
c Delta Delta GS-S = Delta GUH2O(red) - Delta GUH2O(ox) for each enzyme.

The energetic contribution of the disulfide bridge to stability (Delta Delta GUS-S) was calculated using Equation 4 in which a and b are the reduced and oxidized forms of the same protein, respectively. The calculated values in Table II display, within the experimental error, a constant stabilizing effect of 5.3 ± 0.2 kcal·mol-1 for the disulfide bridge in all wild-type and mutant enzymes.

Catalytic Parameters of Wild-type and Mutant Proteins

kcat and Km values for wild-type and mutant enzymes were determined with a specific substrate for 1,3-1,4-beta -glucanases recently developed by our group (33, 34). 4-Methylumbelliferyl 3-O-beta -cellobiosyl-beta -D-glucopyranoside undergoes a single glycosidic bond cleavage upon enzymatic hydrolysis with release of the 4-methylumbelliferone chromophore, which can be continuously monitored at 365 nm. Reactions were done in citrate/phosphate buffer, pH 7.3, 0.1 mM CaCl2. While the optimal temperature for the wild-type enzyme is 55 °C (36), some of the alanine mutants are more thermolabile. 45 °C was found to be the highest temperature for which all the proteins studied showed a linear progress curve during the initial 15 min of reaction. Substrate inhibition was observed at high concentrations, so the data were fitted to an uncompetitive substrate inhibition model by nonlinear regression (36). Calculated values for kcat and Km are summarized in Table III.

Table III. Kinetic parameters for wild-type and mutant 1,3-1,4-beta -glucanases (4-methylumbelliferyl 3-O-beta -cellobiosyl-beta -D-glucopyranoside as substrate)

Conditions were as follows: citrate/phosphate buffer, pH 7.2, 0.1 mM CaCl2; 45 °C; [4-methylumbelliferyl 3-O-beta -cellobiosyl-beta -D-glucopyranoside] = 0.2-15 mM and [enzymes] = 39 nM (wild-type), 104 nM (D51A), 50 nM (G52A), 274 nM (Y53A), 78 nM (S54A), 1.2 mM (N55A), 59 nM (G56A), 49 nM (N57A), 10 nM (M58A), 1.0 mM (F59A), 47 nM (N60A), 59 nM (T62A), 782 nM (W63A), and 57 nM (R64A). Initial rates were fitted to an uncompetitive substrate inhibition model (see "Materials and Methods"). Conditions were as follows: citrate/phosphate buffer, pH 7.2, 0.1 mM CaCl2; 45 °C; [4-methylumbelliferyl 3-O-beta -cellobiosyl-beta -D-glucopyranoside] = 0.2-15 mM and [enzymes] = 39 nM (wild-type), 104 nM (D51A), 50 nM (G52A), 274 nM (Y53A), 78 nM (S54A), 1.2 mM (N55A), 59 nM (G56A), 49 nM (N57A), 10 nM (M58A), 1.0 mM (F59A), 47 nM (N60A), 59 nM (T62A), 782 nM (W63A), and 57 nM (R64A). Initial rates were fitted to an uncompetitive substrate inhibition model (see "Materials and Methods").

Mutant Km kcat kcat/Km kcat/Km relative to wild-type

mM s-1 mM-1·s-1 %
Wild-type 1.8  ± 0.5 4.0  ± 0.4 2.2  ± 0.8 100
D51A 1.8  ± 0.2 2.0  ± 0.2 1.1  ± 0.2 50
G52A 1.7  ± 0.3 2.5  ± 0.2 1.5  ± 0.4 68
Y53A 5.7  ± 1.6 0.2  ± 0.02 0.04  ± 0.01 2
S54A 2.8  ± 0.3 2.5  ± 0.2 0.9  ± 0.1 41
N55A 4.1  ± 0.7 0.14  ± 0.01 0.034  ± 0.008 2
G56A 2.2  ± 0.4 2.7  ± 0.2 1.2  ± 0.3 54
N57A 2.0  ± 0.3 2.8  ± 0.2 1.4  ± 0.3 63
M58A 3.7  ± 0.5 27.8  ± 0.2 7.5  ± 0.9 340
F59A 9.5  ± 3 0.083  ± 0.004 0.009  ± 0.003 0.4
N60A 1.8  ± 0.2 3.1  ± 0.3 1.7  ± 0.4 77
T62A 1.9  ± 0.2 2.8  ± 0.2 1.5  ± 0.3 68
W63A 4.7  ± 0.8 0.21  ± 0.02 0.05  ± 0.01 2
R64A 2.0  ± 0.3 2.8  ± 0.2 1.4  ± 0.3 64

