(Received for publication, November 7, 1994; and in revised form, November 30, 1994)
From the
In -glucans those
-1,4 glycosidic bonds which are
adjacent to
-1,3 bonds are cleaved by
endo-1,3-1,4-
-glucanases (
-glucanases). Here, the
relationship between structure and activity of the
-glucanase of Bacillus macerans is studied by x-ray crystallography and
site-directed mutagenesis of active site residues. Crystal structure
analysis at 2.3-Å resolution reveals a jellyroll protein
structure with a deep active site channel harboring the amino acid
residues Trp
, Glu
, Asp
, and
Glu
as in the hybrid Bacillus
-glucanase
H(A16-M) (Keitel, T., Simon, O., Borriss, R., and Heinemann, U.(1993) Proc. Natl. Acad. Sci. U. S. A. 90, 5287-5291).
Different mutant proteins with substitutions in these residues are
generated by site-directed mutagenesis, isolated, and characterized.
Compared with the wild-type enzyme their activity is reduced to less
than 1%. Several mutants with isosteric substitutions in Glu
and Glu
are completely inactive, suggesting a
direct role of these residues in glycosyl bond hydrolysis. The kinetic
properties of mutant
-glucanases and the crystal structure of the
wild-type enzyme are consistent with a mechanism where Glu
and Glu
are the catalytic amino acid residues
responsible for cleavage of the
-1,4 glycosidic bond within the
substrate molecule.
Endo-1,3-1,4--glucanase (
-glucanase,
1,3-1,4-
-D-glucan 4-glucanohydrolase, EC 3.2.1.73)
hydrolyzes
-1,4-linkages adjacent to
-1,3 linkages in mixed
linked polymeric
-glucans (Anderson and Stone, 1975). Genes
encoding bacterial
-glucanases have been cloned and sequenced from
different Bacillus species (Murphy et al., 1984;
Hofemeister et al., 1986; Tezuka et al., 1989;
Borriss et al., 1990; Lloberas et al., 1991; Gosalbes et al., 1991; Louw and Reid, 1993), Fibrobacter
succinogenes (Teather and Erfle, 1990), Ruminococcus
flavefaciens (Flint et al., 1993), and Clostridium
thermocellum (Schimming et al., 1992). All bacterial
endo-1,3-1,4-
-glucanases (``lichenases'') known to
date share sequence similarities with endo-1,3-
-glucanases
(``laminarinases'') and have been classified into glycosyl
hydrolase family 16 (Henrissat, 1991; Henrissat and Bairoch, 1993).
Like other -glycosidases,
-glucanase acts by a general
acid catalysis in which 2 acidic residues participate in a single or
double replacement reaction, resulting in the inversion, or more
likely, retention of the configuration at the anomeric carbon (Sinnot,
1990). General acid catalysis requires the participation of a proton
donor residue and a catalytic residue that is responsible for
nucleophilic attack on the substrate. A nucleophilic water molecule
completes the pathway by generating reaction products.
The crystal
structure of the hybrid Bacillus -glucanase H(A16-M) (
)(Keitel et al., 1993) suggests that
-glucan
hydrolysis takes place in a deep channel spanning one side of the
molecular surface and that residues Glu
,
Asp
, and Glu
(that correspond to
Glu
, Asp
, and Glu
in Bacillus macerans wild-type enzyme) are oriented toward the
active site cleft. Substitution by site-directed mutagenesis of
Glu
in B. macerans and of the corresponding
Glu
in Bacillus licheniformis
-glucanases
abolishes enzyme activity (Olsen, 1990; Planas et al., 1992).
Epoxybutyl-
-D-cellobioside (G4G-O-C
) has been
identified as the most effective inhibitor of Bacillus
-glucanase (Høj et al., 1992). The structure
analysis of this inhibitor bound to H(A16-M) revealed that the epoxide
is covalently attached to the side chain of Glu
(Keitel et al., 1993). Therefore it was suggested that Glu
acts by forming a covalent glycosyl enzyme intermediate or by
stabilizing the intermediate state through electrostatic interaction
with a transiently formed oxocarbonium ion.
This paper focuses on
the further identification of the catalytic residues in Bacillus -glucanase. In order to clarify the role of residues which
might be involved in the hydrolytic cleavage of
-glucan, we have
constructed different mutants of the B. macerans
-glucanase gene. Following expression in Escherichia
coli, mutant proteins substituted in putative catalytic residues
were characterized with regard to their enzymatic properties. The
results are discussed in relation to the crystal structure of the
enzyme which was determined at 2.3-Å resolution. In addition, the
observed differences in thermostability between B. macerans
-glucanase and H(A16-M) are considered in relation to the
crystal structures.
