The three-dimensional crystal structure of the catalytic core of cellobiohydrolase I from Trichoderma reesei.

Cellulose is the major polysaccharide of plants where it plays a predominantly structural role. A variety of highly specialized microorganisms have evolved to produce enzymes that either synergistically or in complexes can carry out the complete hydrolysis of cellulose. The structure of the major cellobiohydrolase, CBHI, of the potent cellulolytic fungus Trichoderma reesei has been determined and refined to 1.8 angstrom resolution. The molecule contains a 40 angstrom long active site tunnel that may account for many of the previously poorly understood macroscopic properties of the enzyme and its interaction with solid cellulose. The active site residues were identified by solving the structure of the enzyme complexed with an oligosaccharide, o-iodobenzyl-1-thio-beta-cellobioside. The three-dimensional structure is very similar to a family of bacterial beta-glucanases with the main-chain topology of the plant legume lectins.

Crystallographic evidence for substrate ring distortion and protein conformational changes during catalysis in cellobiohydrolase Ce16A from trichoderma reesei.

BACKGROUND: Cel6A is one of the two cellobiohydrolases produced by Trichoderma reesei. The catalytic core has a structure that is a variation of the classic TIM barrel. The active site is located inside a tunnel, the roof of which is formed mainly by a pair of loops. RESULTS: We describe three new ligand complexes. One is the structure of the wild-type enzyme in complex with a nonhydrolysable cello-oligosaccharide, methyl 4-S-beta-cellobiosyl-4-thio-beta-cellobioside (Glc)(2)-S-(Glc)(2), which differs from a cellotetraose in the nature of the central glycosidic linkage where a sulphur atom replaces an oxygen atom. The second structure is a mutant, Y169F, in complex with the same ligand, and the third is the wild-type enzyme in complex with m-iodobenzyl beta-D-glucopyranosyl-beta(1,4)-D-xylopyranoside (IBXG). CONCLUSIONS: The (Glc)(2)-S-(Glc)(2) ligand binds in the -2 to +2 sites in both the wild-type and mutant enzymes. The glucosyl unit in the -1 site is distorted from the usual chair conformation in both structures. The IBXG ligand binds in the -2 to +1 sites, with the xylosyl unit in the -1 site where it adopts the energetically favourable chair conformation. The -1 site glucosyl of the (Glc)(2)-S-(Glc)(2) ligand is unable to take on this conformation because of steric clashes with the protein. The crystallographic results show that one of the tunnel-forming loops in Cel6A is sensitive to modifications at the active site, and is able to take on a number of different conformations. One of the conformational changes disrupts a set of interactions at the active site that we propose is an integral part of the reaction mechanism.

High-resolution crystal structures reveal how a cellulose chain is bound in the 50 A long tunnel of cellobiohydrolase I from Trichoderma reesei.

Detailed information has been obtained, by means of protein X-ray crystallography, on how a cellulose chain is bound in the cellulose-binding tunnel of cellobiohydrolase I (CBHI), the major cellulase in the hydrolysis of native, crystalline cellulose by the fungus Trichoderma reesei. Three high-resolution crystal structures of different catalytically deficient mutants of CBHI in complex with cellotetraose, cellopentaose and cellohexaose have been refined at 1.9, 1.7 and 1.9 A resolution, respectively. The observed binding of cellooligomers in the tunnel allowed unambiguous identification of ten well-defined subsites for glucosyl units that span a length of approximately 50 A. All bound oligomers have the same directionality and orientation, and the positions of the glucosyl units in each binding site agree remarkably well between the different complexes. The binding mode observed here corresponds to that expected during productive binding of a cellulose chain. The structures support the hypothesis that hydrolysis by CBHI proceeds from the reducing towards the non-reducing end of a cellulose chain, and they provide a structural explanation for the observed distribution of initial hydrolysis products.

Activity studies and crystal structures of catalytically deficient mutants of cellobiohydrolase I from Trichoderma reesei.