Analysis of Enzyme Thermal Stability

A measure of the enzyme thermotolerance can be obtained by deducing t50 at a specified temperature. Residual activity of the enzymes was measured after various periods of incubation at a given temperature by steady-state kinetics with 4-methylumbelliferyl 3-O-beta -cellobiosyl-beta -D-glucopyranoside substrate at 45 °C. Preliminary experiments with the wild-type enzyme at 50 and 500 µg·ml-1 at 65 and 70 °C in sodium acetate buffer, pH 6.0, showed that extensive protein aggregation took place at high enzyme concentration and that a very low t50 (<10 min) was obtained at 70 °C. Therefore, we chose a protein concentration of 50 µg·ml-1 and an incubation temperature of 65 °C as standard assay conditions. The plot of residual activity versus incubation time follows a double exponential curve, with the value of t50 being in the first phase of the inactivation decay. Mutants Y53A, N55A, F59A, and W63A could not be analyzed under these conditions due to their low activity, which required enzyme concentrations above 500 µg·ml-1. Values of t50 are summarized in Table I.


DISCUSSION

The carbohydrate-binding cleft of the 1,3-1,4-beta -glucanase of B. licheniformis is partially covered by the surface loop between residues 51 and 67, which is linked to beta -strand-(87-95) of the minor beta -sheet III (18) by the single disulfide bridge at Cys61-Cys90. The technique of alanine scanning mutagenesis has been applied to analyze the role of loop residues (Asp51-Arg64) in substrate binding and stability as well as the contribution of the disulfide bridge to stability.

Equilibrium Urea Denaturation

Unfolding transition curves are described by two parameters in a two-state model: [D]50% is a measure of the midpoint of the transition region, and m is a measure of the steepness of the transition region and reflects the cooperativity of the unfolding process. A clear distinction is observed between reduced and oxidized forms in terms of m values, with the oxidized enzymes having a steeper transition. Common to all models that have been proposed to describe the dependence of the free energy of unfolding on denaturant concentration (37, 38) is the premise that denaturants alter the equilibrium N left-right-arrow  U through a preferential interaction with the denatured state. Schellman (37) proposed that the parameter that can describe the differential interaction of the native and denatured state to denaturants is the different solvent-accessible area between both states, AN and AU, respectively (Equation 5),
&Dgr;G<SUB><UP>U</UP></SUB><SUP>D</SUP>=&Dgr;G<SUB><UP>U</UP></SUB><SUP><UP>H</UP><SUB>2</SUB><UP>O</UP></SUP>−K · (<UP>A</UP><SUB><UP>U</UP></SUB>−<UP>A</UP><SUB><UP>N</UP></SUB>) · [D] (Eq. 5)
where K represents a thermodynamic constant. According to this model, the smaller m value of the reduced enzymes (Table I) must reflect a smaller value of (AU - AN), only explained by a decrease in AU (as large variations in AN are not expected). However, the reduction of the disulfide bridge is more likely to produce an increase in AU, accounted for by a more extended denatured conformation (39, 40). Thus, the results are unlikely to be explained in this way and reveal a more complex meaning of the m parameter.

