©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Tetrad of Ionizable Amino Acids Is Important for Catalysis in Barley -Glucanases (*)

(Received for publication, November 24, 1994; and in revised form, January 19, 1995)

Lin Chen (1)(§) Thomas P. J. Garrett (2)(¶) Geoffrey B. Fincher (3) Peter B. Høj (1) (4)(**)

From the  (1)Department of Biochemistry, La Trobe University, Bundoora, Victoria 3083, Australia, the (2)Biomolecular Research Institute, 343 Royal Parade, Parkville, Victoria 3052, Australia, and the Departments of (3)Plant Science and (4)Horticulture, Viticulture, and Oenology, University of Adelaide, Waite Campus, PMB1 Glen Osmond, South Australia 5064, Australia

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Determination of the crystal structures of a 1,3-beta-D-glucanase (E.C. 3.2.1.39) and a 1,3-1,4-beta-D-glucanase (E.C. 3.2.1.73) from barley (Hordeum vulgare) (Varghese, J. N, Garrett, T. P. J., Colman, P. M., Chen, L., Høj, P. B., and Fincher, G. B.(1994) Proc. Natl. Acad. Sci. U. S. A. 91, 2785-2789) showed the spatial positions of the catalytic residues in the substrate-binding clefts of the enzymes and also identified highly conserved neighboring amino acid residues. Site-directed mutagenesis of the 1,3-beta-glucanase has now been used to investigate the importance of these residues. Substitution of glutamine for the catalytic nucleophile Glu (mutant E231Q) reduced the specific activity about 20,000-fold. In contrast, substitution of glutamine for the catalytic acid Glu (mutant E288Q) had less severe consequences, reducing k approximately 350-fold with little effect on K. Substitution of two neighboring and strictly conserved active site-located residues Glu (mutant E279Q) and Lys (mutant K282M) led to 240- and 2500-fold reductions of k, respectively, with small increases in K. Thus, a tetrad of ionizable amino acids is required for efficient catalysis in barley beta-glucanases. The active site-directed inhibitor 2,3-epoxypropyl beta-laminaribioside was soaked into native crystals. Crystallographic refinement revealed all four residues (Glu, Glu, Lys, and Glu) to be in contact with the bound inhibitor, and the orientation of bound substrate in the active site of the glucanase was deduced.


INTRODUCTION

The enzymic hydrolysis of polysaccharides and glycosides is a critically important process in the metabolism of plants, animals, and microorganisms (Sinnott, 1990). The basic mechanistic assumption, supported with few exceptions by the available data, is that enzyme-mediated glycoside hydrolysis is initiated by protonation of glycosidic oxygen atoms via an active site-located general acid. Following protonation, the aglycon is released, and a positively charged oxycarbonium ion is stabilized by a nucleophilic amino acid, most often a carboxylate (reviewed by Svensson and Søgård(1993)). The exact mode of stabilization is not known but is thought to be either electrostatic or via a covalent enzyme-substrate intermediate. Hydration of the stabilized oxycarbonium ion or, alternatively, hydrolysis of the covalent enzyme-substrate intermediate serves to regenerate the reducing terminus of the glycosyl moiety.

Amino acids mediating in the protonation and stabilization are referred to as the catalytic acid and the catalytic nucleophile, respectively. Identification of such catalytic residues has been attempted in a number of enzymes using chemical modification (Svensson et al., 1990), x-ray crystallography (Anderson et al., 1981), or homology searches coupled with site-directed mutagenesis of residues thought to be involved (Trimbur et al., 1992). Due to theoretical and practical constraints in each of these techniques, a combined approach is required for a reliable definition of the functional importance of individual amino acids at the active sites of enzymes (Schimmel, 1993).

To define the molecular basis for catalysis and substrate specificity in polysaccharide endohydrolases, we have employed a range of mechanism-based inhibitors to define geometric differences within the active sites of a family of beta-glucan endohydrolases (Høj et al., 1989b, 1991, 1992). In particular, detailed investigations of 1,3-beta-D-glucan glucanohydrolases (E.C. 3.2.1.39) and 1,3;1,4-beta-D-glucan 4-glucanohydrolases (EC 3.2.1.73), two classes of polysaccharide hydrolases with closely related substrate specificities, led to the conclusion that the acquisition of distinct substrate specificities in the evolution of these related enzymes did not require the recruitment of novel catalytic amino acids but rather that it involved differences in the positioning of catalytic residues at the active site and/or changes in residues responsible for substrate binding (Chen et al., 1993a).

A 1,3-beta-glucanase, designated isoenzyme GII, (^1)and a 1,3;1,4-beta-glucanase, designated isoenzyme EII, have recently been crystallized (Chen et al., 1993b). Their three-dimensional structures reveal a complete spatial conservation of both the catalytic nucleophile (Glu in the 1,3-beta-glucanase and Glu in the 1,3-1,4-beta-glucanase) and the proposed general acid (Glu in both enzymes) (Varghese et al., 1994). To further investigate the structural basis for catalysis and substrate specificity in these enzymes, we have now established bacterial expression systems for both classes of enzyme and used site-directed mutagenesis of the 1,3-beta-glucanase to determine the relative importance of these putative catalytic residues in enzyme action. Inspection of the three-dimensional structures reveals that the catalytic amino acids Glu and Glu are spatially close to the functional groups of Glu and Lys. We have also tested the contribution of these two neighboring conserved residues (Glu and Lys) and conclude they play an important role in binding and/or catalysis. This conclusion was independently corroborated by structural studies, which revealed that Glu and Lys are in intimate contact with a mechanism-based inhibitor covalently linked to the nucleophile Glu.