The roles of the residues in the catalytic trio Glu212-Asp214-Glu217 in cellobiohydrolase I (CBHI) from Trichoderma reesei have been investigated by changing these residues to their isosteric amide counterparts. Three mutants, E212Q, D214N and E217Q, were constructed and expressed in T. reesei. All three point mutations significantly impair the catalytic activity of the enzyme, although all retain some residual activity. On the small chromophoric substrate CNP-Lac, the kcat values were reduced to 1/2000, 1/85 and 1/370 of the wild-type activity, respectively, whereas the KM values remained essentially unchanged. On insoluble crystalline cellulose, BMCC, no significant activity was detected for the E212Q and E217Q mutants, whereas the D214N mutant retained residual activity. The consequences of the individual mutations on the active-site structure were assessed for two of the mutants, E212Q and D214N, by X-ray crystallography at 2.0 A and 2.2 A resolution, respectively. In addition, the structure of E212Q CBHI in complex with the natural product, cellobiose, was determined at 2.0 A resolution. The active-site structure of each mutant is very similar to that of the wild-type enzyme. In the absence of ligand, the active site of the D214N mutant contains a calcium ion firmly bound to Glu212, whereas that of E212Q does not. This supports our hypothesis that Glu212 is the charged species during catalysis. As in the complex of wild-type CBHI with bound o-iodobenzyl-1-thio-beta-D-glucoside, cellobiose is bound to the two product sites in the complex with E212Q. However, the binding of cellobiose differs from that of the glucoside in that the cellobiose is shifted away from the trio of catalytic residues to interact more intimately with a loop that is part of the outer wall of the active site.

The crystal structure of the catalytic core domain of endoglucanase I from Trichoderma reesei at 3.6 A resolution, and a comparison with related enzymes.

Cellulose is the most abundant polymer in the biosphere. Although generally resistant to degradation, it may be hydrolysed by cellulolytic organisms that have evolved a variety of structurally distinct enzymes, cellobiohydrolases and endoglucanases, for this purpose. Endoglucanase I (EG I) is the major endoglucanase produced by the cellulolytic fungus Trichoderma reesei, accounting for 5 to 10% of the total amount of cellulases produced by this organism. Together with EG I from Humicola insolens and T. reesei cellobiohydrolase I (CBH I), the enzyme is classified into family 7 of the glycosyl hydrolases, and it catalyses hydrolysis with a net retention of the anomeric configuration. The structure of the catalytic core domain (residues 1 to 371) of EG I from T. reesei has been determined at 3.6 A resolution by the molecular replacement method using the structures of T. reesei CBH I and H. insolens EG I as search models. By employing the 2-fold non-crystallographic symmetry (NCS), the structure was refined successfully, despite the limited resolution. The final model has an R-factor of 0.201 (Rfree 0.258). The structure of EG I reveals an extended, open substrate-binding cleft, rather than a tunnel as found in the homologous cellobiohydrolase CBH I. This confirms the earlier proposal that the tunnel-forming loops in CBH I have been deleted in EG I, which has resulted in an open active site in EG I, enabling it to function as an endoglucanase. Comparison of the structure of EG I with several related enzymes reveals structural similarities, and differences that relate to their biological function in degrading particular substrates. A possible structural explanation of the drastically different pH profiles of T. reesei and H. insolens EG I is proposed.

Homologous domains in Trichoderma reesei cellulolytic enzymes: gene sequence and expression of cellobiohydrolase II.

Fungal cellobiohydrolases are unique enzymes capable of degrading highly ordered crystalline cellulose. We present here the isolation and complete sequence analysis of the chromosomal and cDNA copies of the structural gene (cbh2) coding for one of the major cellobiohydrolases (CBH II) of Trichoderma reesei. We also present data on expression of the cbh2 gene and show that the transcription start points of the cbh2 gene are heterogeneous and are located 32 to 52 bp downstream from a putative TATA box. The derived CBH II protein sequence is 471 amino acids long and the coding region is interrupted by three short introns. Most of the CBH II protein bears no apparent resemblance to CBH I and endoglucanase I. However, a short region of extensive homology is found in all Trichoderma cellulases characterized so far, suggesting that this region is important for cellulose hydrolysis. The implications of this information with regard to the evolution of fungal cellulase genes and the enzymology of cellulose hydrolysis are discussed.

