Directed Mutagenesis of Specific Active Site Residues on
Fibrobacter succinogenes
1,3-1,4-
-D-Glucanase Significantly Affects Catalysis
and Enzyme Structural Stability*
Jui-Lin
Chen
,
Li-Chu
Tsai§,
Tuan-Nan
Wen¶,
Jyh-Bing
Tang
,
Hanna S.
Yuan§, and
Lie-Fen
Shyur
From the Institutes of
BioAgricultural Sciences,
§ Molecular Biology, and ¶ Botany, Academia Sinica,
Taipei 115, Taiwan, Republic of China
Received for publication, January 30, 2001, and in revised form, February 19, 2001
 |
ABSTRACT |
The functional and structural significance of
amino acid residues Met39, Glu56,
Asp58, Glu60, and Gly63 of
Fibrobacter succinogenes
1,3-1,4-
-D-glucanase was explored by the approach of
site-directed mutagenesis, initial rate kinetics, fluorescence
spectroscopy, and CD spectrometry. Glu56,
Asp58, Glu60, and Gly63 residues
are conserved among known primary sequences of the bacterial and fungal
enzymes. Kinetic analyses revealed that 240-, 540-, 570-, and 880-fold
decreases in kcat were observed for the E56D, E60D, D58N, and D58E mutant enzymes, respectively, with a similar substrate affinity relative to the wild type enzyme. In contrast, no
detectable enzymatic activity was observed for the E56A, E56Q, D58A,
E60A, and E60Q mutants. These results indicated that the carboxyl side
chain at positions 56 and 60 is mandatory for enzyme catalysis. M39F,
unlike the other mutants, exhibited a 5-fold increase in
Km value. Lower thermostability was found with the
G63A mutant when compared with wild type or other mutant forms of
F. succinogenes 1,3-1,4-
-D-glucanase.
Denatured wild type and mutant enzymes were, however, recoverable as
active enzymes when 8 M urea was employed as the
denaturant. Structural modeling and kinetic studies suggest that
Glu56, Asp58, and Glu60 residues
apparently play important role(s) in the catalysis of F. succinogenes 1,3-1,4-
-D-glucanase.
 |
INTRODUCTION |
1,3-1,4-
-D-Glucanase
(1,3-1,4-
-D-glucan 4-glucanohydrolases, EC 3.2.1.73;
lichenase) specifically hydrolyzes 1,4-
-D-glycosidic bonds adjacent to 1,3-
-linkages in mixed linked
-glucans,
yielding mainly cellobiosyltriose and cellotriosyltetraose (1). This group of enzymes represents a distinct family of glucanohydrolases with
similar substrate specificities widely observed among different organisms, including bacilli, clostridium, ruminal bacteria and fungi,
and higher plants (2-5). The
-D-glucan substrate for the enzyme is a major cell wall component of endosperm tissues in
cereal grains. Chemically it is mostly a linear homopolymer of glucose
molecules linked via
-1,3- and
-1,4-glycosidic bonds at a ratio
of ~1:2.5. Various bacterial or fungal
1,3-1,4-
-D-glucanases show good sequence similarity
with endo-
-1,3-glucanases (laminarases) (see Fig. 1), and together
they are classified as a member of family 16 glycosyl hydrolases (2,
3). A high degree of amino acid sequence homology (70-90%) was
observed among the various 1,3-1,4-
-D-glucanases
isolated from different Bacillus species (6, 7). However,
little or no homology between the bacteria and plant (barley)
1,3-1,4-
-D-glucanases was detected, neither at the
primary nor at the tertiary structure levels of the compared proteins
(4, 8). The bacterial enzymes are, in general, more thermotolerant than
their barley counterparts.
With respect to the kinetic properties of bacterial
1,3-1,4-
-D-glucanases, these enzymes reside with the
retaining glycosidase activity leading to the hydrolysis of glycosidic
bonds with a net retention of the anomeric configuration during
-glucan hydrolysis (9). The 1,3-1,4-
-D-glucanase
enzyme has been proposed to follow a general acid-base catalytic
mechanism in which specific amino acid residues acting as a general
acid or a nucleophile are required for completing a catalytic reaction
of the enzyme (10). A number of methodologies have been previously
applied to the characterization of catalytic mechanism(s) of
Bacillus 1,3-1,4-
-D-glucanases, including
site-directed mutagenesis, affinity labeling, and x-ray crystallography
analysis. The Glu134 and Glu138 amino acid
residues within the Bacillus licheniformis enzyme have been
identified as the nucleophile and the catalytic acid/base, respectively
(11, 12). The Glu105 and Glu109 residues of
Bacillus macerans and
H(A16-M)1,3-1,4-
-D-glucanases have also been shown to
confer a catalytic function similar to that observed for
Glu134 and Glu138 of the B. licheniformis enzyme (4, 12, 13).
A key ruminal bacterial enzyme producer, Fibrobacter
succinogenes, plays a major role in plant fiber degradation in the
rumen of major livestock species. Several of the enzymes related to the
degradation of cellulose or hemicellulose polymers of plant cell walls
from this organism have been isolated and partially characterized (14).