MATERIALS AND METHODS

Bacterial Strains, Plasmids, and Oligonucleotides

Escherichia coli BMH71-18 mutS (thi supEDelta(lac-proAB)(mutS::Tn10) F`(proAB+ lacI^qlacZ DeltaM15)) was supplied by Pharmacia Biotech Inc., and plasmid pMAL-c2 was supplied by New England Biolabs. Oligonucleotides were synthesized on an Applied Biosystems DNA synthesizer.

Construction of Expression Vectors

A cDNA encoding mature 1,3-beta-glucanase isoenzyme GII was generated by PCR amplification of a full-length cDNA (Høj et al., 1989a) using Vent(R) DNA polymerase (New England Biolabs) and two primers (A: 5`-ATCGGCGTGTGCTACGGCGTGATC-3` and B: 5`-GCCGGAATTCCAGCTAGGTAGCTACACTAGAA-3`).

Primer A is identical to the nucleotides encoding Ile of the 1,3-beta-glucanase, while primer B is complementary to the 3`-end of the cDNA (nucleotides TTC to GCT) but is extended to incorporate an EcoRI site in the PCR product. For PCR, reaction mixtures containing 30 ng of template, 150 pmol of each primer, 300 µM each of dNTPs, and 1 unit of Vent(R) DNA polymerase in 50 µl of Vent(R) buffer (consisting of 10 mM KCl, 10 mM (NH(4))(2)SO(4), 20 mM Tris-HCl buffer (pH 8.0), 2 mM MgSO(4), and 0.1% Triton X-100) were heated for 5 min at 94 °C followed by 25 cycles of denaturation (94 °C, 60 s), annealing (45 or 55 °C, 60 s), and extension (72 °C, 90 s). Amplified PCR fragments were purified by agarose gel electrophoresis, cut with restriction enzyme EcoRI, and ligated into the pMAL-c2 vector previously cut with XmnI and EcoRI to generate the 1,3-beta-glucanase isoenzyme GII expression vector pMAL-c2-GII, which was used to transform E. coli strain DH5alpha (supE44DeltalacU169(80lacZDeltaM15)). Colonies were grown on an LB agar plate containing 100 µg/ml ampicillin and restreaked onto a master LB ampicillin plate and an LB ampicillin plate containing 0.02% X-gal and 100 µM IPTG for selection of white colonies. This procedure was adopted because the presence of 100 µM IPTG on the first selection plate drastically reduced the number of transformants. Subsequent experiments revealed that reducing the IPTG concentration to 10 µM diminished this problem. Recombinant clones expressing the MBP-GII fusion protein were further selected by activity measurements and electrophoretic analysis of unpurified bacterial extracts.

Expression vectors directing the synthesis of barley 1,3-1,4-beta-glucanase isoenzymes EI and EII were constructed exactly as described above, using cDNAs encoding the respective enzymes (Fincher et al., 1986; Slakeski et al., 1990). The forward primer A for both cDNAs was 5`-ATCGGGGTGTGCTACGGCATGAGC-3`, and the reverse primers B for isoenzymes EI and EII were 5`-CCGGGAATTCCGTAGCTGCTACCTCATCAGAA-3` and 5`-GGCGGAATTCCCGAGCACGAGCTCCGTCAGAA-3`, respectively. Strains expressing MBP-GII, MBP-EI, and MBP-EII are referred to as DH5alphaMBP-GII, DH5alphaMBP-EI, and DH5alphaMBP-EII, respectively.

Mutagenesis

Site-directed mutagenesis of the 1,3-beta-glucanase cDNA was carried out by the unique restriction enzyme site elimination procedure (Deng and Nickoloff, 1992) using a U.S.E. mutagenesis kit (Pharmacia Biotech Inc.) with double-stranded plasmid DNA (pMAL-c2-GII) as a template. The primers designed to introduce the desired mutations were E231Q, 5`-GTGGTGGTGTCGCAGAGCGGGTGGCCG-3`; E279Q, 5`-GCCATGTTCAACCAGAACCAGAAGACC-3`; E288Q, 5`-GGGGACGCCACGCAGAGGAGCTTCGGG-3`; and K282M, 5`-CGAGAACCAGATGACCGGGGACGCC-3`. The codons mutated are underlined. Two selection primers were made. The primer 5`-CAGACTAATTCGAGGCCTAACAACAACAACAA-3`, which converts the SacI site in the polylinker of the pMAL-c2 to an StuI site, was used to obtain mutants E279Q and E288Q. The primer 5`-CGGTATTTCACACCGGCTAGCGTGCACTCTC-3`, which is designed to convert the unique NdeI site in pMAL-c2 to an NheI site, was used to obtain mutants E231Q and K282M. The mismatched bases in the selection primers are underlined. All primers were phosphorylated at their 5`-end before use, and the mutagenesis procedure was performed essentially as prescribed by the manufacturer. Mutants were confirmed by dideoxynucleotide sequencing using a Sequenase version 2.0 sequencing kit (U. S. Biochemical Corp.).