Efficient secretion of two fungal cellobiohydrolases by Saccharomyces cerevisiae.

Two different cellobiohydrolases, CBHI and CBHII, of the filamentous fungus Trichoderma reesei both hydrolyse highly crystalline cellulose. Cellulolytic strains of the yeast Saccharomyces cerevisiae were constructed by transferring cDNAs coding for these enzymes into yeast on an expression plasmid. These cellulolytic yeasts were able to secrete efficiently the large, heterologous proteins to the culture medium. The recombinant cellulases were observed to be heterogeneous in Mr due, at least partly, to variable N-glycosylation. Recombinant CBHII was able to bind to crystalline cellulose, although slightly less efficiently than the native enzyme. Both of the two recombinant cellulases were able to degrade amorphous cellulose. In a fermenter cultivation, around 100 micrograms/ml of CBHII was secreted into the yeast growth medium.

Efficient secretion of murine Fab fragments by Escherichia coli is determined by the first constant domain of the heavy chain.

Fab fragments of IgG1 and IgG3 subclass antibodies which bind to 2-phenyloxazolone (Ox) were produced in Escherichia coli. The signal sequences of the Fd and L chains were correctly processed, the fragments were secreted into the periplasmic space and released into the culture medium upon prolonged cultivations. The yields of active Ox IgG1 and Ox IgG3 Fab fragments after one-step purification from the culture medium by affinity chromatography were 2 micrograms/ml and 0.5 micrograms/ml, respectively. The majority of the purified Ox IgG1 Fab was properly assembled, but in the case of Ox IgG3, the preparation was found to consist of a complete L chain and C-terminally degraded fragments of the Fd chain. A deletion up to the interchain disulfide bond in the first constant domain (CH1) of the Ox IgG3 Fd chain led to proper assembly of the truncated Fab fragment. The production level of the truncated fragment was comparable to that of the Ox IgG1 Fab and its hapten-binding activity similar to that of the idiotype monoclonal antibody. The temperature stability of the Ox IgG1 Fab was similar to that of the intact antibody. However, both of the Ox IgG3 Fab fragments showed reduced stability, suggesting that the CH1 domain contributes significantly to the thermal stability of the Fab fragment.

EGIII, a new endoglucanase from Trichoderma reesei: the characterization of both gene and enzyme.

A novel endoglucanase from Trichoderma reesei, EGIII, has been purified and its catalytic properties have been studied. The gene for that enzyme (egl3) and cDNA have been cloned and sequenced. The deduced EGIII protein shows clear sequence homology to a Schizophyllum commune enzyme (M. Yaguchi, personal communication), but is very different from the three other T. reesei cellulases with known structure. Nevertheless, all the four T. reesei cellulases share two common, adjacent sequence domains, which apparently can be removed by proteolysis. These homologous sequences reside at the N termini of EGIII and the cellobiohydrolase CBHII, but at the C termini of EGI and CBHI. Comparison of the fungal cellulase structures has led to re-evaluation of hypotheses concerning the localization of the active sites.

Design of a pH-dependent cellulose-binding domain.

Protein-carbohydrate interactions typically rely on aromatic stacking interactions of tyrosine, phenylalanine and tryptophan side chains with the sugar rings whereas histidine residues are rarely involved. The small cellulose-binding domain of the Cel7A cellobiohydrolase (formerly CBHI) from Trichoderma reesei binds to crystalline cellulose primarily using a planar strip of three tyrosine side chains. Binding of the wild-type Cel7A CBD is practically insensitive to pH. Here we have investigated how histidine residues mediate the binding interaction and whether the protonation of a histidine side chain makes the binding sensitive to pH. Protein engineering of the Cel7A CBD was thus used to replace the tyrosine residues in two different positions with histidine residues. All of the mutants exhibited a clear pH-dependency of the binding, in clear contrast to the wild-type. Although the binding of the mutants at optimal pH was less than for the wild-type, in one case, Y31H, this binding almost reached the wild-type level.

A scheme for designating enzymes that hydrolyse the polysaccharides in the cell walls of plants.