A F. succinogenes 1,3-1,4-
-D-glucanase was
isolated and investigated by Erfle and co-workers (15, 16). This enzyme
consists of a protein sequence with a circular permutation in which two
highly conserved catalytic domains (namely A and B) of the enzyme are
in a reverse orientation as compared with that of other
1,3-1,4-
-D-glucanases (6, 8, 16). A 5 times repeated
segment, PXSSSS, was only observed in the C-terminal, nonhomologous region of the amino acid sequence of the
Fibrobacter enzyme relative to the bacilli or other
bacterial and fungal 1,3-1,4-
-D-glucanases. Nevertheless, alignment of the amino acid sequences of the F. succinogenes enzyme with other
1,3-1,4-
-D-glucanases suggests that a number of amino
acid residues in the highly conserved region may play important roles
in catalysis of the enzyme (see Fig. 1). In an attempt to identify the
possible involvement of specific amino acid residues in the catalytic
activity of F. succinogenes 1,3-1,4-
-D-glucanase, we evaluated the potential
functional significance of the Met39, Glu56,
Asp58, Glu60, and Gly63 residues of
the enzyme, using a combination of various approaches including
site-directed mutagenesis, fluorescence spectroscopy, circular
dichroism spectrometry, kinetics, and structural modeling. Several
lines of evidence were obtained, providing some useful information
about the structural and functional relationship of F. succinogenes 1,3-1,4-
-D-glucanase. The most
significant findings of this investigation are: 1) substitutions of
Glu56, Asp58, and Glu60 with either
alanine or glutamine can completely abolish enzymatic activity; 2)
replacement of Gly63 with alanine can greatly reduce
thermostability of the enzyme; and 3) Met39 is essential
for the 1,3-1,4-
-D-glucanase of F. succinogenes to maintain the most effective catalytic efficiency
for the enzyme. This study provides new information that may be used to
improve this ruminal enzyme for industrial utilization as a feed or
food-processing aid, using either rational design or DNA shuffling approaches.
 |
EXPERIMENTAL PROCEDURES |
Subcloning of F. succinogenes
1,3-1,4-
-D-Glucanase Gene--
The full-length
cDNA of F. succinogenes
1,3-1,4-
-D-glucanase
(Fs
-glucanase)1 in a pIJ10
plasmid was amplified and introduced with NcoI and EcoRI restriction enzyme recognition sites at the 5' and 3'
ends, respectively, by using a PCR-based method. The two primers
designed for the NcoI and EcoRI sites were
5'-TCACCACCATGGTTAGCGCAAAG-3' and
5'-GCCACGAATTCTGTTCAAAGTTCAC-3', respectively. The PCR
reaction was performed with a thermocycling program as follows:
94 °C for 5 min, 55 °C for 1 min, and 72 °C for 1 min for 1 cycle; 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 1 min
for 30 cycles; and 94 °C for 1.5 min, 55 °C for 1.5 min, and
72 °C for 10 min for 1 cycle. The resulting amplified DNA fragments
were digested with NcoI and EcoRI, purified, and
ligated onto the pET26b(+) vector, which was predigested with
NcoI and EcoRI. The recombinant gene sequence for
Fs
-glucanase, designated "pFsNcE," was confirmed by DNA
sequencing using the chain termination method (17). In this DNA
construct, a pelB leading peptide at the N terminus plus 19 extra amino acid residues including a His6 tag at the C
terminus to facilitate protein purification were included. The
recombinant plasmid encoding for the wild type enzyme was then
transformed into Escherichia coli BL21(DE3) host.
Site-directed Mutagenesis--
Plasmid pFsNcE was used as the
template for a PCR-based mutation (18) of Fs
-glucanase. For this
purpose, a pair of complementary mutagenic primers was designed for the
mutation of each desired amino acid residue. Eleven pairs of mutagenic
primers, as shown in Table I, were used
to mutate Met39
Phe, Glu56
Ala,
Glu56
Asp, Glu56
Gln, Asp58
Ala, Asp58
Glu, Asp58
Asn,
Glu60
Ala, Glu60
Asp, Glu60
Gln, and Gly63
Ala. The mutagenic PCR reaction
mixture consisted of 10 mM KCl, 10 mM
(NH4)2SO4, 20 mM
Tris-HCl, pH 8.8, 2 mM MgSO4, 0.1% Triton
X-100, 0.1 mg/ml nuclease-free bovine serum albumin, 10 ng of template
DNA, 0.5 mM dNTPs, 0.5 µM each of the
complementary primers, and 2.5 units of cloned Turbo Pfu DNA
polymerase.
Mutant DNA was generated with a thermocycling program of 2 min at
95 °C and 16 cycles of 30 s at 95 °C, 60 s at 55 °C,
and 12 min at 68 °C on a Hybaid TouchDown thermal cycler. The
PCR-generated products were digested with 10 units of DpnI
at 37 °C for 1 h, prior to their use for transformation into
XL-1 Blue cells. Mutations were confirmed by fluorescent dideoxy chain
termination DNA sequencing using T7 promoter and
T7 terminator primers. The mutagenesis plasmid was then
transformed into BL21(DE3) host cells for the overexpression of mutant enzyme.