Purification of Recombinant Fusion Proteins

E. coli strain DH5alphaMBP-GII carrying the pMAL-c2-GII wild-type vector or its mutated derivatives were grown overnight at 37 °C in LB broth containing 100 µg/ml ampicillin, and 10 ml of the culture was used to inoculate 1 liter of LB broth containing 0.2% glucose and 100 µg/ml ampicillin. The culture was grown at 37 °C with vigorous shaking, and when A reached a value of 0.5, IPTG was added to a final concentration of 1 mM to induce synthesis of the fusion proteins. Following 3 h of additional growth, cells were harvested by centrifugation at 4 °C (4000 times g for 30 min), suspended in 3 ml of lysis buffer (50 mM Tris-HCl buffer, pH 8.0, containing 1 mM EDTA and 0.1 M NaCl) per g of wet cells, and lysed with lysozyme (Sambrook et al., 1989) in a modified procedure that incorporated a single freeze-thaw step. The supernatant collected by centrifugation at 12000 times g at 4 °C for 20 min was defined as the crude extract and used in further purification.

For purification of the wild-type enzyme, crude extract from 1 liter of culture was diluted 10-fold with 15 mM Tris-HCl buffer, pH 8.0, and applied at a flow rate of 2.5 ml/min to a DEAE-Sepharose fast flow (Pharmacia) column (3 times 11.5 cm) equilibrated with 25 mM Tris-HCl buffer, pH 8.0. After washing the column exhaustively, bound proteins were eluted with a linear 0-250 mM NaCl gradient in 1.2 liters of equilibration buffer. Fractions containing enzyme activity were pooled, desalted, and adjusted to 25 mM sodium acetate buffer, pH 5.0, by ultrafiltration (Amicon PM10 filter) before application to a CM-Sepharose fast flow (Pharmacia) column (2 times 10.5 cm) equilibrated with 25 mM sodium acetate buffer, pH 5.0. After exhaustive washing, bound proteins were eluted with a linear 0-200 mM NaCl gradient in 1 liter of equilibration buffer. Fractions containing pure protein were pooled to give 5.0 mg of active fusion protein.

Mutant enzymes were purified by a single ion-exchange chromatography step employing a shallow salt gradient elution. The crude extract from 4-5 liters of culture was diluted 10-fold with 15 mM Tris-HCl buffer, pH 8.0, and applied at a flow rate of 2.5-3.0 ml/min to a DEAE-Sepharose column (5 times 21 cm) equilibrated with 12.5 mM Tris-HCl buffer, pH 8.5. After exhaustive washing, bound proteins were eluted with a 1.9-liter linear 0-80 mM NaCl gradient at a flow rate of 2.0 ml/min. Fractions containing pure fusion protein were located by SDS-polyacrylamide gel electrophoresis, pooled, concentrated, and adjusted to 25 mM sodium acetate buffer, pH 5.0, by ultrafiltration before clarification by centrifugation. The yield of pure fusion protein varied between 10 and 20 mg, depending on the mutation introduced.

Soaking and X-ray Analysis

To investigate the complex of native 1,3-beta-glucanase isoenzyme GII and the active site-directed inhibitor(2, 3) -epoxypropyl beta-D-laminaribioside (Rodriguez and Stick, 1990) by x-ray diffraction analysis, native crystals (Chen et al., 1993b) were soaked for 3 months at room temperature in 100 mM sodium acetate buffer, pH 5.0, containing 100 mM(2, 3) -epoxypropyl beta-D-laminaribioside and ammonium sulfate at 55% saturation. Diffraction data to 2.8 Å resolution were collected using a MarResearch detector and processed as described earlier (Varghese et al., 1994). For data 14-2.8 Å resolution and I geq 2, there were 20,800 observations, 13,134 reflections, and R = 0.076. From an F(o) (complex) - F(o) (native) Fourier map, two sites for the inhibitor were located in the 40 Å long glucan-binding cleft. They were the most significant features in the map (represented by all peaks 8-4.5 standard deviations), and one site showed electron density for a glucose unit and the aglycon covalently connected to the enzyme at Glu. The other site appears to bind inhibitor non-covalently, the epoxide group of the aglycon probably being ``inactivated'' by the high level of (NH(4))(2)SO(4) in the crystallization medium during the prolonged soaking. For the initial model, R = 0.217 for data 6-2.8 Å resolution. Cycles of energy minimization and refinement of temperature factors (Brünger, 1992) were indispersed with inspection of difference density and 2F(o) - F(c) maps. During this procedure, additional water molecules were located, and R was reduced to 0.151. After simulated annealing and further energy minimization, the refinement was complete with R = 0.143. In the final model, the rms deviations from ideal bond lengths and angles were 0.017 Å and 2.02°, respectively, and there were 83 water molecules. Refined omit maps were used to confirm the location of inhibitor molecules.