The
structure was solved with the molecular replacement facilities of
X-PLOR (Brünger, 1990) with the structure of the
hybrid -glucanase H(A16-M) (Keitel et al., 1993) as a
search model. Two peaks occurred in the rotation and Patterson
correlation functions that were used to orient the molecules. After
calculation of separate translation functions and combined rigid body
refinement a R-value of 35.4% for x-ray diffraction data
between 16 and 3 Å indicated that the molecules were located
correctly. The model was further refined with the simulated annealing
routine of X-PLOR (Brünger et al., 1990)
making use of non-crystallographic symmetry restraints. Positional and
temperature factor refinement was carried out with X-PLOR and for the
final steps of refinement with the program TNT (Tronrud et
al., 1987). Atomic coordinates and structure amplitudes have been
deposited in the Protein Data Bank, Brookhaven National Laboratory,
Upton, NY (entry code not yet assigned).
The two molecules in the asymmetric unit are
very similar and have identical secondary structure. After a least
squares superposition, the r.m.s. separation is 0.36 Å for
equivalent C and 0.41 Å for equivalent backbone atoms.
Considering the mean error in the atomic coordinates of 0.25 Å as
given by a Luzzati(1952) analysis there are thus no significant
conformational differences between the molecules. They show a
considerable difference of about 10 Å
, however, in
the average thermal parameter indicating a higher flexibility of
molecule 1 in the crystal lattice. This may be related to the higher
number of intermolecular hydrogen bonds formed by molecule 2 in the
crystal (Table 3). In this evaluation, an upper limit of 3.5
Å is used for the donor-acceptor distance. It can be seen that
molecule 1 has seven hydrogen bonds compared with eleven of molecule 2
and, more important, molecule 1 is in contact with only three other
molecules in the lattice, whereas molecule 2 is in contact with six.
The conformational differences between the independent molecules are
most pronounced in the loop regions 51-55 and 94-102. These
residues are without contacts to symmetry-related molecules in the
crystal, but they are poorly defined by the diffraction data and have
large temperature factors. They seem to be flexible parts in the
molecular architecture with little structural influence.
The
-glucanase is an all-
protein with a sandwich-like jellyroll
architecture. The two sheets stacking atop of each other consist of
seven antiparallel strands each that are bent and thus create a channel
on one side of the protein where the substrate is bound and hydrolyzed.
A disulfide bond is formed between Cys
and
Cys
. Loops between the
-strands are mostly stabilized
by
-turns. In addition, the analysis of the secondary structure
and hydrogen bonding with the program DSSP (Kabsch and Sander, 1983)
reveals one turn with
-helical geometry (Fig. 1). On the
convex side of the molecule, remote from the active site, a calcium ion
is bound which has been suggested to play a role in stabilizing the
protein structure (Borriss et al., 1989; Keitel et
al., 1994; Welfle et al., 1994). In
pentahedral-bipyramidal geometry it is coordinated to the carbonyl
oxygen atoms of Pro
, Gly
, and Asp
and the O
1 atom of the latter as well as to three water
molecules. In the related
-glucanase H(A16-M), this binding site
is occupied either by an octahedrally coordinated calcium or a
trigonal-bipyramidally coordinated sodium ion (Keitel et al.,
1994).
Figure 1:
Stereo drawing of the B. macerans -glucanase with
-strands drawn as arrows. The
calcium ion is drawn as a black ball, the S-S bridge between
residues 30 and 59 in ball-and-stick mode, and the
-helical turn between residues 188 and 191 as ribbon.
Some residue numbers are indicated to facilitate following the
polypeptide chain. This figure as well as Fig. 2Fig. 3Fig. 4were prepared with MOLSCRIPT
(Kraulis, 1991).
Figure 2:
Views along the substrate binding channel
of the B. macerans -glucanase. The side chains of the
hydrophilic amino acids Asn, Asp, Glu, Arg, Lys, and His in the channel
are shown in the top drawing. At the bottom the side chains of
the hydrophobic amino acids Phe, Tyr, Trp, Leu, Ala, Val, and Met are
shown.