The spatial distribution of the mutated residues in the crystallographic structure of the wild-type enzyme (18) suggests that the destabilizing effect is larger near the N- and C-terminal ends of the loop (Table IV). This observation is in agreement with the idea that the loop edges are rigid, with a central part being more flexible and the C-terminal end being more tightly packed as judged by the side chain solvent accessibility and van der Waals interaction data shown in Table IV. No correlation was found between the experimental Delta Delta GU values and the free energy of transfer of amino acid side chains from water to octanol (corrected or not for solvent-exposed area) or the number of atoms inside a sphere around C-alpha of the mutated amino acid residue (Table IV). Such correlations have been shown to work properly in a number of proteins (41-44) for series of mutants in hydrophobic regions of the protein structure. This is not the case for the 1,3-1,4-beta -glucanase mutants probably because the loop is partially solvent-exposed and some of the residues are hydrophilic.

Table IV. Structural data from the three-dimensional structure of wild-type B. licheniformis 1,3-1,4-beta -glucanase

Structural analysis was carried out using Insight software (BioSym/MSI, San Diego, CA). Structural analysis was carried out using Insight software (BioSym/MSI, San Diego, CA).

Residue Burieda Hydrogen bond interactionsb van der Waals interactionsc

%
Asp51 74 O-delta 1-N-65 (2.82) Lys49, Tyr53, Trp63, Arg64, Ala65, Trp63
Gly52 N-O-63 (2.86)
Tyr53 43 O-N-63 (2.96) Ala50, Asp51, Asn55, Thr62, Trp63, Arg94
Ser54 82 O-gamma -N-55 (2.96) Gly56, Phe59, Cys61,
Asn55 70 N-O-gamma 54 (2.96) Tyr53, Asn57, Phe59, Trp63
N-delta 2-N-epsilon 163
Gly56 O-N-59 (2.82) Ser54, Met58, Phe59,
Asn57 9 O-delta 1-N-58 (2.80) Asn55, Phe59
Met58 78 N-O-delta 1-57 (2.80) Gly56, Asn60, Gly215, Ala216, Trp221
O-N-216 (2.95)
Phe59 89 N-O-56 (2.82) Ser54, Asn55, Gly56, Asn57, Cys61, Trp63, Trp213
Asn60 100 Met58, Phe88, Asn214, Ala216
Thr62 100 O-N-90 (2.80) Tyr53, Arg64, Phe88, Asp89, Cys90
Trp63 100 O-N-52 (2.86) Ala50, Asp51, Gly52, Tyr53, Asn55 Phe59, Cys90 Gly91, Glu92, Asn211, Leu212
Arg64 71 O-N-67 (2.85) Ala50, Asp50, Thr62, Asn66, Asn67, Asp89, Cys90
N-epsilon -O-delta 1-59 (2.97)

a Percentage of buried amino acid side chain in the wild-type enzyme calculated from Connolly surfaces on the three-dimensional structure.
b Observed hydrogen bond interactions (distance (Å) between heteroatoms in parentheses).
c Amino acid residues having at least one atom with van der Waals contact with the side chain of the residue being mutated.

Contribution of Disulfide Bridge to Protein Stability

The calculated values for Delta Delta GUS-S in Table III indicate a constant stabilizing effect of 5.3 ± 0.2 kcal·mol-1 for the disulfide bridge in all mutant and wild-type enzymes. This value is larger than that previously estimated for the wild-type enzyme (0.7 kcal·mol-1 (25)). The m value for the oxidized wild-type enzyme deviates from the general trend observed for the mutants, and it is much closer to the m value for the reduced wild-type enzyme. In the absence of mutant data, our first estimation was performed using an mav value of 1.31 kcal·mol-1·M-1 for both forms of the wild-type enzyme. However, the large number of mutants studied here clearly shows a significant difference between oxidized and reduced forms. Even though the behavior of the wild-type enzyme might be different, the general trend observed here allows us to conclude that the disulfide bond has a larger contribution to protein stability.