Enzymic Activity and Thermostability

Enzyme activity was assayed at 37 °C with the substrate laminarin (Sigma) as previously described (Chen et al., 1993a). 1 unit of enzyme activity is defined as the amount of enzyme releasing 1 µmol of reducing equivalents per minute. The protein concentration of wild-type enzyme was measured with a BCA protein assay reagent (Pierce) using bovine serum albumin as a standard. The protein concentrations (relative to wt-MBP-GII) of mutant enzymes and the native 1,3-beta-glucanase isoenzyme GII purified from barley were standardized by the method of Ball(1986). Kinetic parameters were determined at 37 °C using laminarin as a substrate over the concentration range 0.4-4.0 mg/ml in 50 mM sodium acetate buffer, pH 5.0. The K(m), V(max), and k values were derived from Lineweaver-Burk plots. The pH dependence of enzyme activities was measured at pH 3.5, 4.3, 5.0, 5.7, and 6.5 (pH 3.5-5.0 in 50 mM sodium acetate buffer; pH 5.7 in 50 mM sodium succinate buffer; and pH 6.5 in 50 mM MES buffer).

Thermostabilities of the purified enzymes were determined by measuring residual activity after incubation in 50 mM sodium acetate buffer, pH 5.0, containing 0.5 mg/ml bovine serum albumin at 50 or 55 °C. Analytical gel filtration chromatography was carried out at room temperature using a Superose-12 HR 10/30 fast protein liquid chromatography column (Pharmacia) operated at a flow rate of 0.5 ml/min in 35 mM sodium acetate buffer, pH 5.0, containing 0.1 M NaCl.


RESULTS AND DISCUSSION

Production of beta-Glucanase Fusion Proteins in E. coli

Attempts to synthesize the authentic, mature form of barley 1,3;1,4-beta-glucanase isoenzyme EII in E. coli have not been successful (Olsen, 1990). For this reason, and to facilitate purification procedures, we decided to express recombinant barley beta-glucanases as fusion proteins. Previously characterized cDNAs encoding mature 1,3-beta-glucanase isoenzyme GII and 1,3-1,4-beta-glucanase isoenzymes EI and EII were inserted downstream from the E. coli malE gene in pMAL-c2 plasmids with the view to employ a one-step purification of the fusion proteins on amylose resin and the subsequent release of beta-glucanases from the fusion proteins by cleavage with factor Xa protease (Maina et al., 1988). All three constructs directed the synthesis of large amounts of a 74-kDa protein species in response to IPTG induction (Fig. 1); this corresponds to a fusion protein consisting of the maltose-binding protein (43 kDa) and the barley beta-glucanase (approximately 32 kDa). The 1,3- and 1,3-1,4-beta-glucanase activities were readily detected in crude bacterial lysates following IPTG induction, but not before induction. One-step purification of the fusion proteins on amylose resins was not successful. Although homogeneous preparations of the fusion proteins were obtained from the amylose column, yields of active enzyme were very low. A simple ion-exchange procedure was therefore employed to prepare larger quantities of active protein (Fig. 1).


Figure 1: Purification of MBP-glucanase fusion proteins synthesized in E. coli. SDS-polyacrylamide gel electrophoresis analysis was performed, and proteins were visualized with Coomassie Brilliant Blue. Lane 1, molecular weight markers; lane 2, extract from non-induced DH5alphaMBP-EI; lane 3, extract from IPTG-induced DH5alphaMBP-EI; lane 4, extract from IPTG-induced DH5alphaMBP-EII; lane 5, extract from IPTG-induced DH5alphaMBP-GII; lane 6, MBP-GII pool after DEAE-Sepharose step; lane 7, MBP-GII pool after CM-Sepharose step, lane 8, extract from IPTG-induced DH5alphaMBP-GII(E279Q); lane 9, purified MBP-GII(E279Q).



Attempts to cleave the purified fusion protein with factor Xa protease were unsuccessful, a result which can now be explained by inspection of the three-dimensional structures of the 1,3- and 1,3-1,4-beta-glucanase. The amino-terminal residue of each enzyme is almost totally buried within the core of the alpha/beta barrel structures (Varghese et al., 1994) (Fig. 2). Access of factor Xa to the scissile bond in the folded fusion proteins is therefore not possible.


Figure 2: Schematic view of barley endoglucanases showing the orientation of the catalytic groove with respect to MBP. The main chain of isoenzyme GII showing secondary structure elements as determined (Varghese et al., 1994) is shaded. The closedarrow points to the amino terminus, the point of attachment of MBP, while the openarrow points to the extended substrate binding cleft and the catalytic site.



Properties of the MBP-Glucanase Fusion Proteins Are Very Similar to Those of Native beta-Glucanases Purified from Germinating Barley

The high levels of active fusion protein and the relative ease of purification make them an attractive choice for the study of glucanase structure-function relationships. Although cleavage by factor Xa was not possible, it is unlikely that the MBP will affect glucanase function because the MBP is attached to the beta-glucanase via the NH(2)-terminal isoleucine situated on the side of the alpha/beta barrel that does not carry the substrate-binding groove (Fig. 2). Indeed, the enzymic and kinetic properties of the fusion proteins appear to accurately reflect the properties of the native enzymes. Thus, 1,3-beta-glucanase isoenzyme GII isolated from germinating barley and MBP-GII do not differ significantly with respect to pH optima (Fig. 3), turnover number, or K(m) values (Table 1). In addition, relative differences in thermostability between 1,3- and 1,3-1,4-beta-glucanases isolated from barley are accurately reproduced when MBP-GII and MBP-EII are compared (Fig. 4). These observations establish the efficacy of the system for introducing mutations into barley beta-glucanases and for the evaluation of their functional consequences. The method has now been used to test the relative importance of four highly conserved amino acid residues, namely Glu, Glu, Lys, and Glu (Fig. 5), as determinants of catalytic activity in 1,3-beta-glucanase isoenzyme GII.


