(Received for publication, February 6, 1995; and in revised form, July 6, 1995)
From the
The amino acid sequences of -glucosidases from Cellvibrio gilvus and Agrobacterium tumefaciens show
about 40% similarity. The pH/temperature optima and stabilities and
substrate specificities of the two enzymes are quite different. C.
gilvus
-glucosidase exhibits an optimum pH of 6.2-6.4
and temperature of 35 °C, whereas the corresponding values for A. tumefaciens are 7.2- 7.4 and 60 °C, respectively. The
substrate specificity of A. tumefaciens enzyme toward
different aryl glycosides is broader than C. gilvus enzyme. To
analyze these properties further, three chimeric
-glucosidases
were constructed by substituting segments from the C-terminal
homologous region of C. gilvus
-glucosidase gene with
that of A. tumefaciens. The chimeric enzymes were
characterized with respect to pH/temperature activity and stability and
substrate specificity. Chimeric enzymes exhibited chromatographic
behavior similar to that of C. gilvus enzyme. However,
enzymatic properties of chimeras were admixtures of those of the two
parents. The chimeric enzymes were optimally active at 45-50
°C and pH 6.6-7.0. K
values of
chimeric enzymes for the various saccharides were admixtures of both
parental enzymes. These results suggest that the two domains of C.
gilvus and A. tumefaciens enzymes probably can fold
independently. The homologous C-terminal region in
-glucosidase
appears to play an important role in determining enzyme
characteristics. Changes in the properties on substitution of segments
in this region might be related to the enzyme specificity, and
-glucosidases with improved properties can be prepared by
manipulating this region.
The enzyme -glucosidase (EC 3.2.1.21) catalyzes the
hydrolysis of alkyl- and aryl-
-D-glucosides
(methyl-
-D-glucoside and p-nitrophenyl-
-D-glucoside) as well as
glycosides containing only carbohydrate residues (Cellobiose). On the
basis of substrate specificity,
-glucosidases can be classified as
aryl-
-glucosidases, cellobiases, and those hydrolyzing both
aryl-
-glucosides and oligosaccharides. The last group is often
found in cellulolytic microorganisms(1, 2) . On the
basis of sequence homology,
-glucosidases have been divided into
two subfamilies(2) : BGA (
-glucosidases and
phospho-
-glucosidases from bacteria to mammals) and BGB
(
-glucosidases from yeasts, molds, and rumen bacteria). It is one
of the components of the cellulase enzyme complex required for the
hydrolysis of cellulose to glucose by catalyzing the final step which
converts cellobiose to glucose(3, 4) .
The study of
these enzymes has been facilitated by the use of recombinant DNA
technology(1, 5, 6) . Although a number of
cellulase genes including several -glucosidases have been cloned
and expressed in both Escherichia coli and Saccharomyces
cerevisiae(7, 8, 9, 10) , their
enzymological properties, especially structure-function relationships,
have not been well understood, partially because most of the cellulases
show little sequence homology. Analysis of structure-function
relationships may be facilitated by the formation of chimeric
genes/enzymes produced by gene fusion(11) .
Cellvibrio
gilvus, a cellulose-metabolizing bacterium, has the unique
property of producing cellobiose in high yields from acid-swollen
cellulose(12) . The isolation and characterization of the
cellulase, xylanase, and -glucosidase systems of this organism (13, 14, 15) as well as the cloning,
analysis, and manipulation of the genes coding these enzymes (16, 17) have been investigated in our laboratory. The
-glucosidases from C. gilvus share conserved regions in
-glucosidases from different organisms. The nucleotide sequence of
the
-glucosidase gene revealed that this enzyme belongs to the BGB
group of
-glucosidases(15) . The amino acid sequences of
the C. gilvus
-glucosidase gene show significant
similarity (about 40%) with those of a
-glucosidase gene from Agrobacterium tumefaciens(18) . Despite this
similarity, their enzymatic properties, especially pH activity, thermal
stability, and substrate specificity, are quite different. To analyze
these properties further, chimeric
-glucosidases were constructed
between them by substituting different segments from one enzyme in the
C-terminal homologous region of the other and comparing the enzyme
characteristics of parental and chimeric enzymes. The C-terminal region
seems to be important for
-glucosidase activity, since deletion of
more than a 70-base pair fragment from the C-terminal part of C.
gilvus
-glucosidase gene resulted in the loss of enzyme
activity. (
)Although, the deletion of about 100 amino acid
residues near the C-terminal region of the
-amylase gene did not
affect enzyme activity(19) , cyclomaltodextrin
glucanotransferases lacking 30 amino acids (20) and an
endoglucanase lacking 75 amino acids (21) from the C-terminal
end showed no enzyme activity. Keeping in mind the importance of the
C-terminal region and the estimated location of the catalytic center of
Asp-291 in the N-terminal region of C. gilvus(15) ,
the C-terminal region was selected for the construction of chimeric
enzymes.
