Domain function in Trichoderma reesei cellobiohydrolases

Journal o f Biotechnology, 24 (1992) 169-176 © 1992 Elsevier Science Publishers B.V. All rights reserved 0168-1656/92/$05.00

169

BIOTEC 00758

Domain function in Trichoderma reesei cellobiohydrolases Tuula T. Teeri a, Tapani Reinikainen a, Laura R u o h o n e n T. Alwyn Jones b and Jonathan K.C. Knowles a,t

a,

a VT"TBiotechnical Laboratory, Box 202, SF-02151 Espoo, Finland b Biornediska Centrum, Box 590, S-75124 Uppsala, Sweden

(Received 21 January 1991; revision accepted 8 May 1991)

Summary The filamentous fungus, Trichoderma reesei produces all of the enzymatic activities required for efficient hydrolysis of highly ordered crystalline cellulose. All the principal enzymes involved in cellulose hydrolysis possess two functional domains, one directly involved in catalysis and the other in substrate recognition and binding. The availability of high-resolution three dimensional structures of the two domains allows genetic engineering to be used for detailed structure-function studies of these important enzymes. Cellulase; Trichoderma; Domains; Catalysis; Substrate binding

Introduction Native cellulose consists of long homopolymers of /3-1,4 linked glucose bound tightly together by regular H-bonding networks. Despite its chemical simplicity, cellulose contains considerable microheterogeneity. It contains both crystal and non-crystal amorphous regions and the /3-1,4 bonds therefore have a number of different conformations. The high degree of crystallinity in native cellulose renders Correspondence to." T.T. Teeri, VTT Biotechnical Laboratory, Box 202, Espoo, Finland. ~Present address." Glaxo Institute for Molecular Biology S.A., Route des Acacias 46, 1211 Geneva 24, Switzerland.

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Catalyticdomain

@

Hinge\

Celdomai lulose-bi n~nding

Fig. 1. Schematicrepresentation of the domain structure of T. reesei cellulases. it very resistant to degradation. Probably due to that efficient biodegradation of cellulose requires the cooperative actions of a number of different enzymes. Fungal cellulolytic enzyme systems typically consist of two different types of activities: endoglucanases, which hydrolyse amorphous or soluble substituted derivatives of cellulose in a random manner and exoglucanases or cellobiohydrolases, which release cellobiose from the nonreducing ends of the cellulose chains. The exoglucanases clearly have a central role in the degradation of crystalline cellulose. However, synergy between the two types of enzymes is seen in the significant increase observed in the rate of the hydrolysis of crystalline cellulose upon the addition of even trace amounts of endoglucanases to a mixture of exoglucanases (F~igerstam and Pettersson, 1980; Henrissat et al., 1985; Wood et al., 1989). The filamentous fungus Trichoderma reesei produces at least two or three different endoglucanases and two cellobiohydrolases. Sequence comparisons of the cloned cellulolytic enzymes (Penttil~i et al., 1986; Teeri et al., 1987; Saloheimo et al., 1988) and biochemical data (Van Tilbeurgh et al., 1986; Tomme et al., 1988) show that the enzymes are composed of two functionally and structurally distinct domains, a large catalytic domain and a much smaller substrate binding domain which are linked to each other by a serine and threonine rich O-glycosylated hinge region (Fig. 1). The primary structures of the large catalytic domains of T. reesei cellulases are generally not conserved though there would appear to be two general classes. The small terminal domains, called tails, which are found at either the C- or N-terminus of all Trichoderma cellulases characterized share 70% amino acid identity (Teeri et al., 1987). A similar two domain structural organization has been observed in almost all cellulolytic enzymes so far studied and seems to be important for the function of enzymes degrading solid substrates. We have undertaken genetic and structural studies of the two cellobiohydrolases, CBHI and CBHII in order to elucidate their mechanism of action on crystalline cellulose.

Active site tunnel of CBHII

The first three dimensional structure of a cellulolytic enzyme to be solved was that of the catalytic core protein of T. reesei CBHII (Rouvinen et al., 1990). The protein folds into a regular all3 barrel essentially similar to the structures of triose

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Fig. 2. The a-carbon backbone of the crystal structure of T. reesei CBHII. An inhibitor diffused in the crystal is seen in the active site tunnel formed by two stable surface loops (shaded) of CBHII. phosphate isomerase or a-amylase except that the 8th a helix and /3 strand are missing. Structures determined with small inhibitors and ligands diffused into the crystal revealed a remarkable active site tunnel enclosing the substrate by two stable surface loops (Fig. 2). It is of interest that in the three bacterial endoglucanases similar in sequence to C B H I I the sequences corresponding to these surface loops are not present (Rouvinen et al., 1990). It is therefore likely that endoglucanases possess a more open active site cleft facilitating the hydrolysis of internal bonds in cellulose chains while the exoglucanases are restricted to endwise hydrolysis due to the tunnel shaped active site. Four distinct subsites for substrate recognition and binding, first suggested by Van Tilbeurgh et al. (1985), can be seen clearly in the tunnel. In each of the subsites the glucose unit is bound.by hydrophobic interactions mediated by Tyr and Trp residues and by a number of H-bonds (Rouvinen, Ruohonen, Teeri, Knowles and Jones, manuscript in preparation). Prediction of the detailed catalytic mechanism of C B H I I from its three dimensional structure is not possible. However, previous work ( T o m m e and Claeyssens,