Figure 3: pH optima for wild-type and mutant enzymes. Isoenzyme GII purified from germinating barley (), MBP-GII(wt) (), MBP-GII(E288Q) (), MBP-GII(K282M) (box), and MBP-GII(E279Q) () were incubated at the indicated pH values, and their relative activities were measured. Background activities were obtained at all pH values tested and subtracted.






Figure 4: Thermostabilities of endoglucanases synthesized in germinating barley or in E. coli as fusion proteins. A, isoenzymes GII (0.7 µg/ml) () and EII (18 µg/ml) () were purified from germinating barley and incubated at 50 °C in 50 mM sodium acetate (pH 5.0) in the presence of bovine serum albumin (0.5 mg/ml). Residual enzyme activities (A) were determined and compared to the initial activity at t = 0 (A). B, as in A, except MBP-GII (3.1 µg/ml) () and MBP-EII (39 µg/ml) () were used.




Figure 5: Invariant active site residues in some plant and fungal endoglucanases. A, relevant segments of the deduced primary structures of barley 1,3-beta-glucanases isoenzyme GI to GVII (Xu et al., 1992; Malehorn et al., 1993), a bean (Phaseolus vulgaris) 1,3-beta-glucanase (Edington et al., 1991), a tobacco (Nicotiana tabacum) 1,3-beta-glucanase (Payne et al., 1990), barley 1,3-1,4-beta-glucanases isoenzyme EI (Slakeski et al., 1990) and EII (Fincher et al., 1986), and a Saccharomyces cerevisiae (Sc) 1,3-beta-glucanase (Klebl and Tanner, 1989) were visually aligned, and residues with positional identity in all of these enzymes are shaded. The position of the catalytic nucleophile (Glu (GII)) is indicated with the arrow (&cjs0435;), and the catalytic acid is indicated with a star (). The mutated residues (Glu, Glu, Lys, and Glu) are indicated with down triangle. B, deduced amino acid sequence for a portion of the anther-specific protein encoded by the B. napus A6 gene (Hird et al., 1993) with no apparent glucanase activity. Residues with positional identity to the invariable positions of the 30-35-kDa endoglucanases shown in panelA are shaded. The non-conserved Gln residue is indicated with (see text).



Mutant Construction and Purification

Four MBP-GII cDNAs encoding the E231Q, E279Q, K282M, and E288Q mutant enzymes were constructed using mutagenic primers and the authenticity of the resulting constructs checked by nucleotide sequencing. No sequence errors were observed. Following expression in E. coli cells and purification of the expressed fusion proteins, about 5 mg of wild-type and 3-5 mg of mutant MBP-GII were obtained per liter of culture medium. The wild-type and mutant enzymes exhibited apparently identical chromatographic and electrophoretic behavior. Their Stokes' radii were indistinguishable as judged from analytical gel filtration, during which the proteins eluted as monomers with an apparent molecular mass of 53 kDa and appeared to adopt a highly globular structure (data not shown). From these observations and from measurements of K(m) values (Table 1), we conclude that the overall topology of the MBP-GII enzyme has not been changed by the mutations introduced at these surface-located residues.

Role of Glu and Glu

We have previously employed mechanism-based inhibitors to identify Glu as the likely catalytic nucleophile at the active site of barley 1,3-beta-glucanase isoenzyme GII and a less specific carbodiimide/glycine ethyl ester-labeling protocol to identify Glu as the putative catalytic acid (Chen et al., 1993a). The assignments were given additional weight by the three-dimensional structure of the enzyme, which showed that Glu and Glu are located in a deep substrate-binding cleft with a distance between their O atoms of 8.6 Å (Varghese et al., 1994) (Fig. 6). If the glycosidic oxygen were positioned midway between the two O atoms, this distance would enable protonation of the glycosidic oxygen by the catalytic acid and subsequent stabilization of the oxycarbonium ion by the catalytic nucleophile (Varghese et al., 1994).


Figure 6: Active site structure of isoenzyme GII and location of a covalently linked substrate analogue. A, the inhibitor is in thickbonds (carbon, gold; oxygen, crimson), and the protein is shown as a smoothed Calpha trace (gray) with active site chains as balls and sticks (carbon, white; nitrogen, paleblue; oxygen, pink). The inhibitor is covalently attached to the catalytic nucleophile (Glu, in red (bottomcenter)) behind the inhibitor, and the oxygen, originally from the epoxide ring, is hydrogen bonded to the catalytic acid (Glu in red) via a water molecule. The glucose moiety modeled is hydrogen bonded via O-6 to Glu and Lys and via O-2 to Asn. There is hydrophobic contact between the face of the glucose and the ring of Tyr. The catalytic acid (Glu) and nucleophile (Glu) are shown in red. Side chains included are Cys^4, Gly^6, Ile^8 (center to upperleft); Tyr, Phe (left); Gly, Asn (frontleft); Asn, Glu (frontright); Asn (behind Glu), Tyr, Phe (right); Glu, Phe, Glu, Lys, Glu, and Phe. B, stereo view of panelA with the non-reducing end of the glucosyl moiety facing North.