C. gilvus, A.
tumefaciens, and chimeric -glucosidase preparations were
partially purified by ion exchange chromatography. C. gilvus and the chimeric enzyme preparations were applied to a FPLC system
(Pharmacia LKB Biotechnology Inc.) using a column of SP Sepharose Fast
Flow HiLoad(TM) 26/10 (bed volume 53-58 ml) equilibrated with
25 mM acetate buffer (pH 5.0). The proteins were eluted with a
linear gradient of 0-1 M NaCl in the same buffer. In the
case of
-glucosidases of A. tumefaciens, the enzyme
preparation was applied to a column of Q Sepharose Fast Flow
HiLoad
26/10 (bed volume 53-58 ml) equilibrated with
20 mM bis-tris propane (pH 6.5). The proteins were eluted with
a linear gradient of 0-1 M NaCl in the same buffer. The
partially purified fractions were used for the determination of enzyme
characteristics.
For determination of kinetic parameters, C.
gilvus -glucosidase and chimeric CHBSM
-glucosidase were
further purified on a large scales. Ten liters of cultures were
centrifuged at 5000
g for 10 min, and cells were
suspended in 100 ml of 25 mM MOPS buffer (pH 6.5). The enzyme
solution was obtained after sonification of the cells and removal of
the cell debris by centrifugation. The enzyme solution was applied to a
column of SP Sepharose Fast Flow. The enzymes were eluted with a linear
gradient of 0-1 M NaCl in 20 mM acetate buffer,
pH 5.0. Active fractions were pooled, dialyzed, and applied to a column
of Mono Q (Pharmacia). The enzymes were eluted with a linear gradient
of 0-1 M NaCl. The active fractions were pooled,
concentrated, and finally applied to a gel filtration column of
Superose 6 (Pharmacia). The enzymes were eluted with 25 mM acetate buffer (pH 5.0) containing 0.15 M NaCl.
Homogeneity of the purified enzyme preparations was monitored by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis on a PhastSystem
(Pharmacia).
The amino acid sequences of -glucosidases from C. gilvus and A. tumefaciens show significant
similarity on most of the parts. In particular, the region from Ala-541
to Pro-811 of
-glucosidase from A. tumefaciens is quite
similar to the region from Ala-472 to Pro-741 of
-glucosidase from C. gilvus (Fig. 1). Considering the translation frame
and similar regions of both genes, three regions in the C. gilvus
-glucosidase gene (plasmid pCG5) were selected for
substitution with the A. tumefaciens
-glucosidase gene.
Schematic representation of the structure of the chimeric
-glucosidase gene is shown in Fig. 2. The region between
the NdeI and HinfI sites starting from Cys-517 and
over stop codon in pcbg1 were substituted at the BsmI site of
pCG5 to obtain the chimeric enzyme CHBSM. Similarly, two other chimeric
enzymes were obtained by substituting the region between two AvaII sites starting from Ile-594 in pcbg1 at the AgeI site of pCG5 (CHAGE) and the region between SfiI
and HinfI sites starting from Asp-660 in pcbg1 at the BsaBI site of pCG5 (CHBSA). The clones expressing chimeric
enzymes were purified, and their plasmids were characterized by
restriction analysis. Plasmid pCHBSM encoding chimeric enzyme CHBSM has
two SfiI sites in the inserted fragment of pcbg1, whereas
pCHAGE1 and pCHBSAB1 encoding chimeras CHAGE and CHBSA, respectively,
have two EcoRV sites in the inserted fragments of pcbg1 (Fig. 3).
Figure 1:
Homology in amino acid sequences of
-glucosidases from C. gilvus and A. tumefaciens. AT and CG represent A. tumefaciens and C. gilvus, respectively. A, schematic representation
of sequence homology in N-terminal (solid) and C-terminal (hatched) regions of
-glucosidase genes. B,
amino acid sequences of A. tumefaciens and C. gilvus
-glucosidases in the C-terminal region. Identical and similar
amino acid residues are designated by
and
, respectively.
Chimeric enzymes were constructed by shuffling the regions marked by arrowheads.
Figure 2:
Schematic representation of parental and
chimeric -glucosidase genes. Light and dark bars represent regions derived from C. gilvus and A.
tumefaciens, respectively. Restriction enzymes used for the
construction of chimeric enzymes are shown with open
arrowheads, whereas the restriction enzymes used for the
confirmation of chimeric plasmids are shown with filled
arrowheads.