Figure 3:
Comparison of conformation and
intramolecular hydrogen bonding of the NH-terminal region
of B. macerans
-glucanase and H(A16-M). Residues
15-212 of the former and 17-214 of the latter protein which
have identical sequence were matched in a least squares fit to
superimpose the NH
-terminal peptide portions. Residue
labels refer to the B. macerans protein (thick
lines). Hydrogen bonds are shown as dashed
lines.
Figure 4:
Active site geometry of B. macerans -glucanase. On the left, the cellobiose part of an
inhibitor bound covalently to H(A16-M) (Keitel et al., 1993)
has been fitted into the active site channel by least squares
superposition of the two proteins. In the blow-up on the right, looking through the channel the side chains of those
amino acids that are involved in catalysis are drawn in ball-and-stick representation with hydrogen bonds indicated as dashed lines.
At the bottom of the groove mainly polar side chains are located, especially acidic amino acid residues. They are thought to interact with the polar groups of the polysaccharide and to position it for cleavage. In contrast, the upper and lower rims of the crevice are lined with hydrophobic amino acid residues presumed to contribute to substrate binding through van der Waals interactions (Fig. 2).
The secondary structure of the wild-type and hybrid
enzymes differs in their NH-terminal region (Fig. 3). In both proteins the termini are close together in
space. The NH
-terminal
-strand of the wild-type enzyme
reaches from Ser
to Glu
, in H(A16-M) it is
shorter by 2 residues reaching from Phe
to Glu
.
In the B. macerans
-glucanase five main chain hydrogen
bonds are formed between residues 2, 4, and 6 of the
NH
-terminal strand and residues 211, 209, and 207 of the
COOH-terminal strand, respectively. Furthermore, the
NH
-terminal loop is stabilized internally by hydrogen bonds
from Asn
to Thr
and Trp
.
Conversely, in H(A16-M) only four main chain hydrogen bonds are found
between the NH
- and COOH-terminal strands and an additional
one between the NH
terminus and the loop from residue 67 to
residue 72, one of the four loops that connect the two seven-stranded
sheets. Thus, just by counting hydrogen bonds the small differences in
thermal stability cannot be explained. The size of a ``loop''
closed by a hydrogen bond may also play a role as well as subtle
differences in cation binding.
Hydrogen
bonds are formed between O1 of Glu
and N
1 of
Trp
and between O
2 of Glu
and O
1
of Asp
. In the absence of bound substrate a proton has to
be bound between the side chains of Glu
and Asp
to allow the formation of this hydrogen bond which is conceivable
at the pH of crystallization of 4.3. However, the conformation and
hydrogen bonding pattern of these amino acids is identical in the
wild-type enzyme and in hybrid H(A16-M), even though the latter was
crystallized from a neutral buffer. Therefore, the active site geometry
shown here appears to be representative for the entire family of
bacterial
-glucanases. In a similar way as Glu
, the
side chain of Glu
is involved in stabilizing hydrogen
bonding via its carboxyl oxygen to N
2 of Gln
which
in turn hydrogen bonds to Trp
N
1 via its O
1
atom.
Figure 5:
Mutations within the active site region of B. macerans -glucanase. A silent mutation was introduced
at residue 108 by site-directed mutagenesis to yield an EcoRI
restriction site. The four degenerated oligonucleotides shown were used
as primers to introduce changes in Trp
,
Glu
, Asp
, and Glu
. Altered
nucleotides are underlined and indicated by bold
letters. MAC-W101-Rev: W = 50% T, 50% A; MAC-E103-Rev: W
= 75% T, 25% A; MAC-D105-Rev: W = 75% A, 25% T;
MAC-E107-Dir: W = 75% A, 25% T and S = 75% G, 25%
C.
Figure 6:
Immunoblot of B. macerans wild-type and mutant -glucanases expressed in E.
coli. Extracts of DH5
cells harboring recombinant and vector
plasmid pTZ19R grown in LB medium were prepared and loaded onto
SDS-PAGE (15% acrylamide). Detection of
-glucanases was with
polyclonal antibodies raised against purified B. macerans
-glucanase.
Figure 7:
Circular dichroism spectra of B.
macerans wild-type and mutant -glucanases. The
samples were buffered in 2 mM cacodylate, pH 6.0, 1 mM CaCl
, at a protein concentration of 0.5 mg/ml. The
measurements were performed with a J720 spectrometer
(Jasco).