However, as pointed out by Schimmel(1993), what appear to be straightforward rationales for the conservation of residues at a particular location may be secondary to alternative and more sophisticated considerations and that no matter how obvious the role of a particular residue may seem, it pays to make substitutions at such positions to test the effect on function, as illustrated by the analysis of Asn in thymilidate synthase (Liu and Santi, 1993). For these reasons the putative catalytic amino acid residues of the barley 1,3-beta-glucanase were subjected to site-directed mutagenesis, the enzymes were expressed in E. coli, and the kinetic properties of the mutant enzymes were examined. When the putative catalytic nucleophile Glu was changed to a Gln residue in the E231Q mutant, the specific activity was reduced about 20,000-fold. At this level of activity, reliable estimates of K(m) and k could not be obtained. The effect of mutating the postulated general acid (Glu) to a glutamine residue was less dramatic. The E288Q mutant exhibited a 350-fold reduction in k with no significant change in K(m) (Table 1). Both mutant enzymes exhibited bell-shaped pH profiles similar to that of the wild-type MBP-GII (Fig. 3), although one might anticipate that removal of either of the catalytic amino acids would alter the shape of the pH rate curve dramatically. This observation raised the possibility that the activity measured could be due to wt activity. The likelihood of contamination of the expressed enzyme with wt enzyme appears to be remote, given that individual cDNA clones were used in expression work. Translational misreading is a possibility (Schimmel, 1989), but spontaneous deamidation of the introduced Gln residue might be more likely (Planas et al., 1992). To address these possibilities, the thermal inactivation of the E288Q mutant was compared with that of the wt enzyme (Fig. 7). The activity loss of the E288Q mutant does not parallel that of the wt enzyme, and we therefore conclude that the activity measured in the mutant preparation is due to the abundant, low specific activity MBP-GII(E288Q) mutant enzyme itself and not due to a small amount of wt enzyme with a high specific activity.


Figure 7: The effect of amino acid substitutions on the thermostability of MBP-GII. Thermostabilities of MBP-GII(wt) (box), MBP-GII(E288Q) (), MBP-GII(K282M) (), and MBP-GII(E279Q) () were tested at 55 °C as described in the legend to Fig. 4.



The relative impact on activity of mutations in the catalytic acid and the catalytic nucleophile was unexpected and not in line with commonly observed effects (Svensson and Søgård, 1993; Legler, 1993). Replacement of the general acid usually results in near complete loss of activity, as observed for Tyr in beta-galactosidase (Ring and Huber, 1990), Glu in hen egg white lysozyme (Malcolm et al., 1989), and Glu in Aspergillus niger glucoamylase (Sierks et al., 1990), while low to significant residual activity usually remains after mutation of catalytic nucleophiles. For example, the D52N mutation in hen egg white lysozyme, in which Asp is the nucleophile, reduces k to 5% of wt activity on Micrococcus cells (Malcolm et al., 1989). In the case of MBP-GII, this trend is reversed. Substitution of the nucleophile led to an almost complete loss of activity, while significant levels of activity remained following mutation of the catalytic acid. Nevertheless, similar results have been recently recorded. The substitution of a Gln residue for the nucleophilic Glu in an Agrobacterium beta-glucosidase (Withers et al., 1992) and Glu in a Bacillus licheniformis 1,3-1,4-beta-glucanase (Planas et al., 1992) resulted in dramatic loss of activity, while the substitution of a potential, as yet unconfirmed, proton donor (Asp) in the glucosidase had a somewhat less dramatic effect (Trimbur et al., 1992).

As expected for catalytic amino acid residues, Glu and Glu are highly conserved in the primary structures of a wide range of eukaryotic 30-35-kDa beta-glucanases (Chen et al., 1993a) (Fig. 5A). One exception is the anther-specific 53-kDa protein encoded by the developmentally regulated Brassica napus and Arabidopsis thaliana A6 gene, which despite a 30% sequence identity with isoenzyme GII is predicted to contain a glutamine residue (Gln) at the equivalent position of Glu in isoenzyme GII (Hird et al., 1993) (Fig. 5B). Significantly, to date, no glucanase activity has been associated with the A6 protein when expressed prematurely in tobacco anthers or in E. coli (Hird et al., 1993). Therefore, these data support rather than undermine the importance of Glu and its equivalents for activity. If the A6 protein is not active, what could the purpose of a glutamine residue in place of the Glu equivalent be? Since premature expression of beta-glucanase activity in anthers of transgeneic tobacco results in male sterility (Worrall et al., 1992), it is possible that A6 activity is either required to be very low or alternatively regulated by deamidation. It has been previously suggested that structure-dependent deamidation of proteins serve as in vivo molecular clocks that control development and aging (Robinson and Robinson, 1991). Neighboring Thr and Ser residues have a catalytic effect on Asn and Gln deamidation, and this may be why the three least probable side-by-side residue pairs involving amides in proteins are Gln-Ser, Asn-Thr, and Thr-Gln (Robinson and Robinson, 1991). We note that the B. napus A6 protein contains the sequence Thr-Gln. Expression in anthers of a mutated A6 gene (Q341E) could be most informative.