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1989) has suggested that cellulases act by acid catalysis and close to the putative scissile bond between the sugar residues at subsites B and C, two carboxylic acid residues, Asp175 and Asp221 can be found. In order to study their role in the catalytic events we have made two different mutants in which these residues have been mutated each to an alanine. Preliminary analysis of the activities of the mutant enzymes produced in yeast reveals that the mutant Asp221Ala is apparently totally inactive while some residual activity on barley/3-glucan is detected in the mutant Asp175Ala. These results suggest that Asp221 has a central role in the catalysis acting, e.g., as the proton donor and that Asp175 is more indirectly involved, possibly keeping Asp221 in protonated state (Rouvinen et al., 1990).

Role of tail domain of CBHI

Proteolytic removal of the small terminal domain from either bacterial or fungal cellulases reduces dramatically their activities on crystalline cellulose, while the activities of these truncated proteins on small soluble substrates remain largely unaffected (Van Tilbeurgh et al., 1986; Gilkes et al., 1988; St~hlberg et al., 1988; Tomme et al., 1988). The three-dimensional structure of the terminal domain of T. reesei CBHI has been determined by NMR and shown to fold into a wedge-shaped molecule with one face hydrophilic and the other face more hydrophobic (Kraulis et al., 1989; Fig. 3). Two hypotheses have been proposed concerning the function

Fig. 3. The three dimensional structure of the tail domain involved in cellulose binding of T. reesei CBHI. The two residues studied by site directed mutagenesis are shown as solid spheres.

173

A

13 Fig. 4. Two hypotheses have been presented concerning the function of the cellulase tail domains: A) The terminal domain binds to cellulose thereby increasing the enzyme concentration on cellulose surface. B) The terminal domain participates in single glucose chain liberation from the cellulose crystal. of the terminal domains. According to the first hypothesis (Fig. 4A) .the terminal domain acts merely as a substrate binding domain which anchors the enzyme onto the solid substrate thereby increasing the effective substrate concentration with respect to the enzyme. According to the second hypothesis (Fig. 4B) the tail has a more active role facilitating the release of single cellulose chains from the crystalline surface (Knowles et al., 1987). Closer examination of the tail structure reveals three conserved tyrosine residues dominating the formation of the hydrophilic surface (Fig. 3). Tyrosines are known to be involved in protein-sugar interactions in general and chemical modification has been used to show that tyrosines are important for the binding of CBHI in particular (Claeyssens and Tomme, 1990). It is therefore possible that the hydrophilic surface binds to cellulose. One way to examine the two hypotheses experimentally is to investigate whether both of the surfaces must be intact for full enzymatic activity on crystalline cellulose or whether the integrity of only the hydrophilic surface is sufficient. We have introduced specific amino acid changes onto both surfaces of the wedge by site directed mutagenesis of the corresponding cDNA. The mutagenized proteins were produced in the yeast, Saccharomyces cerevisiae, as described by Penttil~i et al. (1988) and their activities on different substrates analysed (Table 1). The mutation at Tyr31 changes the properties of the very tip of the wedge (Fig. 3) while the mutation Prol6Arg was designed to break the hydrophobic surface (Fig. 3)

174 TABLE 1 Comparison of the activities on 4-methylumbelliferyl-/3-D-lactoside, M U L (0.25 mm) and on crystalline bacterial cellulose (1 mg m l - ~ ) of CBHI native and mutant enzymes purified from Trichoderma or the yeast, Saccharomyces cerecisiae Enzyme

MUL nkat mg 1

Crystalline cellulose (pkat mg -~) ~

2.1 2.8

970 250

1.7 2.3 2.8 2.7 2.7 2.3

367 233 250 250 250 217

Trichoderma Wild type Core Yeast Wild type Core Y31A Y31H Y31D P16R

~ Expressed as glucose equivalents of reduCing sugars.

(Reinikainen, Ruohonen, Laaksonen, Nevanen, Knowles and Teeri, manuscript in preparation). After mutagenesis indirect evidence of the structural integrity of the mutant domains was obtained by molecular dynamic simulations and by the positive reaction of all of the mutant proteins by at least one of the two different monoclonal antibodies specific for the terminal domains (Reinikainen et al., supra). As seen in Table 1, mutations at either of the two sites chosen destroy the enzymatic activity of CBHI on crystalline cellulose. While alternative explanations are still possible our results would seem to suggest that both surfaces of the wedge may be functionally important and, therefore, that it is possible that the terminal domain not only binds to cellulose but perhaps also participates in its solubilization.