Protein Production and Cellular Localization of Fs
-glucanase
in E. coli--
Optimal protein production conditions and cellular
localization of Fs
-glucanase in E. coli cells were
investigated. 5 ml of pregrown culture of the BL21(DE3) bacterial
strain carrying pET26b(+) containing the Fs
-glucanase gene was added
to 500 ml of fresh LB broth containing 30 µg/ml kanamycin. The
culture was shaken vigorously at 33 °C until the
A600 nm reached 0.4-0.6. Addition of 1 mM isopropyl-
-D-thiogalactopyranoside (IPTG)
to the culture then was performed, and the culture was further
incubated for 12-24 h at 33 °C. Small amounts of culture aliquots
were collected with a constant time interval after the IPTG induction.
The culture medium and cell extract prepared from the collected cell
pellet at different time periods of IPTG induction were then employed for enzymatic activity assay, SDS-polyacrylamide gel
electrophoresis (PAGE) according to Laemmli (19), and zymogram analysis.
Purification of Wild Type and Mutant Fs
-glucanase--
The
wild type or mutant forms of Fs
-glucanase produced in the
above-described procedure were further purified. Approximately 80-85%
of the total Fs
-glucanase expressed in E. coli host cells was secreted into the culture medium. The extracellular secreted enzyme
was collected by centrifugation at 8,000 × g for 15 min at 4 °C and concentrated 10 times by volume using a Pellicon
Cassette concentrator (Millipore, Bedford, MA) with a 10,000 Mr cut-off membrane. The concentrated
supernatant was then dialyzed against 50 mM Tris-HCl
buffer, pH 7.8 (buffer A), and loaded onto a Q-Sepharose FF (Amersham
Pharmacia Biotech) column pre-equilibrated with the same buffer.
1,3-1,4-
-D-Glucanase proteins, either wild type or
mutants, were collected from eluants of the column using a 0-1
M NaCl salt gradient in buffer A. A second
nickel-nitrilotriacetic acid affinity column equilibrated with 50 mM sodium phosphate, pH 8.0, 0.3 M NaCl, and 10 mM imidazole buffer (buffer B) was then employed for
further purification of the enzymes. From a 10-300 mM
imidazole gradient eluant, a homogeneous enzyme preparation was
obtained, as verified by SDS-PAGE. Protein concentration was quantified
as described by Bradford (20) with bovine serum albumin as the standard.
Zymogram Analysis--
A zymogram was used to measure the
enzymatic activity of the wild type and mutant forms of
Fs
-glucanase, which was performed essentially according to a
reported method (21) with minor modifications. A 12%
SDS-polyacrylamide gel containing lichenan (1 mg/ml) and protein
samples in sample buffer (19) pretreated at 90 °C for 10 min was
prepared for zymogram analysis. After electrophoresis, the gel was
rinsed twice with 20% isopropyl alcohol in 50 mM sodium citrate buffer, pH 6.0, for 20 min to remove SDS, and then equilibrated in 50 mM sodium citrate buffer for 20 min. Before staining
with Congo red solution (0.5 mg/ml), the gel was preincubated at
40 °C for 10 min. The protein bands with
1,3-1,4-
-D-glucanase activity were then visualized
using the Congo red staining.
N-terminal Amino Acid Sequencing--
Protein samples for
N-terminal sequence determination were resolved on a 12%
SDS-polyacrylamide gel followed by electrophoretic transfer onto a
polyvinylidene difluoride membrane, using a Mini-Trans-Blot cell system
(Bio-Rad). Transferred protein bands on the membrane were visualized
using the 0.1% Amido Black staining and then excised with a clean
sharp razor blade. N-terminal amino acid sequencing was carried out on
an Applied Biosystems model 492 gas phase sequencer equipped with an
automated on-line phenylthiohydantoin analyzer.
Kinetic Studies--
The enzymatic activity of
1,3-1,4-
-D-glucanase was measured by determining the
rate of reducing sugar production from the hydrolysis of substrate
(lichenan). The reducing sugar was measured and quantified by the
method of Miller (22) with glucose as the standard. A standard enzyme
activity assay was performed in a 0.3-ml reaction mixture containing 50 mM sodium citrate buffer (pH 6.0) and 2.7-8 mg/ml lichenan
by starting the reaction with the enzyme addition. After incubation at
40 °C for 10 min, the reaction was terminated by the addition of a
salicylic acid solution (22). One unit of enzyme activity was defined
as the amount of enzyme required for releasing 1 µmol of reducing
sugar (glucose equivalent). The specific activity is expressed as
µmol of glucose/min/mg of protein. Various amounts of the purified
enzymes (0.24-82.7 µg/ml) were used in each kinetic assay reaction,
depending on the enzymatic activity of the enzyme. Kinetic data were
analyzed using the computer program ENZFITTER (Biosoft) and using
enzyme kinetics.
Circular Dichroism Spectrometry--
CD studies on the wild type
and mutant forms of F. succinogenes glucanase were carried
out in a Jasco J715 CD spectrometer and a 1-mm cell at 25 °C.
Spectra were collected from 200 to 260 nm in 1.3-nm increments, and
each spectrum was blank collected and smoothed by using the software
package provided with the instrument.
