©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Crystal Structure and Site-directed Mutagenesis of Bacillus macerans Endo-1,31,4--glucanase (*)

(Received for publication, November 7, 1994; and in revised form, November 30, 1994)

Michael Hahn (1) Ole Olsen (2) Oliver Politz (3) Rainer Borriss (3) Udo Heinemann (1)(§)

From the  (1)Forschungsgruppe Kristallographie, Max-Delbrück-Centrum für Molekulare Medizin, Robert-Rössle-Strasse 10, D-13122 Berlin, Federal Republic of Germany, the (2)Department of Physiology, Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-2500 Copenhagen-Valby, Denmark, and the (3)Fachbereich Biologie, Humboldt-Universität, Warschauer Strasse 43, D-10243 Berlin, Federal Republic of Germany

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

In beta-glucans those beta-1,4 glycosidic bonds which are adjacent to beta-1,3 bonds are cleaved by endo-1,3-1,4-beta-glucanases (beta-glucanases). Here, the relationship between structure and activity of the beta-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 beta-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 beta-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 beta-1,4 glycosidic bond within the substrate molecule.


INTRODUCTION

Endo-1,3-1,4-beta-glucanase (beta-glucanase, 1,3-1,4-beta-D-glucan 4-glucanohydrolase, EC 3.2.1.73) hydrolyzes beta-1,4-linkages adjacent to beta-1,3 linkages in mixed linked polymeric beta-glucans (Anderson and Stone, 1975). Genes encoding bacterial beta-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-beta-glucanases (``lichenases'') known to date share sequence similarities with endo-1,3-beta-glucanases (``laminarinases'') and have been classified into glycosyl hydrolase family 16 (Henrissat, 1991; Henrissat and Bairoch, 1993).

Like other beta-glycosidases, beta-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 beta-glucanase H(A16-M) (^1)(Keitel et al., 1993) suggests that beta-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 beta-glucanases abolishes enzyme activity (Olsen, 1990; Planas et al., 1992). Epoxybutyl-beta-D-cellobioside (G4G-O-C(4)) has been identified as the most effective inhibitor of Bacillus beta-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 beta-glucanase. In order to clarify the role of residues which might be involved in the hydrolytic cleavage of beta-glucan, we have constructed different mutants of the B. macerans beta-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 beta-glucanase and H(A16-M) are considered in relation to the crystal structures.


MATERIALS AND METHODS

Crystallization, Diffraction Data Collection, and Structure Analysis

The B. macerans beta-glucanase was concentrated to 6 mg/ml and stored in a 10 mM sodium acetate buffer, pH 4.3, with 5 mM CaCl(2). The precipitant solution additionally contained 30%, w/v, ammonium sulfate and 20 mM MgCl(2). Crystals were grown by mixing equal volumes of protein and precipitant solution in a hanging-drop vapor diffusion setup at room temperature. They were enlarged by macroseeding to approximately 0.3 times 0.3 times 0.1 mm^3 within 2 or more weeks. Two diffraction data sets with a resolution to 2.3 Å were collected on a MAR-Research imaging plate detector mounted on an Enraf-Nonius FR571 rotating anode x-ray generator. The space group was identified by the autoindexing routine XDS (Kabsch, 1993) as C2 with cell constants a = 203.68 Å, b = 42.41 Å, c = 60.70 Å, and beta = 101.27°, suggesting a V(m) value (Matthews, 1968) of 2.7 Å^3/Da with two molecules in the asymmetric unit. The data were processed with MOSFLM as part of the CCP4 program suite (Collaborative Computational Project, Number 4, 1994).

The structure was solved with the molecular replacement facilities of X-PLOR (Brünger, 1990) with the structure of the hybrid beta-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).

Strains and Vectors

E. coli DH5alpha, vector plasmid pTZ19R, and expression plasmid pUC13-M harboring the B. macerans beta-glucanase gene have been described (Olsen et al., 1991). Expression plasmid pTZ19MAC is identical to pUC13-M except for the vector sequences. Cells were grown in LB medium supplemented with 100 µg/ml ampicillin when necessary.

Mutagenesis

Site-directed mutagenesis was performed by the polymerase chain reaction as described by Kadowaki et al. (1989). The mutated sequences were obtained by mutagenic primer-directed fragment amplification. Afterwards, an amplified sequence was introduced by replacing a cassette within the B. macerans gene. Plasmids pUC13-M and pTZ19-H(A16-M) were used as template DNA for mutagenesis. In order to simplify site-directed mutagenesis, an additional EcoRI site was created at residue 108. The sequence of the primer P1-EcoRI-Dir also covers the unique EcoRV restriction site at residues 105-106: 5`-gat atc gaa ttc tta gga aaa gac aca acg. The amplified DNA was cloned as EcoRV/HindIII fragment into the B. macerans gene cut at the same restriction sites. Primers DIR and dIII-LS specifying the 5` and 3` ends have been described (Politz et al., 1993). Plasmids containing the mutated sequences were checked by DNA sequencing.