Mutation of Two Charged Residues in the Vicinity of the Catalytic Acid

In attempting to define other amino acid residues that are important during catalysis in the 1,3-beta-glucanases, it was expected that strictly conserved residues that line the active site cleft in close spatial proximity to Glu and Glu would be most likely to be involved ( Fig. 5and Fig. 6).

Earlier biochemical and structural data did not implicate Glu in the catalytic process, but its strict conservation between related eukaryotic beta-glucanases and its location within one of the most conserved stretches of primary structure nevertheless points to a crucial function (see Fig. 5). The potential importance of Glu was therefore tested through its mutation to the sterically similar Gln residue. MBP-GII(E279Q) exhibited a 240-fold reduction in k with a small increase in K(m) (Table 1), suggesting that a negative charge at this position might be required for optimal enzyme activity. Because the decrease in activity is comparable with that observed when the catalytic acid is removed (mutant E288Q), Glu might influence the ionization status of Glu. However, the considerable distance between these residues suggests that the effect would be most likely to be exerted through Lys, which is located between Glu and Glu (Fig. 6). The spatial relationship between these residues in the wt enzyme is illustrated in Fig. S1. It is possible that Glu ensures Lys exists in a protonated state, thus allowing Glu to retain its proton for catalytic purposes (Fig. S1, panelA). In the absence of Glu, the long flexible side chain of Lys might promote the deprotonation of Glu. The conversion of Glu to its carboxylate form through the protonation of Lys would therefore prevent protonation of the glycosidic oxygen, and activity would be lost as a result (Fig. S1, panelB). Such a possibility could be tested with a double mutant, MBP-GII(E279Q,K282M), only if Lys individually is non-essential for activity. To explore this further, MBP-GII(K282M) was constructed and purified. The use of methionine in place of lysine was based on the three-dimensional structure, which indicated such a change would maintain the packing, and van der Waals' contacts contributed by the four methylene groups of the lysine residue in the wt enzyme. As shown in Table 1, a 2500-fold reduction in specific activity and a modest increase in K(m) resulted from this mutation. Due to the critical nature of the K282M mutation alone, a MBP-GII (E279Q,K282M) double mutant could therefore not give critical information in regard of our hypothesis and was therefore not constructed.


Scheme 1: Scheme 1Proposed mechanism of action of isoenzyme GII and the effect of introduced mutations (see text for details). Note the mechanism shown in this scheme occurs with retention of configuration at the anomeric carbon, as has been deduced from NMR.(^3)



The results discussed above strongly indicate that a tetrad of ionizable amino acids (Glu, Glu, Lys, and Glu) is of crucial importance for the catalytic mechanism of this polysaccharide endohydrolase. While the catalytic role of Glu and Glu now appears well established, the mechanism(s) through which Glu and Lys exert their influence remain(s) unclear at this stage, although it is possible that these two residues form an ion pair or a salt bridge, presenting Glu with the positive side of a dipole. This dipole may help stabilize the negative charge developing on Glu during protonation of the glycosidic oxygen atom (Fig. S1A). Such a mechanism would be analogous to that of T4 lysozyme, in which residues especially sensitive to mutational alteration include those involved in buried salt bridges near the catalytic site (in particular, a salt bridge involving the presumed catalytic acid Glu and Arg (Rennel et al., 1991)). T4 lysozyme contains an additional salt bridge between Asp and His, which contributes to the enzyme's thermal stability, as judged from the much lower T(m) of the D70N and H31N mutants (Anderson et al., 1990). Similarly, the MBP-GII(E279Q) and MBP-GII(K282M) mutants were significantly less stable than wt MBP-GII (Fig. 7). While this points to the presence of a salt bridge between these two residues, it is not certain whether it actually contributes to the stability of the enzyme or rather minimizes the potential instability caused by the introduction of unmatched charges in the two mutant enzymes. However, the enhanced stability of the E288Q mutation indicates that the overall number of ionizable residues per se does not influence the stability and again shows that the residual activities measured in the four mutant enzymes are not due to contaminating wt-enzyme activity. It is noteworthy that a similar enhancement of stability has been observed in the E358Q mutant of an Agrobacterium beta-glucosidase (Withers et al., 1992).

To further investigate the potential influence of Glu and Lys on the ionization state of Glu, the pH-rate profiles of MBP-GII(E279Q), MBP-GII(K282M), and wt MBP-GII were compared (Fig. 3). Small but significant differences were observed, and these are consistent with the mechanisms presented in Fig. S1. Thus, the pH optimum of MBP-GII(K282M) was clearly shifted toward higher pH values, and the activity/pH profile broadened, consistent with an increase of the pK(a) for Glu in this mutant. The pH-rate profile of the E279Q mutant was virtually identical to that of the wt enzyme, except for a small but noticeable broadening of the pH-rate profile toward a more acidic pH.

Additional experimental evidence in support of the ionization state of the residues shown in Fig. S1is the observation that Glu but not Glu is modified by carbodiimide/glycine ethyl ester (Chen et al.,1993a) and that Glu sits in a relatively hydrophobic environment (Fig. 6), which could lead to an increase in the pK(a) of the nearby Glu residue. It is possible that the strategy employed by the glucanases is to raise the pK(a) of Glu through the positioning of hydrophobic residues in its vicinity (Urry et al., 1993) and at the same time to facilitate deprotonation due to the neighboring dipole of Lys and Glu. Such an arrangement may ensure that the proton on Glu is exchanging frequently, a prerequisite for catalysis. Such a delicate balance between protonation states may well be influenced by the presence of bound substrate.