Fluorescence Spectroscopy--
The fluorescence emission spectra
of the wild type and mutant forms of F. succinogenes
1,3-1,4-
-D-glucanase were taken on an Amico-Bowman
series 2 spectrometer (Spectronic Instruments, Inc.) at 25 °C with
1 × 1-cm cuvette. Excitation spectra were taken at 282 nm, and
emission spectra were recorded at 302-440 nm, with a 4-nm slit for
both spectra. Protein samples were treated with 4-8 M urea
or without any pretreatment (native form) in 50 mM
phosphate pH 7.0 buffer before the spectra were recorded. The urea-denatured samples were followed with a renaturation procedure by
dialysis against 50 mM phosphate pH 7.0 buffer at 4 °C
for 24 h, and the fluorescence emission spectra of the protein
samples were then taken at the same parameters. The protein
concentration for the wild type and mutant forms of the enzyme was 30 µg/ml for each measurement.
 |
RESULTS |
Expression and Cellular Localization of the Recombinant
Fs
-glucanase in E. coli Cultures--
The conditions for expression
of Fs
-glucanase in engineered E. coli cells were
optimized in this study. The wild type and mutant forms of the enzyme
were effectively expressed and secreted into the LB medium as a soluble
protein when host cells were grown at 33 °C with IPTG induction.
Production of the enzyme in the whole E. coli culture was
detected 2 h after IPTG induction and reached a plateau of maximum
activity 8-16 h postinduction (data not shown). It was also found that
enzymatic activity was detected primarily in the cell-associated
fraction (cytosolic form) during the early stage (2-8 h) of induction,
and upon prolonged IPTG induction for up to 16 h, enzymatic
activity was mostly detected in the conditioned culture medium
(extracellular form). The pelB leader sequence in the
plasmid DNA construct was apparently fully functioning and facilitated
the effective secretion of Fs
-glucanase into the culture medium.
Approximately 60% of the extracellular secretion form of the proteins
was found as Fs
-glucanase after IPTG induction for 16-20 h, as
determined using SDS-PAGE and zymogram analyses (data not shown). The
results from SDS-PAGE and zymogram analyses are also in good agreement
with data from enzymatic activity assays (data not shown), suggesting
that the E. coli expressed enzyme can be collected as either
a cytosolic or an extracellular form from the host bacterial culture.
Purification and Biochemical Characterization of Wild Type and
Mutant Forms of Fs
-glucanase--
Homogeneous preparations of
various recombinant enzymes were obtained by fractionation with a
Q-Sepharose cation exchange column and followed by a separation with
nickel-nitrilotriacetic acid affinity column, as described under
"Experimental Procedures." The first 25-amino acid sequence at the
N terminus of the purified Fs
-glucanase was determined to be
MVSAKDFSGAELYTLEEVQYGKFEA, which represents a matured form of the
Fs
-glucanase enzyme without the presence of a pelB leader
peptide at the N terminus. The wild type enzyme has a molecular mass of
37,669 Da, as determined by mass spectrometry. The estimated
isoelectric point of the recombinant enzyme is pH 6.7, as analyzed by a
Genetics Computer Group, Inc. (Madison, WI) computer program. The
three-dimensional structure of this enzyme, to our knowledge, has not
been solved so far. Therefore, for the current study the target amino
acid residues for mutation were chosen based on the evaluation and
comparison of the amino acid sequence of the
1,3-1,4-
-D-glucanases isolated from different organisms
and on the prediction of their possible roles in catalysis. Fig.
1 shows that several of the amino acid residues of Fs
-glucanase, including Glu56,
Asp58, Glu60, and Gly63, are all
conserved in the compared amino acid sequences. Methionine 39 is the
only nonconservative amino acid residue observed in Fs
-glucanase; in
other words, the equivalent residues to position 39 of Fs
-glucanase
among other compared bacterial or fungal enzymes are all hydrophobic
residues, including phenylalanine, isoleucine, and leucine. The
expression conditions and purification protocol for the 11 mutant
enzymes, namely M39F, E56A, E56D, E56Q, D58A, D58E, D58N, E60A, E60D,
E60Q, and G63A, were similar to that of the wild type enzyme. Purity of
the wild type and mutant enzymes was evaluated by SDS-PAGE analysis
(Fig. 2). The wild type and the 11 mutant
enzymes exhibited identical mobility and are present as greater than
96% homogeneity when using electrophoresis as a criterion. Zymogram
analysis revealed that the mutant enzymes showed a similar or reduced
level of enzymatic activity as compared with the wild type enzyme (data
not shown). Similar protein expression profiles and yield levels were
obtained from the culture supernatants collected for the wild type and
the 11 mutant forms of Fs
-glucanase, as judged by SDS-PAGE analysis,
and protein concentrations were determined by Bradford assay.

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Fig. 1.
Amino acid sequence alignment of the putative
catalytic domain of
1,3-1,4- -D-glucanases
(Lic) and
1,3- -D-glucanases
(Lam). Tn-Lam, Thermotoga
neapolitana (30); Tm-Lam, Thermotoga
maritima (31); Pf-Lam, Pyrococcus furiosus
(32); Rm-Lam, Rhodothermus marinus; Rm-Lic,
R. marinus (33); Ox-Lam, Oerskovia
xanthinedytica (34); Bm-Lic, B. macerans
(35); Bp-Lic, Bacillus polymyxa (36);
Ba-Lic, B. amyloliquefaciens (37);
Bs-Lic, B. subtilis (38); Bl-Lic,
B. licheniformis (39); Ct-Lic, Clostridium
thermocellum (6); Op-Lic, Orpinomyces strain
PC-2 (5); Bb-Lic, Bacillus brevis (7);
Fs-Lic, F. succinogenes (16). The alignment was
optimized by introducing gaps, denoted by a dot, and
residues that are highly conserved in all sequences are
shaded. Numbers on the right are the
residue numbers of the last amino acid in each line.