A scheme is proposed for designating enzymes that hydrolyse the polysaccharides in the cell walls of plants. These enzymes are predominantly beta-1,4-glycanases. The scheme is based on the classification of the catalytic domains of glycoside hydrolases into families of related amino acid sequences. The new designation for an enzyme indicates its family and, because all members of a family have these characteristics in common, its three-dimensional fold and stereospecificity of hydrolysis. The scheme is intended to simplify comparison of the systems of enzymes produced by different microorganisms for the hydrolysis of plant cell walls.

The difference in affinity between two fungal cellulose-binding domains is dominated by a single amino acid substitution.

Cellulose-binding domains (CBDs) form distinct functional units of most cellulolytic enzymes. We have compared the cellulose-binding affinities of the CBDs of cellobiohydrolase I (CBHI) and endoglucanase I (EGI) from the fungus Trichoderma reesei. The CBD of EGI had significantly higher affinity than that of CBHI. Four variants of the CBHI CBD were made in order to identify the residues responsible for the increased affinity in EGI. Most of the difference could be ascribed to a replacement of a tyrosine by a tryptophan on the flat cellulose-binding face.

Tryptophan 272: an essential determinant of crystalline cellulose degradation by Trichoderma reesei cellobiohydrolase Cel6A.

Trichoderma reesei cellobiohydrolase Cel6A (formerly CBHII) has a tunnel shaped active site with four internal subsites for the glucose units. We have predicted an additional ring stacking interaction for a sixth glucose moiety with a tryptophan residue (W272) found on the domain surface. Mutagenesis of this residue selectively impairs the enzyme function on crystalline cellulose but not on soluble or amorphous substrates. Our data shows that W272 forms an additional subsite at the entrance of the active site tunnel and suggests it has a specialised role in crystalline cellulose degradation, possibly in guiding a glucan chain into the tunnel.

Site-directed mutagenesis of the putative catalytic residues of Trichoderma reesei cellobiohydrolase I and endoglucanase I.

Site directed mutagenesis has been performed to test hypotheses concerning the putative active sites of Trichoderma reesei cellobiohydrolase I and endoglucanase I. It is shown that mutagenesis of the residue E126, previously proposed to be the proton donor in CBHI, did not totally inactivate the enzyme while mutagenesis of the residue E127 in the homologous enzyme EGI resulted in complete loss of activity. These results are compared with those obtained in similar studies of other glucanases and the effects on enzymatic activity of hyperglycosylation of the yeast produced cellulases are discussed.

Directed immobilization of recombinant staphylococci on cotton fibers by functional display of a fungal cellulose-binding domain.

The immobilization of recombinant staphylococci onto cellulose fibers through surface display of a fungal cellulose-binding domain (CBD) was investigated. Chimeric proteins containing the CBD from Trichoderma reesei cellulase Cel6A were found to be correctly targeted to the cell wall of Staphylococcus carnosus cells, since full-length proteins could be extracted and affinity-purified. Furthermore, surface accessibility of the CBD was verified using a monoclonal antibody and functionality in terms of cellulose-binding was demonstrated in two different assays in which recombinant staphylococci were found to efficiently bind to cotton fibers. The implications of this strategy of directed immobilization for the generation of whole-cell microbial tools for different applications will be discussed.

Trichoderma reesei cellobiohydrolase I with an endoglucanase cellulose-binding domain: action on bacterial microcrystalline cellulose.

Cellulolytic enzymes consist of distinct catalytic and cellulose-binding domains (CBDs). The presence of a CBD improves the binding and activity of cellulases on insoluble substrates but has no influence on their activities on soluble substrates. Structural and biochemical studies of a fungal CBD from Trichoderma reesei cellobiohydrolase I have revealed a wedge shaped structure with a flat cellulose binding surface containing three essential tyrosine residues. The face of the wedge is strictly conserved in all fungal CBDs while many differences occur on the other face of the wedge. Here we have studied the importance of these differences on the function of the T. reesei CBHI by replacing its CBD by a homologous CBD from the endoglucanase, EGI. Our data shows that, apart from slightly improved affinity of the hybrid enzyme, the domain exchange does not significantly influence the function of CBHI.