Linker The role of the linker region joining together the two cellulase domains is also interesting in terms of crystalline cellulose hydrolysis. In most eellulases, both bacterial and fungal, this region is characterized by the abundance of Serine or Threonine residues which are O-glycosylated. Reasons for the glycosylation are still unclear but in the case of Cellulomonas fimi cellulases it has been shown to protect an unbound enzyme from proteolysis (Miller et al., 1988). It has also been shown that the activities on soluble substrates of many of the truncated cellulases lacking the tail are somewhat increased when compared to the intact enzymes (Ghangas and Wilson, 1988; Gilkes et al., 1988; Nitisinprasert, 1990). These observations would seem to support an earlier suggestion that the activities of at least some of the cellulases are modulated by specific proteolysis in the different

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stages of growth (Teeri, 1987). In this way enzymes with higher specific activities on soluble substrates could be generated towards the end of the hydrolysis when most of the solid substrate has already been degraded to soluble oligosaccharides. The role of the linker must however also be considered in the light of the two hypotheses concerning crystalline cellulose degradation by these enzymes. If the role of the tail is simply to facilitate the binding of cellulases onto the substrate it is quite possible that the linker acts as spacer between the functional domains. However, if the second hypothesis is correct, coordinated, very specific interactions of the two functional domains may be postulated and the linker may then have a role as a mediator of these interactions. Experimental work is in progress in our laboratory to test these hypotheses.

Acknowledgements The Nordic Industrial Foundation and Alko Ltd. are gratefully acknowledged for financial support, and Research Professor Sirkka Ker~inen for fruitful discussions.

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176 Penttil~i, M.E., Andr6, L., Lehtovaara, P., Bailey, M., Teeri, T.T. and Knowles, J.K.C. (1988) Efficient secretion of two fungal cellobiohydrolases by Saccharomyces cerevisiae. Gene 63, 103-112. Rouvinen, J., Bergfors, T., Teeri, T., Knowles, J.K.C. and Jones, T.A. (1990) The three dimensional structure of cellobiohydrolase II from Trichoderma reesei. Science 249, 380-386. Saloheimo, M., Lehtovaara, P., Penttil~i, M., Teeri, T.T., St]hlberg, J., Johansson G., Pettersson, G., Claeyssens, M., Tomme, P. and Knowles, J.K.C. (1988) EGIII, a new endoglucanase from Trichoderma reesei: the characterization of both gene and enzyme. Gene 63, 11-21. St~hlberg, J., Johansson, G. and Pettersson, G. (1988) A binding-site-deficient, catalytically active, coreprotein of endoglucanase III from the culture filtrate of Trichoderma reesei. Eur. J. Biochem. 173, 179-183. Teeri, T.T. (1987) The cellulolytic enzyme system of Trichoderma reesei. Molecular cloning, characterization and expression of the cellobiohydrolase genes. Doctoral thesis, Technical Research Centre of Finland Publications, 38. Teeri, T.T., Lehtovaara, P., Kauppinen, S., Salovuori, I. and Knowles, J. (1987) Homologous domain in Trichoderma reesei cellulolytic enzymes: gene sequence and expression of cellobiohydrolase II. Gene 51, 43-52. Tomme, P. and Claeyssens, M. (1989) Identification of a functionally important carboxyl group in cellobiohydrolase I from Trichoderma reesei. FEBS Lett. 243, 239-243. Tomme, P., Van Tilbeurgh, H., Pettersson, G., Van Damme, J., Vandekerchove, J., Knowles, J., Teeri, T. and Claeyssens, M. (1988) Studies of the cellulolytic system of Trichoderma reesei QM 9414. Analysis of domain function in two cellobiohydrolases by limited proteolysis. Eur. J. Biochem. 170, 575-581. Van Tilbeurgh, H., Pettersson, G., Bhikhabhai, R., De Boeck, H. and Claeyssens, M. (1985) Studies of the cellulolytic system of Trichoderma reesei QM 9414. Reaction specificity and thermodynamics of interactions of small substrates and ligands with the 1,4-/3-glucan cellobiohydrolase II. Eur. J. Biochem. 148, 329-334. Van Tilbeurgh, H., Tomme, P., Claeyssens, M., Bhikhabhai, R. and Pettersson, G. (1986) Limited proteolysis of the cellobiohydrolase I from Trichoderma reesei. FEBS Lett. 204, 223-227. Wood, T.M., McCrae, S. and Bhat, K.M. (1989) The mechanism of fungal cellulase action. Synergism between enzyme components of Penicillium pinophilum cellulase in solubilizing hydrogen bond ordered cellulose. Biochem. J. 260, 37-43.