Asterisks denote the candidate residues for mutation in this
study (R. Borriss and M. Krah, unpublished data; EMBL Data Base
accession number O52754.sp_ bacteria).
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Fig. 2.
SDS-polyacrylamide gel electrophoresis of the
purified wild type and mutant forms of F. succinogenes
1,3-1,4- -D-glucanase.
A 12% SDS-polyacrylamide gel was used and stained with Coomassie
Brilliant Blue R-250. Lane M, molecular mass standards;
lane 1, wild type; lanes 2-12, M39F, E56A, E56D,
E56Q, D58A, D58E, D58N, E60A, E60D, E60Q, and G63A mutants,
respectively.
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Fluorescence and Secondary Structure Analysis--
The emission
fluorescence spectra of the native, urea-denatured, and
denatured-renatured wild type and mutant forms of Fs
-glucanase were
analyzed. Fig. 3 shows the superimposed
emission spectra of both native wild type and G63A enzymes, with a
maximum emission peaked at 336 nm. The emission spectra of 8 M urea-denatured wild type and mutant enzymes were
bathochromic (red) shift and also superimposed with a maximum emission
at 350 nm and a slight shoulder peak at 376 nm (Fig. 3). Fluorescence
spectra similar to those of the native enzyme were also observed for
the wild type and mutant enzymes pretreated with 8 M urea
and followed with a renaturation protocol (via dialysis against
phosphate buffer for 24 h). In this case, the maximum emission
spectra for the wild type and G63A proteins have been found to shift
back to 336 nm, and the spectra were very similar to those of native
enzymes. These phenomena on emission fluorescence spectra of native,
denatured, and denatured-renaturated forms of the protein for the wild
type and G63A enzymes were also obtained for the E56Q mutant, which is
an enzymatically inactive Fs
-glucanase.

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Fig. 3.
Fluorescence emission spectra of wild type
and G63A mutant F. succinogenes
1,3-1,4- -D-glucanase.
Each enzyme was 30 µg/ml in 50 mM phosphate buffer, pH
7.5, or in 8 M urea-phosphate buffer. Excitation wavelength
was 282 nm. The fluorescence emission spectra for the native form of
wild type ( ) and G63A ( ) enzymes, the 8 M
urea-denatured wild type ( ) and the G63A ( ) enzymes, and the 8 M urea denatured-renatured wild type ( ) and G63A ( )
enzymes are shown in this figure.
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CD spectrometry was employed for analyzing the performance of the
native and urea-denatured then renatured wild type or mutant enzymes.
All of the tested native forms of the wild type and the 11 mutant
enzymes were shown to exhibit a maximum CD absorbance at 225 nm (data
not shown). The renatured forms of the wild type, E56Q, and G63A
glucanases showed CD spectra between 200 and 260 nm similar to their
correspondent native forms of the proteins (data not shown). These
results suggest that the single amino acid substitution of wild type
Fs
-glucanase in this study did not cause a global conformational
change or aberrant folding of the enzyme. Folded structures were
observed for the individual mutant enzymes as evidenced by analysis of
the fluorescent and CD spectra, relative to those of the wild type
enzyme. Therefore, the observed differences in the kinetic properties
between the mutant and wild type forms of the Fs
-glucanase enzyme
apparently are not due to the disruption of the structural integrity of
the enzyme.
Enzymatic activity assays were further carried out for the native and
renatured form of the wild type and mutant enzymes. After renaturation,
the wild type and mutant enzymes showed a recovery of ~85% activity
relative to their original native forms of the enzyme. The results from
the fluorescence, CD, and activity assays suggest that a great majority
of the denatured Fs
-glucanases, either as wild type or mutants, was
able to effectively reassociate to their correspondent native forms of
the protein. This study hence concludes that a reversible denaturation
capability was observed for Fs
-glucanase after the treatment with a
high concentration of urea.
Kinetic Analysis of Wild Type and Mutant Forms of
Fs
-glucanase--
Experiments on kinetic studies were mainly
performed by using lichenan as the substrate in standard enzyme assays,
as described under "Experimental Procedures." The specific activity
of the recombinant, wild type Fs
-glucanase expressed in E. coli cells is 1388 ± 82 units/mg, estimated with lichenan at
40 °C in 50 mM sodium citrate buffer, pH 6.0. This value
is slightly higher than that (960 units/mg) reported for the native
enzyme (15). The affinity for lichenan (Km), the
turnover number (kcat), and the catalysis
efficiency (kcat/Km) of the
wild type enzyme were 1.91 mg/ml, 871 s
1, and 456 s
1 (mg/ml)
1, respectively.
