ELSEVIER Biochimica et B iophysica Acta 1253 (1995) 112-116

BH Biochi~ic~a et Biophysica P~ta

Stereochemical course of glucan hydrolysis by barley( 1 3)- and (1 > 3,1 4)-/3-glucanases

Lin Chen a, Maruse Sadek b, Bruce A. Stone a, Robert T.C. Brownlee b, Geoffrey B. Fincher c, Peter B. Hcj d,,

a School of Biochemistry, La Trobe UnicersiO', Bundoora, Vic. 3083. Australia b School of Chemistry, La Trobe UnicersiO', Bundoora, Vic. 3083, Australia

c Department of Plant Science, Universio, of Adelaide, Waite Campus, PMB1, Glen Osmond, S.A. 5064, Australia d Department of Horticulture, Viticulture and Oenology, University of Adelaide, Waite Campus, PMB1, Glen Osmond, S.A. 5064, Australia

Received 10 April 1995; accepted 10 July 1995

Abstract

The stereochemical course of hydrolysis of Laminaria digitata laminarin and barley (1 -~ 3,1 -~ 4)-fl-glucan by barley (1 ~ 3)-/3- glucanase (E.C. 3.2.1.39) isoenzyme GII and (1 --* 3,1 -~ 4)-fl-glucanase (EC 3.2.1.73) isoenzyme EII, respectively, has been determined by I H-NMR. Both enzymes catalyse hydrolysis with retention of anomeric configuration (e -~ e) and may therefore operate via a double displacement mechanism. We predict that all other members of Family 17 of/3-glycosyl hydrolases also follow this stereochemical course of hydrolysis.

Keywords: /3-Glucanase, (1 ~ 3)- ; fl-Glucanase, (1 ~ 3,1 ~ 4)- ; Reaction mechanism; NMR; (Barley); (E. coli); (L. digitata)

I. Introduct ion

The (1 ~ 3)-fl-glucanases (EC 3.2.1.39) and the (1 3,1 ~ 4)-fl-glucanases (EC 3.2.1.73) are fl-glucan endohy- drolases with closely related but distinct substrate speci- ficities (for a general review, see [1]). Thus, the (1 ~ 3)-fl- glucanases hydrolyse internal (1 ~ 3)-/3-glucosyl linkages in /3-glucans containing a contiguous region of unsubsti- tuted (1 ~ 3)-/3-1inked glucosyl residues whilst the (1 --* 3,1 ~ 4)-fl-glucanases catalyse the hydrolysis of (1 ~ 4)- fl-glucosyl linkages only where the glucosyl residue is itself linked at the (0 )3 position [2-4]. Despite their distinct substrate specificities, the (1 ~ 3)- and (1 ~ 3,1

4)-/3-glucanases share a high degree of similarity at the level of amino-acid sequence and three-dimensional struc- ture, and there is now compelling evidence that these two

Abbreviations: (1 --* 3)-fl-glucanase, (1 ~ 3)-/3-D-glucan 3-glucano- hydrolase; (1 ~ 3,1 ~ 4)-fl-glucanase, (1 ~ 3,1 ~ 4)-fl-D-glucan 4- glucanohydrolase; MBP-GII, fusion protein of Escherichia coli maltose- binding protein and (I ~ 3)-/3-glucanase isoenzyme GII ; MBP-EII, fusion protein of Escherichia coli maltose-binding protein and (1 ~ 3,1 --* 4)-/3-glucanase isoenzyme Eli.

* Corresponding author. Fax: +61 8 3037117.

0167-4838/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 1 6 7 - 4 8 3 8 ( 9 5 ) 0 0 1 5 7 - 3

enzyme classes have evolved via duplication of a common ancestral gene [5].

As part of an ongoing program aimed at defining the molecular basis for catalysis and for the evolution of distinct substrate specificities in the barley (1 -~ 3)- and (1 -~ 3,1 -~ 4)-fl-glucanases, we previously identified their catalytic amino acids [6] and also prepared diffracting crystals of both (1 ~ 3)-/3-glucanase isoenzyme GII and ( 1 - ~ 3,1 ~ 4)-/3-glucanase isoenzyme EII [7]. Both en- zymes fold into a / f l -bar re l structures with their two cat- alytic glutamic acid residues located within deep grooves which extend across the enzymes [8]. Although detailed knowledge of the enzyme structure and identity of the catalytic amino acids greatly aids the understanding of the mechanistic aspects of glucanase action, further progress requires knowledge of the stereochemical course of glyco- side hydrolysis, an aspect of hydrolysis which has yet to be established for any plant fl-glucan endohydrolase.