Spatial Relationship between Mutated Residues and a Bound Substrate Analogue

To gain structural insight into the roles of the residues close to the catalytic acids, the active site-directed inhibitor 2,3-epoxypropyl-beta-D-laminaribioside (Rodriguez and Stick, 1990; Chen et al., 1993a) was soaked into native 1,3-beta-glucanase crystals, which were subsequently examined by x-ray diffraction. In the resulting structure, the position of the aglycon and one attached glucosyl residue could be modeled (Fig. 6). As predicted (Høj et al., 1992; Chen et al., 1993a; Keitel et al., 1993), a covalent adduct was observed between the inhibitor and the catalytic nucleophile (in this case, Glu). The point of nucleophilic attack appears to have taken place exclusively at the C-3 position of the epoxypropyl-aglycon as hypothesized earlier for the action of this inhibitor class on polysaccharide hydrolases (Høj et al., 1989).

Apart from the nucleophilic action of Glu, the structural analysis of the inhibitor-enzyme complex also supports other aspects of Fig. S1. The orientation of the inhibitor within the active site indicates that substrate binding occurs with the non-reducing end protruding toward the Western exit (as seen in Fig. 6A) and the reducing end facing the Eastern exit. Furthermore, since the length of the substrate binding cleft (40 Å) is sufficient to accommodate about eight residues of an extended glucan chain and since the two catalytic glutamic residues lie about one third of the way along the cleft (as counted from the Western end in Fig. 6) (Varghese et al., 1994), it appears that cleavage could occur between the third and fourth glucosyl binding subsite (numbering from the non-reducing end of the substrate), a conclusion that has been supported by independent mapping of binding subsites by kinetic analysis. (^2)Excitingly, the refined structure also clearly indicates the presence of a water molecule bound via a hydrogen bond to the catalytic acid (Glu). The positioning of such a molecule would allow it to regenerate the active enzyme following release of the aglycon, as outlined in panelA of Fig. S1. Finally, the glucose moiety is hydrogen bonded via O-6 to both Glu and Lys. Thus, while Glu and Lys, as discussed above, may well enhance catalytic efficiency of MBP-GII through their influence on the ionization state of Glu, this is unlikely to constitute their only role. Indeed, the fact that MBP-GII(E288Q) exhibits 7-fold higher activity than MBP-GII(K282M) in particular points to additional crucial roles for Lys, for example, binding of substrate. Such a role would be consistent with the 240% increase in K(m) seen of MBP-GII(K282M) for the polymeric laminarin substrate. Therefore, in the absence of Lys, substrate bound at the active site cleft may be positioned in a sub-optimal fashion in relation to the catalytic nucleophile and the catalytic acid, thus leading to a substantial loss of activity (Fig. S1C) (see also Høj et al. (1989b)). The sub-optimal binding could be due to an effect on the substrate-enzyme interaction only and/or to a change in active site conformation induced by the absence of the putative Glu-Lys salt bridge. We cannot discriminate between these possibilities at present, but note that a cluster of ionizable amino acids also is found in the alpha-amylases, which invariably contains Asp, Glu, and Asp (barley alpha-amylase numbering) at the active site. As seen for the beta-glucanases, alpha-amylase catalysis requires all three groups, and stabilization of the transition state is mediated by the neighboring and invariant His, which is believed to hydrogen bond to the 6-OH of the substrate glycon ring adjacent to the cleavage site (Søgaard et al., 1993). Further studies are needed to establish whether such clusters of charged amino acids in distinct classes of enzymes reflect a basic requirement for polysaccharide hydrolysis.


FOOTNOTES

*
This work was supported by grants from the Australian Research Council. 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.

§
Recipient of a La Trobe University Postgraduate Research Award.

Supported by a Queen Elizabeth II Fellowship.

**
To whom reprint requests should be addressed.

(^1)
The abbreviations used are: isoenzyme GII, 1,3-beta-D-glucan glucanohydrolase isoenzyme GII from barley (Hordeum vulgare); isoenzyme EI and EII, 1,3-1,4-beta-D-glucan 4-glucanohydrolase isoenzymes EI and EII from barley (H. vulgare); IPTG, isopropyl-1-thio-beta-D-galactopyranoside; Mes, 2-(N-morpholino)ethanesulfonic acid; MBP-EI, MBP-EII, and MBP-GII, fusion proteins of the E. coli maltose-binding protein and isoenzymes EI, EII, and GII, respectively; wt, wild-type; X-gal, 5-bromo-4-chloro-3-indoyl beta-D-galactoside; PCR, polymerase chain reaction.

(^2)
M. Hrmova and G. B. Fincher, unpublished results.

(^3)
L. Chen., M. Sadek, R. Brownlee, B. A. Stone, G. B. Fincher, and P. B. Høj, unpublished results.


ACKNOWLEDGEMENTS

We thank Drs. E. B. Rodriguez, R. V. Stick, and Prof. B. A. Stone for the generous donation of inhibitors. The continuous support, encouragement, and constructive suggestions from Dr. J. N. Varghese, Dr. P. M. Colman, and Prof. B. A. Stone are gratefully acknowledged.


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