In an attempt to evaluate the effect of specific amino acid
substitutions on enzyme activity or other functions, the kinetic properties of the mutant enzymes were characterized. A dramatic change
in the turnover rate was found for the mutations on amino acid residues
Glu56, Asp58, and Glu60. For the
E56A, E56Q, D58A, E60A, and E60Q mutants, we showed that no enzymatic
activity was detectable for these variant protein forms. 2.5-, 241-, 570-, 880-, 540-, and 2.8-fold decreases in kcat
were observed for M39F, E56D, D58N, D58E, E60D, and G63A, respectively,
relative to the kcat value of the wild type
enzyme. These results suggest that amino acid residues
Glu56, Asp58, and Glu60 may play an
important role(s) for enzymatic activity and may be directly involved
in the catalysis of Fs
-glucanase. Small changes in
Km values for lichenan were observed for the E56D,
D58E, D58N, E60D, and G63A mutant enzymes; however, a 5.3-fold higher
Km for lichenan was found for M39F as compared with
the wild type enzyme. Comparison of the specificity constants, kcat/Km, is shown in Table
II. The specificity constants decreased
4.5- and 14-fold in G63A and M39F, respectively. However, more
significant changes (~210-970-fold decrease) were observed in the
single mutations of Glu56, Asp58, and
Glu60, relative to the wild type enzyme. These results
indicate that the three acidic amino acid residues in Fs
-glucanase
may play important roles in the catalysis of the enzyme. We have also
examined the substrate specificity of the wild type and mutant forms of Fs
-glucanase, by using lichenan, barley
-glucan, larminarine, carboxymethyl cellulose, and xylan as test substrates in enzymatic activity assays. No detectable binding affinity was observed for the
wild type or mutant Fs
-glucanase enzymes when larminarine, carboxymethyl cellulose, or xylan was used as the substrate.
Various buffers at different pH values were employed for evaluating the
optimum pH and pH tolerance of the Fs
-glucanase enzyme. Mutant and
wild type enzymes exhibited similar pH response profiles between pH 4 and pH 10 with the pH optimum value being between 6 and 8 (conferring
~100% enzyme activity). At pH 5.0 and pH 10.0, the wild type and
mutant enzymes were shown at only ~20 and 40% of their optimal
activities, respectively (data not shown). The optimum temperatures for
the wild type and D58N Fs
-glucanase were observed at 50 °C;
however, for the M39F, E56D, D58E, and E60D mutants the optimum
temperatures were found to have shifted to 40 °C as compared with
the wild type enzyme (data not shown). The G63A mutant shows an optimum
temperature at 30 °C, which is 20 °C lower than that of the wild
type enzyme.
Temperature Sensitivity of Wild Type and Mutant
Fs
-glucanases--
The temperature sensitivity of wild type and
mutant Fs
-glucanases was investigated to further characterize the
effect of introduced mutations. Replacement of amino acid residues in
Met39, Glu56, Asp58, and
Glu60 did not cause significant changes in thermostability,
as evaluated with a temperature range between 30 and 90 °C. The wild
type and mutant enzymes with specific mutations of acidic amino acid
residues were shown to exhibit similar enzymatic activity between 20 and 45 °C, but a dramatic loss of enzymatic activity was observed at
temperatures higher than 50 °C. In contrast, the G63A variant exhibited a greatly impaired thermostability, i.e. only 26%
of the original activity was observed when the enzyme was treated at
35 °C, and less than 10% of original activity was obtained when
treated at a temperature higher than 40 °C (Fig.
4).

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Fig. 4.
Temperature sensitivity of wild type and
mutant forms of F. succinogenes
1,3-1,4- -D-glucanase.
The purified wild type ( ), M39F ( ), E58Q ( ), and G63A ( )
enzymes at a enzyme concentration of 0.007-1.24 mg/ml were incubated
for 10 min at 35, 40, 45, 50, 55, 60, 65, 70, 75, and 80 °C,
respectively, in 50 mM citrate buffer, pH 6.0. Enzyme
activity was assayed by the method of Miller (22), as described under
"Experimental Procedures," immediately after incubation and is
expressed as a percentage of the relative activity at 40 °C. The
protein concentration was 0.24-82.7 µg/ml in each assay. Each assay
was performed either in duplicate or in triplicate.
|
|
Homologous Modeling--
F. succinogenes
1,3-1,4-
-D-glucanase is the only native, circular
permuted protein reported so far in the family 16 endoglucanase in
which the primary amino acid sequences of the enzyme family are
arranged in the order of domain A followed by domain B. However, a
reverse orientation of the two domains (B to A) was observed for
Fs
-glucanase. The three-dimensional structure of a de
novo circularly permuted variant, cpA16M-59, was reported by Hahn
et al. (23). This cpA16M-59
1,3-1,4-
-D-glucanase variant was generated by PCR
mutagenesis on the gene encoding an H(A16-M) hybrid enzyme with a
sequence organization corresponding to the F. succinogenes protein. In this study, a structural model of Fs
-glucanase was built
by Modeller 4 (24), using the homologous mimic enzyme cpA16M-59 with
Protein Data Bank entry 1cpm (23) as a template. The entire structural
model was briefly energy-minimized, and the final model has 96% of the
nonglycyl and nonpropyl residues falling in the most favored or in the
additionally allowed regions in the Ramachandran plot, as analyzed by
the PROCHECK program (25). The modeled Fs
-glucanase structure
consists mainly of two antiparallel
-sheets with seven and eight
strands, respectively (Fig. 5), arranged
atop each other to form a compact, sandwich-like structure. The overall
-sheet model structure of the Fs
-glucanase is similar to that of
cpA16M-59 with only minor changes in the surface loop regions. The two
-sheets are bent to give rise to a concave and a convex side of the
molecule. The Glu56, Asp58, and
Glu60 amino acid residues are located at the cleft on the
concave side of the protein molecule (Fig. 5), and this cleft was
likely the substrate binding side as previously suggested based on the
structure analysis of a protein-inhibitor complex of the
Bacillus 1,3-1,4-
-D-glucanase with
epoxyalkyl
-oligoglucosides (4).