Thermotolerance

Enzyme thermotolerance was determined at 65 °C as the incubation time required to irreversibly inactivate the enzyme to 50% of its initial activity (t50). A good correlation was observed between Delta GU (from equilibrium urea denaturation) and t50 for the mutants in their oxidized form (Fig. 3), except for N57A, which is surprisingly more thermotolerant than the wild-type enzyme. Even though direct comparison of thermal stability and urea denaturation is not possible in general (kinetic versus equilibrium experiments), the results may be rationalized considering a fast irreversible process from the denatured state at high temperature (Reaction 1),
<UP>N</UP> <LIM><OP><ARROW>⇌</ARROW></OP><LL>k<SUB>2</SUB></LL><UL>k<SUB>1</SUB></UL></LIM><UP> U </UP><LIM><OP><ARROW>→</ARROW></OP><UL>k<SUB>3</SUB></UL></LIM> <UP>I</UP>
<UP>R<SC>eaction</SC></UP> 1
where k1, k2, and k3 are rate constants and I is the irreversibly denatured state. Recent examples have shown that mutations in mobile loops or at labile residues may yield proteins that are more resistant to thermal denaturation, whereas reversible denaturation is similar to the wild-type proteins (45-47). The higher thermotolerance of the N57A mutant and its deviation from the above correlation may be the result of lowering the irreversible denaturation rate constant k3 as a consequence of removing a labile solvent-exposed Asn residue. Asparagine residues are often involved in several degradative covalent reactions in proteins such as deamidation, isoaspartate formation, and peptide bond cleavage at high temperatures or in low pH environments, with the effect being more pronounced when the Asn residue is next to a Gly residue in the amino acid sequence (48). Asn57 is next to Gly56 in a highly solvent-exposed region of the loop (Table IV). Another Asn mutant in the loop (N60A) fits the correlation between Delta GU and t50, but it is a buried residue with no Gly residue next to it. Finally, Asn55 is also solvent-exposed, but the thermotolerance of the mutant N55A could not be determined due to its low activity. For the purpose of engineering more heat-stable enzymes, preventing irreversible thermal inactivation may be more important than increasing stability.


Fig. 3. Stability versus thermotolerance plot for the oxidized enzyme forms. Delta GU values are from equilibrium urea denaturation (data from Table II), and t50 values are the enzymatic half-lives at 65 °C (data from Table I). wt, wild-type enzyme.
[View Larger Version of this Image (14K GIF file)]