Protein Expression and Purification of Wild-type and Mutant Enzymes

Proteins were expressed from E. coli cells harboring plasmids encoding the wild-type and mutant enzymes essentially as described previously (Politz et al., 1993). Whole cell extracts were prepared by ultrasonication and diaultrafiltrated against 50 mM sodium acetate, pH 5.0, 10 mM CaCl(2). The sample was applied to a column containing CM-Sepharose CL-6B (Pharmacia) and eluted with a linear gradient of 0-400 mM sodium chloride. Fractions containing beta-glucanase were collected and further purified by gel chromatography using Sephacryl S-200 (Pharmacia). The proteins obtained were separated by SDS-polyacrylamide gel electrophoresis and blotted onto nitrocellulose filters. These were incubated with serum containing antibodies raised against B. macerans beta-glucanase. Proteins reacting with the antibodies were detected using the Western blot AP System (Promega).

Characterization of beta-Glucanases

CD spectra of wild-type and mutant beta-glucanases were recorded on a J720-spectrophotometer (Jasco, Tokyo, Japan) between 180-260 and 250-340 nm. Enzyme was dissolved in 2 mM cacodylate, pH 6.0, 1 mM CaCl(2) at a final concentration of 0.5 mg/ml. beta-Glucanase activity of purified wild-type and mutant proteins was assayed by the release of reducing sugars from lichenan (Olsen et al., 1991). Other biochemical analyses and isoelectric focussing were performed as described (Politz et al., 1993).


RESULTS

Crystal Structure of the Endo-1,3-1,4-beta-glucanase of B. macerans

Structure refinement against all structure amplitudes > 2 between 8.0 and 2.3 Å converged at a R-value of 16.3%. One Ca ion per enzyme molecule and a total of 175 water molecules were located. The diffraction data and refinement results are summarized in Table 1and Table 2. In both independent molecules in the asymmetric unit, residues 96-99 show temperature factors up to 90 Å^2 and are poorly defined by electron density. 87 and 85%, respectively, of the residues are in the most favorable regions of the Ramachandran diagram as defined by Laskowski et al. (1993).





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 Calpha 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^2, 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 beta-glucanase is an all-beta 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 beta-strands are mostly stabilized by beta-turns. In addition, the analysis of the secondary structure and hydrogen bonding with the program DSSP (Kabsch and Sander, 1983) reveals one turn with alpha-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^7, Gly, and Asp and the O1 atom of the latter as well as to three water molecules. In the related beta-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 beta-glucanase with beta-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 alpha-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 beta-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(2)-terminal region of B. macerans beta-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(2)-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 beta-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).

Comparison with H(A16-M) and Origin of Different Thermostability

The hybrid H(A16-M) differs from the wild-type B. macerans beta-glucanase only by the 16 NH(2)-terminal amino acids that are derived from the Bacillus amyloliquefaciens protein. As expected, the three-dimensional structures of the proteins are very similar (see Table 2). This holds true although three protein molecules in three different crystal environments are compared. It confirms the remarkable conformational rigidity of the bacterial beta-glucanases observed earlier (Hahn et al., 1994). On the other hand, differences in thermostability have been described for various hybrid and parental beta-glucanases (Politz et al., 1993). For example, the temperature required to reduce the enzymatic activity to 50% within 10 min is 72.4 °C for the wild-type B. macerans beta-glucanase studied here, but 75.0 °C for H(A16-M). Is it possible to explain this small difference in stability from the crystal structures?

The secondary structure of the wild-type and hybrid enzymes differs in their NH(2)-terminal region (Fig. 3). In both proteins the termini are close together in space. The NH(2)-terminal beta-strand of the wild-type enzyme reaches from Ser^2 to Glu^6, in H(A16-M) it is shorter by 2 residues reaching from Phe^6 to Glu^8. In the B. macerans beta-glucanase five main chain hydrogen bonds are formed between residues 2, 4, and 6 of the NH(2)-terminal strand and residues 211, 209, and 207 of the COOH-terminal strand, respectively. Furthermore, the NH(2)-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(2)- and COOH-terminal strands and an additional one between the NH(2) 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.

Active Site Structure

The geometry of the active site of B. macerans beta-glucanase is shown in Fig. 4. For a better orientation, the cellobiose moiety of an inhibitor bound covalently in the crystal of H(A16-M) (Keitel et al., 1993) has been fitted into the active site channel by least squares superposition of the protein atoms. Near the presumed catalytic site, the side chains of Trp, Glu, Asp, and Glu point toward the channel, whereas those of Asp, Ile, and Ile point into the hydrophobic interior of the enzyme. Trp plays an important role in enzyme activity (see below), although it is located in a flexible surface loop. The remaining catalytic site residues are on one contiguous stretch of beta-strand.

Hydrogen bonds are formed between O1 of Glu and N1 of Trp and between O2 of Glu and O1 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 beta-glucanases. In a similar way as Glu, the side chain of Glu is involved in stabilizing hydrogen bonding via its carboxyl oxygen to N2 of Gln which in turn hydrogen bonds to Trp N1 via its O1 atom.