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Fig. 5.
Ribbon diagram of the F. succinogenes
1,3-1,4- -D-glucanase
structure model. Two views of the model are shown here with the
second view rotated ~90° counterclockwise to the first one.
-Strands are drawn as arrows, and the residues subjected
for mutational analysis are displayed by ball-and-stick
diagrams. The active site of the enzyme is proposed to be located
at the concave surface of the central -sheet based on the protein
structure of cpA16M-59, which was used as the template for generating
the Fs -glucanase model. This figure was prepared using the program
MOLSCRIPT (40).
|
|
 |
DISCUSSION |
Bacterial 1,3-1,4-
-D-glucanases belong to the
category of retaining enzymes and are classified into family 16 endoglucanases (3). More than 50% protein sequence homology was found
among the bacterial enzymes, including those from
Clostridium and Bacilli and the ruminal fungus
Orpinomyces (5). In comparison, only ~30% homology was
found for the F. succinogenes
1,3-1,4-
-D-glucanase with respect to the other
bacterial enzymes. In addition, a naturally occurring circular
permutation structure was only found for the F. succinogenes
1,3-1,4-
-D-glucanase. Although the biochemical properties of the native F. succinogenes enzyme (15) and its encoding cDNA sequence have been characterized (16), further studies on the structure-function relationship were not reported. In
this study, a number of conserved amino acid residues, as revealed by
sequence comparison studies, were investigated for their potential involvement in the catalysis of the enzyme.
Studies on the three-dimensional structure of a covalent
protein-inhibitor complex of H(A16-M) with
3,4-epoxybutyl-
-D-cellobioside (4) have shown that the
inhibitor binds to the Glu105 amino acid residue, which
corresponds to the Glu56 residue of the
Fs
-D-glucanase enzyme, based on the amino acid sequence
alignment of these two proteins. Site-directed mutagenesis and chemical
modification of the glutamic acid residues, e.g. Glu105 and Glu109 in H(A16-M),
Glu105 in Bacillus amyloliquefaciens (26),
Glu103 in B. macerans (27), and
Glu134 and Glu138 in B. licheniformis (11), suggested that the glutamic acid residues are
crucial to catalysis of 1,3-1,4-
-D-glucanase. In this
study, possible functions of three acidic amino acid residues, including Glu56, Asp58, and Glu60
were investigated. These three residues are located at the cleft of the
concave side of the Fs
-glucanase protein molecule, as shown in the
structure model in Fig. 6. Dramatic
decreases in the turnover rate (kcat) of
Fs
-glucanase and minor changes in lichenan affinity relative to that
of wild type enzyme were found for the single mutations of
Glu56, Asp58, and Glu60 (Table II).
These results suggest that the structure of these mutant forms of
Fs
-glucanase remain undisturbed, thus allowing the substrates to
bind to the enzyme. The results from the fluorescence and circular
dichroism spectrometric studies also show evidence that the mutant
enzymes can retain a folded protein structure similar to that of the
wild type enzyme without undergoing significant global structural
changes (Fig. 3). However, severe disruption of the enzymatic activity
of Fs
-glucanase demonstrates that these three acidic residues may
play important roles in the catalytic mechanism.

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Fig. 6.
Stereoview of the putative active site of
F. succinogenes
1,3-1,4- -D-glucanase.
The side chain of Glu56, Asp58, and
Glu60 amino acid residues that are involved in the
catalysis are drawn in ball-and-stick diagrams with hydrogen
bonds networking (indicated as dashed lines) among the
residues of in the F. succinogenes
1,3-1,4- -D-glucanase structure model.
|
|
Structural modeling of the mutant enzymes was also undertaken to gain
more insight into the effect of mutation of Glu56,
Asp58, and Glu60. In this model, the carboxyl
group (O
-1 atom) of Glu56, corresponding to
Glu105 of cpA16M-59, is hydrogen bonded to the carboxyl
group (O
-1) of Asp58 (Fig. 6). Isofunctional
replacement of Glu56, Glu60, and
Asp58 with aspartate or glutamate, as in mutants E56D,
D58E, and E60D, yield low but detectable activities. This indicates
that a similar hydrogen-bonding network may be still maintained in the
mutant Fs
-glucanases with only minor local structural rearrangements in the side chains, which do not change the substrate binding affinity
in the mutant enzymes. Isosteric replacement of the Glu56
and Glu60 with glutamine rendered the enzyme inactivated,
although very low level residual enzymatic activity was still
detectable (e.g. 0.2-0.4% activity relative to the wild
type enzyme; Table II) when the glutamic acid residues were substituted
with aspartate. It has been suggested that the ionizable carboxylic
acid groups in the catalytic site, acting as a general acid for proton
transfer or as a nucleophile in their anionic form, were found to be
essential for the catalysis of Bacillus
1,3-1,4-
-D-glucanase. These observations are in good
agreement with the previous studies on Glu134 and
Glu138 of B. licheniformis (11, 12) and on
Glu105 and Glu109 of B. macerans
enzymes (26, 27), using site-directed mutagenesis and chemical rescue
approaches. The residue equivalent to Glu56 was proposed to
act as a catalytic nucleophile, and the Glu60 counterpart
was proposed to function as a general acid in the Bacillus
1,3-1,4-
-D-glucanases. Moreover, a comparison between the activity of E56Q (not detectable), D58N (0.2%), and E60Q (not detectable) further shows that Glu56 and Glu60
may be directly involved in catalysis and play important roles in the
enzymatic reaction. Detectable residual activity in D58N demonstrates
that the carboxyl group is not mandatory for the enzymatic reaction. It
is speculated here that Asp58 may play a structural role in
stabilizing the active site structure as well as an important
electrostatic role in affecting the pKa of the
nucleophilic residue of Glu56, as was previously suggested
for the equivalent residue of Asp58 in B. licheniformis glucanase (Asp136) (11). The enzymatic
activities of E56A, D58A, and E60A were not detected, suggesting that
the alanine side chain is neither capable of making hydrogen bonds with
the neighboring residues nor capable of serving in an electrostatic
role or acting as a general acid-base in the hydrolysis reaction of
Fs
-glucanase, which are important for maintaining the catalytic
function of the enzyme.