Figure 3:
Restriction analysis of chimeric plasmids
on agarose gel electrophoresis. Lane 1, HindIII
digest of DNA marker; lane 2, BioMarker; lane 3, SfiI-digested pCHBSM1; lane 4, BamHI/EcoRV-digested pCHAGE1; lane 5, BamHI-digested pCHBSAB1; lane 6, SfiI-digested pCG5; and lane 7, BamHI-digested pCG5. The 1.2-kilobase band in pCHBSM1 and
0.7-kilobase band in pCHAGE1 and pCHBSAB1 were created by the SfiI and EcoRV sites, respectively, in the inserted
gene.
The pH optima for C. gilvus and A. tumefaciens enzymes are 6.2-6.4 and 7.2-7.4,
respectively. These enzymes also show marked differences in their
temperature optima. -Glucosidase from C. gilvus is
optimally active at 35 °C, whereas that of A. tumefaciens exhibits maximum activity at 60 °C. With regard to heat
stability,
-glucosidase from C. gilvus shows complete
activity up to 30 °C, retains about 80% of its maximum activity at
35 °C, and inactivates completely at 55 °C. On the other hand, A. tumefaciens enzyme is stable up to 55 °C, and, even at
65 °C, it retains 60% of its maximum activity. A. tumefaciens enzyme specificity toward aryl-glycoside substrates is broader
than of C. gilvus enzyme.
The pH activity profiles of
chimeric -glucosidases are shown in Fig. 4. Chimeric
enzymes showed intermediate profiles of their parents. CHBSM enzyme
exhibited the maximum activity at pH 6.6-7.0, about 40% at pH
8.0, and no activity at pH 10.0. The optimum pH of CHAGE enzyme was
6.8-7.0 with about 35% activity at pH 8.0. CHBSA enzyme was
optimally active at pH 6.6 and inactivated at pH 9.0. All the chimeras
were stable between pH 4 and 9, whereas the
-glucosidases from C. gilvus and A. tumefaciens were stable at pH
4-8 and pH 5-10, respectively. Substitution of segments in
the homologous C-terminal region seems to have a marked influence on pH
activity and stability. In Bacillus cyclomaltodextrin
glucanotransferase (20) and cellulase(24) , pH activity
profiles were found to be influenced by the N- and the C-terminal
parts.
Figure 4:
pH
activity (A) and pH stability (B) profiles of
chimeric and parental -glucosidases. The pH was adjusted with
buffers: citrate (pH 3.0-4.0), MES (pH 5.0-6.8), MOPS (pH
7.0-8.0), and CHES (pH 9.0-10.0). For pH stability
experiments, enzyme was incubated at different pH values for 1 h at 25
°C. The residual activities were measured under standard assay
conditions.
--
, CHBSM;
--
, CHAGE;
--
, CHBSA;
--
, C. gilvus;
--
, A.
tumefaciens.
The chimeric -glucosidases also exhibited a significant
variation in temperature optimum from their parent enzymes (Fig. 5). The chimeric enzymes were optimally active at
45-50 °C, showing an intermediate temperature optimum between C. gilvus and A. tumefaciens enzymes. CHBSM exhibited
maximum activity at 50 °C, and 61% of its maximum activity at 60
°C. On the other hand, CHAGE was optimally active at 50 °C and
exhibited 52% of its maximum activity at 60 °C. CHBSA showed the
temperature optima of 45 °C with no activity at 70 °C. Heat
stability experiments revealed that CHBSM enzyme was completely active
up to 45 °C, retained about 65% of its maximum activity at 55
°C, and was completely inactivated at 55 °C. CHAGE enzyme was
stable up to 40 °C, and, even at 55 °C, 50% of its maximum
activity was retained. CHBSA was least stable among the three chimeras.
It was stable up to 40 °C, and retained only 20% of its maximum
activity at 55 °C. The temperatures at which 50% loss of the enzyme
activities occurred were 41, 67, 57, 55, and 50 °C for C.
gilvus, A. tumefaciens, CHBSM, CHAGE, and CHBSA enzymes,
respectively. Thus heat stability of chimeric enzymes was increased by
9-16 °C as compared to C. gilvus enzyme.
Figure 5:
Temperature optima (A) and heat
stability (B) profiles of chimeric -glucosidases. For
heat stability experiments, each enzyme at its optimum pH was treated
at different temperatures for 1 h. The residual activities were
measured under standard assay conditions.
--
, CHBSM;
--
,
CHAGE;
--
, CHBSA;
--
, C. gilvus;
--
, A.
tumefaciens.
Heat
stability may be influenced by only a few amino acid substitutions (17, 25) . In general, protein stability increases
with the insertion into an -helix of helix-forming amino acids
(alanine, glutamic acid etc.) and decreases with the insertion of
helix-breaking amino acids (proline, glycine etc.). The secondary
structures of the parental and chimeric enzymes were predicted by
Robson's method(26) . There were similar numbers of
helix-breaking but more helix-forming amino acid residues in the
-helix regions of chimeric enzymes than C. gilvus enzyme,
suggesting that it could be one of the factors influencing the heat
stability of chimeras. Hydrophobic interaction inside the protein
molecule is another important factor in stabilizing protein structure.