Construction of Mutant Proteins

Single substitutions of Trp, Glu, Asp, and Glu were achieved by cassette mutagenesis of the gene taking advantage of either a unique EcoRV restriction site in the vicinity of the site of the mutational event or an artificial EcoRI restriction site introduced at the codon of residue 108. In order to obtain substitutions in Trp, Glu, and Asp mutation primers containing degenerated oligonucleotides in the desired mutation site and a silent mutation creating an additional EcoRI restriction were used as reverse primers (Fig. 5). DNA fragments encoding the NH(2)-terminal part with the mutated sequence in the active site region of the beta-glucanase were amplified, cut by BstEII and EcoRI, and ligated back into the beta-glucanase gene sequence with the appropriate deletion. The expected substitutions were screened by DNA sequencing. Mutants W101F, W101Y, E103D, E103Q, E103K, D105N, and D105K were isolated. Mutations within Glu were generated by using primer Mac-E107-Dir as direct primer for amplification of a DNA fragment encoding the COOH-terminal part of the B. macerans beta-glucanase. DNA sequencing revealed mutations E107H, E107D, and E107Q. All mutant genes obtained in this way were cloned into pTZ19R vector plasmid and transformed into E. coli DH5alpha cells.


Figure 5: Mutations within the active site region of B. macerans beta-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.



Characterization of Mutant Proteins

Analysis of mutant and wild-type beta-glucanases using polyclonal antibodies raised against B. macerans beta-glucanase revealed that E. coli cells expressed approximately the same amount of precipitating material (Fig. 6). The mutant proteins were purified from E. coli and their enzymatic properties were compared. To ensure that the protein structures are not affected by site-directed mutagenesis, CD spectra of the wild-type and mutated variants were obtained. These revealed only subtle differences in secondary structure (Fig. 7). Temperature optima of the enzyme reactions catalyzed by the wild-type and the active mutant proteins were found to be essentially similar except for mutant E105H in which the optimum is slightly decreased.


Figure 6: Immunoblot of B. macerans wild-type and mutant beta-glucanases expressed in E. coli. Extracts of DH5alpha cells harboring recombinant and vector plasmid pTZ19R grown in LB medium were prepared and loaded onto SDS-PAGE (15% acrylamide). Detection of beta-glucanases was with polyclonal antibodies raised against purified B. macerans beta-glucanase.




Figure 7: Circular dichroism spectra of B. macerans wild-type and mutant beta-glucanases. The samples were buffered in 2 mM cacodylate, pH 6.0, 1 mM CaCl(2), 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(m). Replacement of Trp with another aromatic residue results in a beta-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 beta-glucan hydrolysis by mutant D105K is no longer measurable. Hybrid H(A16-M) beta-glucanase is identical with the B. macerans enzyme except for 16 amino-terminal residues derived from B. amyloliquefaciens beta-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. (^2)




DISCUSSION

The crystal structure of B. macerans beta-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 beta-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 beta-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 beta-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(m). The such defined catalytic site is quite unusual in that all four residues are on one contiguous stretch of beta-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 beta-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 beta-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 beta-glucan cleavage by the homologous B. licheniformis beta-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 beta-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 beta-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 beta-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 beta-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-beta-glucanases which reflects the regular way in which side chains subtend from a beta-strand toward the hydrophobic interior or the surface of the protein. This pattern is disturbed in the endo-1,3-beta-glucanases by insertion of a methionine residue between Ile and Glu of B. macerans beta-glucanase. Assuming generally similar active site geometry and catalytic mechanism this residue must be accommodated by formation of a beta-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 beta-1,3 linkages of the laminarinases. However, B. macerans beta-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 beta-glucanases.


Figure 8: Conserved region of bacterial endo-1,3-1,4-beta-glucanase and endo-1,3-beta-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).




FOOTNOTES

*
This study was made possible by grants from the Deutsche Forschungsgemeinschaft (to U. H. and R. B.), by the Fonds der Chemischen Industrie, and by Grant 6028 from the Danish Program for Food Technology to D. von Wettstein (to O. O.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 49-30-9406-3420/2270; Fax: 49-30-9406-2548; uh{at}orion.rz.mdc-berlin.de.

(^1)
The abbreviations used are: H(A16-M), hybrid beta-glucanase with residues 1-16 derived from the B. amyloliquefaciens enzyme and residues 17-214 from the B. macerans enzyme; W101F . . . etc. (see ``Construction of Mutant Proteins'' under ``Results''), mutant B. macerans beta-glucanase with a single site substitution of Trp with Phe . . . etc.; r.m.s., root mean square.

(^2)
K. Piotukh and R. Borriss, unpublished data.


ACKNOWLEDGEMENTS

We are grateful to W. Schwarz for communicating the sequence of the laminarinase (licA) prior to publication. W. Saenger, O. Simon, and H. Welfle are thanked for providing laboratory facilities and for support in purification and CD analysis of wild-type and mutant beta-glucanases. Cloning of active site mutants into hybrid H(A16-M) glucanase by K. Piotukh and helpful comments on the manuscript by D. von Wettstein, J. J. Müller, and Ch. Förster are gratefully acknowledged.


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