In the present Fs
-glucanase structure model, Met39 is
located at a position pointing away from the active site and is buried in the hydrophobic core of the enzyme. Based on the amino acid sequence
comparison of the bacterial and fungal
1,3-1,4-
-D-glucanases (Fig. 1), the corresponding
position to the amino acid residue Met39 of Fs
-glucanase
is phenylalanine or other hydrophobic residues (e.g. Ile or
Leu) in the compared bacterial or fungal enzymes. In this study, we
intended to mutate Met39 of Fs
-glucanase to
phenylalanine, and that resulted in 2-, 5-, and 14-fold decreases in
kcat, affinity for lichenan, and
kcat/Km, respectively, as
compared with that of the wild type enzyme. These results indicate that
the methionine at position 39 is essential for the Fs
-glucanase to
maintain the most effective catalytic efficiency for the enzyme.
Moreover, this mutation apparently has resulted in a more
heat-sensitive Fs
-glucanase; only ~30% of original activity was
detected after a heat treatment at 45 °C for 10 min. With the same
heat treatment, ~80% of original activity was maintained for the
wild type and the D58N mutant enzymes. However, this mutation is
tolerated in the folding of the entire structure, and this
interpretation also supported the results of fluorescence spectrometry
assays (data not shown).
Slight decreases in specific activity and substrate binding affinity
were observed for the replacement of Gly63 with alanine;
however, significant reduction of the enzyme stability was also
observed for this mutant. After incubation at 35 °C for 10 min, the
G63A mutant only retains ~25% of its original enzymatic activity.
Therefore, the conserved Gly63 residue among various
bacterial and fungal 1,3-1,4-
-D-glucanases (Fig. 1) is
first reported in this study as an important residue for enzyme
stability. This residue is located at the end of a
-strand, which
contains three active site residues, Glu56,
Asp58, and Glu60. It has been shown that the
thermal stability of the Fis protein (28) and the Arc repressor (29)
was largely reduced when glycines located in turn regions were mutated
to alanines. Therefore, it is possible that the Gly63 plays
a similar role in 1,3-1,4-
-D-glucanases in that it
stabilizes the folding of the entire protein. We found that only ~5%
residual activity was detected in the mutant enzyme treated with
40 °C for 10 min, whereas the wild type was still fully active after the same treatment.
In summary, this report first identified several key amino acid
residues that are involved in the catalysis of F. succinogenes 1,3-1,4-
-D-glucanase. It is proposed
here that Glu56 and Glu60 may function as
general acid/base residues based on the detailed comparison in
kinetics. We also demonstrated that Gly63 is important for
the stability of the enzyme. Our study has provided useful information
on the structure-function relationship of the naturally occurring,
circular permutated protein structure of a bacterial
1,3-1,4-
-D-glucanase isolated from rumen F. succinogenes. Further studies of the three-dimensional structure
of the enzyme using the x-ray crystallography approach are in progress.
 |
ACKNOWLEDGEMENTS |
We are grateful to Dr. Ning-Sun Yang
(Institute of BioAgricultural Sciences, Academia Sinica, Taiwan) for
encouragement and critical reading of this manuscript. We thank Dr.
L. B. Selinger and Dr. K. J. Cheng for providing us with a
pUC plasmid carrying the wild type F. succinogenes
1,3-1,4-
-D-glucanase gene.
 |
FOOTNOTES |
*
This work was supported in part by Research Grant
NSC88-2311-B-001-031 from the National Science Council and by Academia
Sinica, Taiwan.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.
To whom correspondence should be addressed: Inst. of
BioAgricultural Sciences, Academia Sinica, Taipei, Taiwan, 11529. Tel. or Fax: 886-2-27899322; E-mail:
lfshyur@ccvax.sinica.edu.tw.
Published, JBC Papers in Press, March 2, 2001, DOI 10.1074/jbc.M100843200
 |
ABBREVIATIONS |
The abbreviations used are:
Fs
-glucanase, F. succinogenes 1,3-1,4-
-D-glucanase;
PCR, polymerase chain reaction;
IPTG, isopropyl-
-D-thiogalactopyranoside;
PAGE, polyacrylamide
gel electrophoresis.
 |
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Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
Copyright © 2001 by the American Society for Biochemistry and Molecular Biology.