In this study we employed recombinant fusion proteins produced in Escherichia coli and 1H-NMR to define the stereochemical course of hydrolysis of the natural poly- meric substrates Laminaria digitata laminarin and barley (1 -~ 3,1 -~ 4)-/3-glucan by (1 ~ 3)-fl-glucanase isoen- zyme GII and (l ~ 3,1 ~ 4)-/3-glucanase isoenzyme Eli,

L Chen et al. /Biochimica et Biophysica Acta 1253 (1995) 112-116 113

respectively. We conclude that both enzymes catalyse hydrolysis with retention of anomeric configuration and predict this to be the case for all members of the family 17 of glycosidases [9]. (e) t= 110 min

2. Materials and methods

2.1. Enzymes and substrates

The (1 ~ 3)-/3-glucanase isoenzyme GII [10] and the (1 --* 3,1 ~ 4)-/3-glucanase isoenzyme EII [11] used in this study were produced in Escherichia coli as fusion proteins with maltose binding protein [12]. Briefly DNA fragments encoding mature (1 ~ 3)-B-glucanase isoenzyme GII and (1 ---, 3,1 ~ 4)-/3-glucanase, isoenzyme Eli were generated by PCR amplification of the respective cDNAs [10,13] using Vent R TM DNA polymerase (New England Biolabs). The PCR fragments were ligated into the pMAL-c2 plas- mids [14] to generate ez~pression vectors encoding the fusion proteins MBP-GII and MBP-EII, respectively. MBP-GII and MBP-EII were produced in the E. coli strain DH5ce and purified by standard chromatography. The two fusion proteins behaved like the authentic barley enzymes with respect to specific activities, pH optima and substrate specificities [ 12]. For NMR experiments, purified enzymes in 50 mM sodium acetate buffer, pH 5.0 (typically 100/,1) were freeze-dried, redissolved in D20 and freeze-dried three more times before finally being dissolved in D20 (99.96 atom % D, Cambridge Isotopes).

Laminarin from Laminaria digitata (Sigma) and barley (1 ~ 3,1 ~ 4)-/3-glucan (BioCon) were used as substrates for the enzymes MBP-GII and MBP-EII, respectively. These substrates were dis:solved in 100 tzl NaOAc buffer (1 M), pH 5.0 and freeze-dried. After three further freeze- dryings from D20, the solids were redissolved in D20 (99.96% atom% D, CI) in preparation for NMR. The laminarin/NaOAc (10 mg polysaccharide) was redis- solved in 0.7 ml D20 to give a solution with a final pH 6.0. Freeze-dried barley /3-glucan (5 mg) was redissolved in 0.7 ml D20 and the pH adjusted to 5.0 by the addition of 20% DC1 in D20.

2.2. I H-NMR spectroscopy

(d) t= 60 min

_ )~,

c(

(c) t= 16 min

Hill

(a) t ; 0 rain

' sJ2 ' ~'.0 ' 4'.8 ' 18 ' 1.4 ' 4'.2 ~ (ppm)

Fig. 1. 1H-NMR spectra showing the stereochemical course of hydrolysis of Laminaria digitata laminarin by (1 ~ 3)-fl-glucanase MBP-GII. Spec- tra are for the anomeric proton region before (a) and 3.2 rain (b), 16.1 rain (c), 60 min (d) and 110 rain (e) after addition of enzyme.

est. The spectra were referenced to the residual HDO at 4.72 ppm (this chemical shift was measured indirectly with respect to sodium 2,2-dimethyl-2-silapentane-5-sulfonate at 303 K). Datapoints (4000) over the 2404 Hz spectral width were acquired by the Bruker sequential method of quad-detection, with 16 transients (and 8 dummy tran- sients) per spectrum. The recycle time was 860 ms. Data were processed with a 90 ° shifted square sine bell window and spectra baseline corrected via interactive polynomial baseline correction. The plotted spectra were normalised to the peak height of the HDO resonance.

Substrate solutions were brought to 303 K, and after optimising the NMR conditions, the initial spectra of sub- strates were recorded. Enzymes (56 /xg MBP-GII, 375 /xg MBP-EII) were added, mixed, and the stereochemical course of the reactions monitored by collecting 1H-NMR spectra at suitable time intervals.