Effects on Enzyme Kinetics

The kcat and Km values of the 1,3-1,4-beta -glucanase mutants (Table II) show that most of the mutations have an effect on enzyme activity. It could be a direct effect of removing an amino acid side chain involved in substrate binding (or interacting with an essential catalytic residue) or an indirect effect of local rearrangements produced by the mutation (which are also reflected in a decrease in protein stability). kcat/Km values are plotted against stability data (as Delta GUH2O values) for the oxidized enzyme forms in Fig. 4. Inspection of this plot suggests that the mutants can be classified in four groups. Group A (wild-type, D51A, G52A, G56A, N60A, T62A, and R64A) is formed by those enzymes showing a good correlation between catalytic efficiency and enzyme stability. For these mutants, the decrease in catalytic efficiency is mainly due to kcat since Km values are <2-fold larger than the wild-type Km value. Moreover, the mutated residues in this group have no specific role in substrate binding as proposed from the structure of the modeled enzyme-substrate complex. Therefore, the reduction in kcat/Km is interpreted as the result of local rearrangements in the protein structure induced by the mutations, which also have a proportional effect on protein stability. Group B mutants (S54A and N57A) slightly deviate from the correlation. They show <2-fold reduction in kcat and almost no effect on Km as compared with the wild-type enzyme. Group C (Y53A, N55A, F59A, and W63A) is composed of mutants that have a pronounced deleterious effect on enzyme activity, with Km > 2-fold and kcat < 5% of the wild-type kinetic parameters. These mutated residues are directly involved in substrate binding or in hydrophobic packing of the loop.2 Tyr53 forms a hydrogen bond with the 3-OH of the glucopyranose unit of the substrate in subsite -III, whereas the amide nitrogen of Asn55 hydrogen-bonds with the 6-OH of the glucopyranose unit in subsite -II according to the modeled enzyme-substrate complex for the B. licheniformis enzyme2 or the modeled complex for the hybrid H(A16M) beta -glucanase (23). On the other hand, Phe59 has the aromatic side chain pointing toward the core of the protein, and it has a strong hydrophobic (stacking) interaction with Trp213, which belongs to a beta -chain of the major beta -sheet on the concave face of the molecule. This interaction might be important to position the loop and to create a hydrophobic environment in this portion of the cleft when the substrate binds. Trp63 also has the aromatic side chain interacting with the main core and very close to Phe59, contributing to the structural integrity of the loop. Finally, group D comprises the single mutant M58A, which is more active, with a kcat value 7-fold higher than that of the wild-type enzyme. This surprising result was unpredictable from a simple structural analysis since the side chain of Met58 does not interact with the substrate or with any essential catalytic residue in the three-dimensional structure of the free enzyme. However, replacement of the side chain by a smaller methyl group (M58A) might allow some readjustments of the loop in the enzyme-substrate complex that may have a favorable effect on transition state stabilization. Up to date, only the structure of a covalent complex between the hybrid H(A16M) 1,3-1,4-beta -glucanase and epoxybutyl beta -cellobioside has been solved by x-ray crystallography (19). Inhibitor binding has no significant effect on the active-site geometry as observed by comparing the structures of the covalent complex and the free H(A16M) enzyme. However, the cellobiose unit of this suicide inhibitor only fills subsites -II and -III, with subsite -I being occupied by an alkyl chain instead of a glucopyranose ring. Since ring distortion in subsite -I is expected, the structure of this covalent complex is not a good model to analyze the structural effects that the reported mutations may have on substrate binding and on transition state stabilization. New three-dimensional structures of enzyme-inhibitor complexes (or inactive mutant-substrate complexes) are required to evaluate small but significant structural changes that might occur upon ligand binding.


Fig. 4. Catalytic efficiency (kcat/Km) versus enzyme stability (Delta Delta GU) plot for wild-type and mutant 1,3-1,4-beta -glucanases (oxidized forms). kcat/Km values are presented in percentage relative to the wild-type values (data from Table III), and Delta Delta GU values are from Table II. wt, wild-type enzyme.
[View Larger Version of this Image (18K GIF file)]

When applying an alanine scanning mutagenesis strategy, comparison between catalytic efficiency and enzyme stability provides a useful method to identify those residues that have an important role in ligand binding or in structural packing of the protein. Taking as a reference the mutants for which the reduction in catalytic efficiency is proportional to the loss of protein stability, mutations that deviate from this correlation indicate that these residues are involved in substrate binding or in maintaining the active-site structure. It is remarkable that two mutants, N57A with increased thermostability and M58A with higher catalytic efficiency, have been obtained. The effects of these mutations were unpredictable with the current knowledge of protein structure/function relationships, supporting the fact that scanning and random mutagenesis strategies are useful approaches to obtain proteins with improved properties for biotechnological applications.


FOOTNOTES

*   This work was supported in part by Grants BIO94-0912-C02 and BIO97-0511-C02 from the Ministerio de Educación y Ciencia, Spain.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
   Recipient of a doctoral fellowship from Ministerio de Educación y Ciencia, Spain.
par    To whom correspondence should be addressed: Inst. Químic de Sarrià, Universitat Ramon Llull, Via Augusta 390, 08017 Barcelona, Spain. Fax: 34-3-205-62-66; E-mail: aplan{at}iqs.url.es.
1   The abbreviations used are: 1,3-1,4-beta -glucanase, 1,3-1,4-beta -D-glucan 4-glucanohydrolase; PCR, polymerase chain reaction.
2   The x-ray structure (code 1GBG) of the B. licheniformis 1,3-1,4-beta -glucanase has been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.

ACKNOWLEDGEMENT

We are indebted to Teresa Dot for the molecular modeling analysis.


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