The mutations introduced in
sequence positions 101, 103, 105, and 107 are mostly conservative,
changing Trp to Phe or Tyr, Glu to Asp, Gln or His, and Asp to Asn or
Lys. Nevertheless, all result in a drastic loss in enzymatic activity
by a factor of at least 300 (Table 4). With the exception of
mutant E107D the change of active site residue affects mostly k and not K
. Replacement of
Trp
with another aromatic residue results in a
-glucanase variant with reduced, but clearly measurable, activity.
For glutamic acids 103 and 107 the isosteric replacement with a
glutamine side chain abolishes activity. Significantly reduced turnover
is observed when they are exchanged for aspartates preserving the
carboxylate function or, in the case of mutant E107H, with histidine.
The presence of ionizable groups at positions 103 and 107 thus appears
to be crucially important for catalysis. In contrast, the isosteric
replacement of the Asp
carboxylate with an amide function
in variant D105N leaves residual activity whereas
-glucan
hydrolysis by mutant D105K is no longer measurable. Hybrid H(A16-M)
-glucanase is identical with the B. macerans enzyme
except for 16 amino-terminal residues derived from B.
amyloliquefaciens
-glucanase (Olsen et al., 1991).
This polypeptide segment does not influence the enzymatic properties as
shown by introducing identical changes into the active site residues of
the hybrid H(A16-M) enzyme. The properties were found to be identical
with those of the B. macerans mutants. (
)
The crystal structure of B. macerans -glucanase
has been determined, and a number of key residues have been exchanged
in order to gain a basic understanding of the enzymatic function. The
three-dimensional structure of a Bacillus
-glucanase has
been described previously for the hybrid H(A16-M) (Keitel et
al., 1993), and site-directed mutagenesis experiments have been
reported for the B. licheniformis enzyme (Planas et
al., 1992; Juncosa et al., 1994). In the present study
x-ray crystallography and site-directed mutagenesis have been applied
to the same enzyme for the first time to define a framework for the
discussion of Bacillus
-glucanase function.
Enzyme
variants W101F, W101Y, E103D, E103Q, D105N, D105K, E107D, E107Q, and
E107H resulting from site-directed mutagenesis of the active site
residues Trp, Glu
, Asp
, and
Glu
show small or nonmeasurable residual activity. The
differential effects of the mutations can be discussed in the light of
the crystal structure. As indicated by the crystal structure of a
covalent H(A16-M)-inhibitor complex, the
-glucan substrate binds
to a pronounced channel on the molecular surface where the 4 residues
analyzed here form the catalytic site. For the wild-type B.
macerans enzyme studied here this view is supported by the
observation that those mutations in these residues which display
measurable residual activity tend to differ in k
much more than in K
. The such defined
catalytic site is quite unusual in that all four residues are on one
contiguous stretch of
-strand with connecting loop
(Trp
) where Glu
is tied in place by
hydrogen bonds to both Trp
and Asp
.
In
mutants W101F and W101Y a hydrogen bond to Glu can no
longer be formed, and consequently the enzyme activity is reduced.
However, a conservation of function at this sequence position is not
mandatory for enzymatic activity. A similar reduction to less than 1%
of wild-type activity is brought about by the isosteric replacement of
Asp
by Asn, whereas the mutant D105K is inactive. This
seems to indicate that a conservation of shape is required at position
105, but not necessarily of function. We note that a hydrogen bond to
Glu
may very well be formed by the Asn
amide group. At positions 103 and 107 mutations tend to cause
more drastic reductions in catalytic efficiency suggesting a direct
role of these residues in catalysis. Isosteric exchanges of the two
glutamic acid residues with glutamine as in mutants E103Q and E107Q
lead to enzymatically inactive protein variants whereas the
isofunctional replacements with aspartic acid as in mutants E103D and
E107D yield low, but measurable, activities. This reduction in
enzymatic activity may arise from the shortening of the side chains by
one methylene group and connected rearrangements in the active site.
Interestingly, mutant E107H shows slightly higher activity than E107D
and a shift in pH optimum. Residue His
is closer in shape
to glutamic acid and can also take up and release a proton. In summary,
the enzymatic activity of
-glucanase appears to require ionizable
groups at positions 103 and 107 as well as conservation of shape and/or
hydrophobicity for sequence positions 101 and 105.
A possible
mechanism for -glucanase action must take into account two general
considerations (Sinnot, 1990; Svensson and Søgaard, 1993).