~H-NMR spectra were recorded on a Bruker AM- 400WB spectrometer with dedicated 5 mm I H-probe, op- erating at 400 MHz at a probe temperature of 303 K. This temperature was chosen to strategically position the HDO resonance to reduce the overlap with resonances of inter-

3. Results

Fig. 1 a and Fig. 2a show partial 1H-NMR spectra of the polymeric substrates, laminarin (degree of polymerisation = 30) and barley (1 --+ 3,1 --+ 4)-/3-glucan (degree of poly- merisation > 1000), respectively. The spectra of both substrates, but in particular that of the (1 ~ 3,1 ~ 4)-/3- glucan, show characteristics of high molecular weight polysaccharides. Thus the H-I resonances characteristic of a- and /3-anomers of reducing glucosyl residues, posi-

114 L. Chen et al. / Biochimica et Biophysica Acta 1253 (1995) 112-116

(e) t= 139 min

(d) t= 62 min

a

(c) t= 32 min

(b) t-- 8 min

(a) t= 0 min

5'.2 ' 5'.o 4~e

(ppm)

4'.e 414

Fig. 2. J H-NMR spectra showing the stereochemical course of hydrolysis of barley (1 ~ 3,1 ~ 4)-fl-glucan by (1 ~ 3,1 ~ 4)-fl-glucanase MBP-EII. Spectra are for the anomeric proton region before (a) and 8 min (b), 32 min (c), 62 min (d) and 139 min (e) after addition of enzyme.

tioned at 5.23 ppm (H-lc~) and 4.67 ppm (H-l/3) [15], were barely detectable. The broad bands in the (1 ~ 3,1 4)-/3-glucan spectrum at 4.77 ppm and 4.53 ppm can be assigned to inter-residue anomeric protons in (1 ~ 3)- and (1 ~4)-/3-glucosidic linkages, respectively [15]. In the laminarin spectrum, the doublet at 4.79 ppm corresponds to inter-residue anomeric protons in (1 ~ 3)-/3-glycosidic linkages [15].

Following addition of MBP-GII to laminarin (Fig. 1) , the intensity of a doublet at 4.67 ppm (3JHH 8.2 Hz) due to H-1/3 increases instantaneously. The intensity increase of resonance lines at 5.23 ppm assigned to H-I a (3JHH 3.8 Hz) becomes noticeable only much later during the NMR monitoring of enzymic reaction. After 110 min the relative intensities of a- and /3-anomer resonance lines (ca. 1:3) are far from the expected a / / 3 ratio of 2:3 at mutarota- tional equilibrium [15]. However, in one trial, 50 /zl of 10 % DC1 was added to the NMR tube after 32 min incuba- tion of the laminarin/MBP-GII reaction mixture. This resulted in a fast increase in the intensity of the doublet at 5.23 ppm and after 80 min incubation the ratio expected for mutarotational equilibrium was reached. Taken to-

gether, these data clearly show that the enzymic hydrolysis catalysed by MBP-GII and therefore barley (1 ~ 3)-/3- glucanase GII proceeds with retention of configuration.

Also, the spectrum of the barley (1 ~ 3,1 ~ 4)-/3-glucan substrate changed dramatically upon addition of the (1 3,1 ~ 4)-/3-glucanase (Fig. 2). The doublet (3JHH 7.4 Hz) at 4.66 ppm, corresponding to the H-1/3 on reducing ends of newly formed hydrolysis products, is detectable within a few minutes of enzyme addition and steadily increases in intensity with time. A low intensity doublet (3JHH 4.7 HZ) at 5.23 ppm attributable to H - l a appears after a delay of about 40 min.. This signal therefore almost certainly re- suits from the mutarotation at the anomeric carbon of hydrolysis products initially released in the fl-anomeric configuration. Indeed, after more than two hours (Fig. 2e) the relative intensities of H-1 of ct- and /3-anomers (ca. 1:4) are still not at the expected mutarotational equilibrium of about 2:3. This clearly indicates that enzymic hydrolysis of the (1 ~ 4)-r-linkages in the (1 ~ 3,1 ~ 4)-/3-glucan also proceeds with the retention of configuration at the anomeric carbon.

In both cases, apart from the rapid appearance of the H-l/3 signals during enzymolysis of the /3-glucans, other major spectral changes were the sharpening of resonance lines and the formation of complex multiples, due to cleavage of the relatively large polymeric precursors into more mobile oligoglucosides (Figs. 1 and 2).