Efficient enantioselective separation of drug enantiomers by immobilised antibody fragments.

There is an increasing need for methods for efficient enantioselective separation and purification of chiral drugs. Genetic engineering provides the means for generating recombinant antibodies exhibiting extremely high specificity for even small molecular mass compounds. Here, recombinant antibody fragments have been generated for the drug diarylalkyltriazole that contains two chiral centres. Immobilised antibody fragments has been used successfully for efficient, step-wise separation of two enantiomers of the drug. Owing to the antibody specificity, one enantiomer came out in the flow-through, while the bound enantiomer could be specifically eluted. One of the antibodies tolerated solvents required both for dissolving the target molecules and for their elution for extended times and was shown to function over multiple cycles of the separation process.

Saccharomyces cerevisiae cells secreting an Aspergillus niger beta-galactosidase grow on whey permeate.

We describe the construction of a lactose-utilizing Saccharomyces cerevisiae that expresses the cDNA for a secreted, thermostable beta-galactosidase (lacA) from Aspergillus niger. Yeast cells expressing the lacA gene from the yeast ADH1 promotor on a multicopy plasmid secrete up to 40% of the total beta-galactosidase activity into the growth medium. The secreted product is extensively N-glycosylated, and cells expressing the lacA gene grow on whey permeate (4% w/v lactose) with a doubling time of 1.6 hours. Such strains may offer a solution to the increasing problem of waste whey disposal.

The cellulose-binding domain of the major cellobiohydrolase of Trichoderma reesei exhibits true reversibility and a high exchange rate on crystalline cellulose.

Cellulose-binding domains (CBDs) bind specifically to cellulose, and form distinct domains of most cellulose degrading enzymes. The CBD-mediated binding of the enzyme has a fundamental role in the hydrolysis of the solid cellulose substrate. In this work we have investigated the reversibility and kinetics of the binding of the CBD from Trichoderma reesei cellobiohydrolase I on microcrystalline cellulose. The CBD was produced in Escherichia coli, purified, and radioactively labeled by reductive alkylation with 3H. Sensitive detection of the labeled CBD allowed more detailed analysis of its behavior than has been possible before, and important novel features were resolved. Binding of the CBD was found to be temperature sensitive, with an increased affinity at lower temperatures. The interaction of the CBD with cellulose was shown to be fully reversible and the CBD could be eluted from cellulose by simple dilution. The rate of exchange measured for the CBD-cellulose interaction compares well with the hydrolysis rate of cellobiohydrolase I, which is consistent with its proposed mode of action as a processive exoglucanase.

Characterization of Trichoderma reesei cellobiohydrolase Cel7A secreted from Pichia pastoris using two different promoters.

Heterologous expression of T. reesei cellobiohydrolase Cel7A in a methylotrophic yeast Pichia pastoris was tested both under the P. pastoris alcohol oxidase (AOX1) promoter and the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter in a fermentor. Production of Cel7A with the AOX1 promoter gave a better yield, although part of the enzyme expressed was apparently not correctly folded. Cel7A expressed in P. pastoris is overglycosylated at its N-glycosylation sites as compared to the native T. reesei protein, but less extensive than Cel7A expressed in Saccharomyces cerevisiae. The k(cat) and K(m) values for the purified protein on soluble substrates are similar to the values found for the native Trichoderma Cel7A, whereas the degradation rate on crystalline substrate (BMCC) is somewhat reduced. The measured pH optimum also closely resembles that of purified T. reesei Cel7A. Furthermore, the hyperglycosylation does not affect the thermostability of the enzyme monitored with tryptophane fluorescence and activity measurements. On the other hand, CD measurements indicate that the formation of disulfide bridges is an important step in the correct folding of Cel7A and might explain the difficulties encountered in heterologous expression of T. reesei Cel7A. The constitutive GAP promoter expression system of P. pastoris is nevertheless well suited for activity screening of cellulase activities in microtiter plates. With this type of screening method a faster selection of site-directed and random mutants with, for instance, an altered optimum pH is possible, in contrast to the homologous T. reesei expression system.