First, the hydrolysis of the glycosyl bond can proceed either under
overall retention or inversion of the configuration at the anomeric
carbon C1. Since NMR analysis of the products of
-glucan cleavage
by the homologous B. licheniformis
-glucanase has proven
retention of configuration (Malet et al., 1993), we may safely
assume the same stereochemical course for the B. macerans enzyme. Overall retention of configuration requires two functional
groups of the enzyme present in an appropriate spatial setting and
acting as nucleophile (or by providing electrostatic assistance) and as
general acid, respectively, in a double displacement reaction. Second,
the reaction may proceed via an oxocarbonium ion or a covalently
enzyme-bound intermediate. This ambiguity cannot be resolved based on
the available data, although the covalent binding of epoxyalkyl
cellobioside inhibitors to Bacillus
-glucanases
(Høj et al., 1989, 1992; Keitel et al., 1993)
lends some support to the latter possibility.
The results of the
structural and mutational analysis of B. macerans -glucanase are consistent with a model that assigns a role as
general acid to Glu
and a role as catalytic nucleophile
or in electrostatic stabilization of an oxocarbonium ion to
Glu
. The crucial importance of the latter residue is also
evident from chemical modification studies of Bacillus
-glucanases which show it to be the site of inhibitor
attachment (Høj et al., 1992; Keitel et al.,
1993) and from further mutagenesis studies (Juncosa et al.,
1994). In this view, the general acid Glu
would activate
a water molecule for binding to the anomeric carbon. In the
three-dimensional structure, the networks of hydrogen bonds apparently
serving to position both the Glu
and Glu
carboxylate groups underline their functional importance.
The
involvement of two carboxylate functions in glycosylases working under
retention of configuration is rather common. It has been demonstrated,
for instance, for Agrobacterium glycosylase (Whithers et
al., 1990), cellobiohydrolases I and II and endoglucanase III from Trichoderma reesei (Divne et al., 1994; Rouvinen et al., 1990; Macarron et al., 1993), endocellulase
E2 from Thermomonospora fusca (Spezio et al., 1993),
and hen egg white as well as phage T4 lysozyme to which the bacterial
-glucanases appear to have some resemblance (Borriss et
al., 1990; Planas et al., 1992).
A comparison of the
sequences of bacterial lichenases and laminarinases suggests a set of
amino acids essential for catalysis and supports the previous findings.
Glu, Asp
, Glu
, and
Gly
are conserved throughout the compared sequences (Fig. 8, numbering according to B. macerans protein).
In the case of Gly
no function in catalysis is assumed,
but glycine tends to be conserved when space is scarce. Other amino
acids like isoleucines 102 and 104 are also highly invariant. The
changes from Ile to Val observed in the F. succinogenes and B. circulans enzymes maintain the apolar character of the side
chain. Starting with Glu
and ending with Phe
there is a strict alternation of polar (acidic) and nonpolar side
chains in the catalytic sites of the
endo-1,3-1,4-
-glucanases which reflects the regular way in
which side chains subtend from a
-strand toward the hydrophobic
interior or the surface of the protein. This pattern is disturbed in
the endo-1,3-
-glucanases by insertion of a methionine residue
between Ile
and Glu
of B. macerans
-glucanase. Assuming generally similar active site geometry
and catalytic mechanism this residue must be accommodated by formation
of a
-bulge (Richardson et al., 1978) which would allow
the Met side chain to point toward the hydrophobic core and the Glu to
subtend into the channel and to participate in catalysis. The
concomitant structural rearrangements of the active site are proposed
to cause the changed substrate specificity toward
-1,3 linkages of
the laminarinases. However, B. macerans
-glucanase
variants carrying an inserted methionine in the active site region were
found to be enzymatically inactive, suggesting that further
modifications are necessary to extend the substrate specificity of Bacillus
-glucanases.
Figure 8:
Conserved region of bacterial
endo-1,3-1,4--glucanase and endo-1,3-
-glucanase
sequences. The numbering is according to the B. macerans enzyme. Lichenases (Lic) are from B. macerans (Borriss et al., 1990), B. amyloliquefaciens (Hofemeister et al., 1986), B. licheniformis (Lloberas et al., 1991), Bacillus subtilis (Murphy et al., 1984), Bacillus polymyxa (Gosalbes et al., 1991), B. brevis (Louw and
Reid, 1993), C. thermocellum (Schimming et al.,
1992), R. flavefaciens (Flint et al., 1993) and F. succinogenes (Teather and Erfle, 1990).
``Laminarinases'' (Lam) are from Rhodothermus marinus (Spilliaert et al., 1994), C. thermocellum (W.
Schwarz, personal communication) and B. circulans (Yahata et al., 1990).