4. Discuss ion

A complete understanding of enzyme action at the molecular level requires, amongst other things, knowledge of the three-dimensional structure of the enzyme, identifi- cation of amino acids important for catalysis and a precise definition of stereochemical events during catalysis. Since Eveleigh and Perlin's pioneering NMR studies on fl-glu- cosidase action [16], this technique has been employed to determine the stereochemical course of a number of glyco- side hydrolase mediated reactions (see [1] for further refer- ences). For example, using cellotetraose as a substrate Withers et al. [17] were able to demonstrate that a (1 -o 4)- fl-glucan hydrolase from Cellulomonas f imi catalyses hy- drolysis with inversion of configuration (e ~ a) and Barras et al. [18] demonstrated that a (1 ~ 4)-/3-glucanase (endo- glucanase Z) from Erwinia chrysanthemi catalysed hydro- lysis of the same substrate with retention of configuration (e ~ e). Notwithstanding this success, short oligosaccha- rides are likely to be hydrolysed slowly by many endoglu- canases and would therefore not be suitable for NMR analysis, because the rates of hydrolysis and mutarotation would be comparable. Thus, (1 ~ 3)-/3-glucanase isoen- zyme GII has recently been shown to hydrolyse only laminaridextrins of DP > eight efficiently [19] and accord- ingly preliminary NMR experiments using borohydride-re- duced laminariheptaose as a substrate were unsuccessful.

L. Chen et al. / Biochimica et Biophysica Acta 1253 (1995) 112-116 115

I o~C~,o

CI

(.

I 0%

z :ro

as catalytic acid and stabilising residue, respectively [6,12,22]. Although the results herein clearly rule out the possibility that the stabilising residue (Glu-231 in (1 ~ 3)- /3-glucanase GII) operates as a general base, the mode of stabilisation afforded by this residue during catalysis is not yet certain, but it is generally thought to be either electro- static or via a covalent enzyme intermediate (see [21]). Since Glu-231, through nucleophilic-attack, forms covalent adducts with mechanism-based inhibitors such as 2,3- epoxypropyl-laminaribioside [6], an SN2 mechanism in which this residue serves as a true nucleophile, attacking the anomeric carbon to generate a covalent intermediate, is conceivable. This intermediate is then hydrolysed by a water molecule which may be activated by the base-form of the acid catalyst as outlined in Scheme 1.

Based on sequence comparisons, Henrissat and Bairoch [9] have classified more than 300 glycosyl hydrolases into 35 distinct families. The two barley /3-glucanases are disposed to family 17. Since representatives of a given family appear to exhibit the same stereoselectivity [23], we predict that all members of family 17 catalyse glycoside hydrolysis with retention of configuration.

I °6? !I

Scheme 1. Proposed mechanism of action of barley (1 ~ 3)- and (1 3,1 ~ 4)-/3-glucanases. In this case the scheme indicates the mechanism employed by barley (1 ~ 3)-/3-glucanase isoenzyme GII during the hy- drolysis of laminarin. E288 [6] or E93 [22] has previously been assigned as the likely catalytic acid and E231 as the likely catalytic nucleophile of this enzyme [6]. See text for further details.

Encouraged by a recent report from Malet et al. [20] in which it was shown that a Bacillus lichiniformis (1 ~ 3,1

4)-fl-glucanase catalyses hydrolysis of barley (1 ~ 3,1 --* 4)-fl-glucan with retention of configuration, we there- fore employed the natural polymeric substrates laminarin and barley fl-glucan without prior reduction. This ap- proach was straightforward and demonstrated unequivo- cally that the two members of the barley fl-glucanase families operate with retention of configuration. This sug- gests a double displacement mechanism (e ~ e) involving two catalytic residues responsible for protonation of the glycosidic oxygen and stabilisation of a carbocationic reac- tion intermediate, respectively [21]. Such catalytic residues are, in polysaccharide hydrolases, most often contributed by Glu and Asp residues. In the case of the barley /3- glucanases, both biochemical and structural evidence has indicated that two highly conserved glutamic acids serve

Acknowledgements

We thank Drs. Hugues Driguez and Marc Clayessens for valuable discussions during the course of these experi- ments. This work was supported by grants from the Aus- tralian Research Council and Department of Industry, Technology and Commerce. Lin Chen acknowledges re- ceipt of a La Trobe University Postgraduate Research Award.

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