Hydrophobic cluster analysis (27, 28) of native and
chimeric enzymes revealed that the amino acid substitution from C.
gilvus to A. tumefaciens significantly increased the
hydrophobic properties of the chimeric enzymes. These substitutions
might be important for heat stability of
-glucosidase.
Thus,
the pH activity and heat stability were changed distinctly by
substituting different segments of C. gilvus -glucosidase
gene with that of A. tumefaciens. It is interesting to note
that these changes were more pronounced with the increased size of the
insertion fragment. For example, CHBSM containing the largest insertion
fragment from A. tumefaciens
-glucosidase exhibited
broader pH optima than the other two chimeras. Thermal stability was
also found to be in the order of CHBSM > CHAGE > CHBSA. In
chimeric isopropylmalate dehydrogenase from an extreme thermophile, Thermus thermophile, and a mesophile, Bacillus
subtilis, the stability of each chimeric enzyme was approximately
proportional to the content of the amino acid sequence from the T.
thermophile enzyme(29) .
While the K value of the
chimeric citrate synthases similarly have been found to be lower than
those of the parental enzymes(30) , substrate affinity
decreased by about 2-fold in active human-yeast chimeric
phosphoglycerate kinase engineered by domain interchanges(31) .
However, no significant differences were found between the K
values of parental and chimeric
isopropylmalate dehydrogenases(23) . Replacement of the
catalytic base Glu-400 by glutamine in Aspergillus niger glucoamylase was found to affect both substrate ground-state
binding and transition state stabilization(32) . K
values for maltose and maltoheptaose
were 12- and 3- fold higher for the Glu-400
Gln mutant, with K
values 35- and 60-fold lower, respectively, as
compared with those of the wild type enzyme. Similarly, in Aspergillus awamori glucoamylase mutants, Ser-119
Tyr,
Gly-183
Lys, and Ser-184
His, slightly higher activity
for maltose hydrolysis and lower activity for isomaltose as compared
with the wild type enzyme was observed by Sierks and Svensson (33) . The observed increase in selectivity was attributed to
the stabilization of the maltose transition-state complex for each
enzyme. Modulation of binding energy by mutation could be attributed to
modification in hydrogen
bonding(32, 34, 35) .
The relative rates
of hydrolysis of cello-oligosaccharides by parental and chimeric
enzymes are shown in Table 2.
4-Methylumbelliferyl--glucoside was found to be the best substrate
for all of the enzymes. Both C. gilvus and A. tumefaciens
-glucosidases releases 4-methylumbelliferone and glucose in
parallel from 4-methylumbelliferyl-
-glucoside (data not shown). C. gilvus enzyme hydrolyzes cellobiose only 11.9% as fast as
4-methylumbelliferyl-
-glucoside, whereas hydrolysis rates of
cellobiose by A. tumefaciens enzyme was 20.2%. Hydrolysis
rates of cellotetraose, cellopentaose, and cellohexaose by C.
gilvus enzymes were 3-5 times higher than that of the A.
tumefaciens enzyme. The hydrolysis rates of these oligosaccharides
by chimeric enzymes were similar to that of the C. gilvus enzyme and higher than that of the A. tumefaciens enzyme.
The products released by enzymatic action on different
cello-oligosaccharides were analyzed by HPLC. The C. gilvus enzyme hydrolyzed each oligosaccharide into smaller
oligosaccharides. However, cellobiose was hardly hydrolyzed. On the
other hand, the A. tumefaciens enzyme hydrolyzed each
oligosaccharide into glucose and the oligosaccharides smaller than the
original ones by one glucose unit. The chimeric enzymes more or less
exhibited patterns similar to that of C. gilvus enzyme.
Genetic construction of chimeric enzymes from two functionally
related proteins, sharing extensive sequence similarity, is expected
not only to provide valuable information on the structure-function
relationship of the parent proteins, but also to prepare enzymes with
improved properties. Enzymatic activities are one of the sensitive
criteria for judging the correct folding of engineered proteins. Our
results demonstrate that different combinations of homologous
C-terminal regions of -glucosidases from C. gilvus and A. tumefaciens resulted in the formation of enzymatically
active chimeric species. The C-terminal region in the
-glucosidase
gene plays an important role in determining enzyme characteristics, and
the changes in enzymatic properties on substitution of the C-terminal
segments might be related to enzyme specificity. Chimeric
-glucosidases with improved enzymatic properties can be prepared
in a convenient and effective way by manipulating this region.