Three-dimensional structure of cellobiohydrolase II from Trichoderma reesei.

The enzymatic degradation of cellulose is an important process, both ecologically and commercially. The three-dimensional structure of a cellulase, the enzymatic core of CBHII from the fungus Trichoderma reesei reveals an alpha-beta protein with a fold similar to but different from the widely occurring barrel topology first observed in triose phosphate isomerase. The active site of CBHII is located at the carboxyl-terminal end of a parallel beta barrel, in an enclosed tunnel through which the cellulose threads. Two aspartic acid residues, located in the center of the tunnel are the probable catalytic residues.

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.

Expression of dicistronic transcriptional units in transgenic tobacco.

We investigated whether the two cistrons of a dicistronic mRNA can be translated in plants to yield both gene products. The coding sequences of various reporter genes were combined in dicistronic units, and their expression was analyzed in stably transformed tobacco plants at the RNA and protein levels. The presence of an upstream cistron resulted in all cases in a drastically reduced expression of the downstream cistron. The translational efficiency of the gene located downstream in the dicistronic units was 500- to 1,500-fold lower than that in a monocistronic control; a 500-fold lower value was obtained with a dicistronic unit in which both cistrons were separated by 30 nucleotides, whereas a 1,500-fold lower value was obtained with a dicistronic unit in which the stop codon of the upstream cistron and the start codon of the downstream cistron overlapped. As a strategy to select indirectly for transformants with enhanced levels of expression of a gene which is by itself nonselectable, the gene of interest can be cloned upstream from a selectable marker in a dicistronic configuration. This strategy can be used provided that the amount of dicistronic mRNA is high. If, on the other hand, the expression of the dicistronic unit is too low, selection of the downstream cistron will primarily give clones with rearranged dicistronic units.

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.

Crystallization and preliminary X-ray studies on the core proteins of cellobiohydrolase I and endoglucanase I from Trichoderma reesei.

The catalytic core domains of cellobiohydrolase I (CBHI) and endoglucanase I (EGI) from Trichoderma reesei have been crystallized using the hanging drop vapour diffusion method. In the case of CBHI, use of polyethylene glycol 20,000, and calcium chloride at low pH produced good quality single crystals suitable for X-ray studies. The crystals belong to a primitive orthorhombic space group with unit cell dimensions a = 84.0 A, b = 86.2 A, c = 111.8 A, and diffract beyond 2.0 A resolution. Bipyramidal crystals of EGI core were grown from ammonium sulphate at pH 7.5. The crystals are tetragonal, either P4(1)22 or the enantiomorph P4(3)22, with cell dimensions a = b = 101.8 A and c = 198.0 A, and at best diffract to a resolution of 2.5 A.

Crystallization of the core protein of cellobiohydrolase II from Trichoderma reesei.

Single crystals of the core protein of the cellulase cellobiohydrolase II have been grown in polyethylene glycol 6000 with the hanging drop method. Successful crystallization occurred only when 82 amino acids were removed from the N terminus by papain cleavage. Crystals belong to the space group P2(1) and have cell constants a = 49.1 A, b = 75.8 A, c = 92.9 A, beta = 103.2. The diffraction pattern extends to better than 2.0 A.

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.

Cloning, nucleotide sequence and characterization of genes encoding naphthalene dioxygenase of Pseudomonas putida strain NCIB9816.

We have cloned the naphthalene dioxygenase(ND)-coding genes from Pseudomonas putida strain NCIB9816 based on their ability to convert indole to indigo. The region coding for this enzyme activity was sequenced and three successive open reading frames were found. The corresponding gene products were identified using the T7 polymerase/promoter system. All of them are necessary for the ND activity. A comparison of the ND-coding genes with the ones coding for benzene dioxygenase revealed significant homology which was more pronounced at the nucleotide level than at the amino acid level.

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.

Molecular analysis of a Phanerochaete chrysosporium lignin peroxidase gene.

The basidiomycete fungus Phanerochaete chrysosporium produces a number of extracellular peroxidases which appear to be important for lignin degradation. We present here the isolation and complete nucleotide (nt) sequence of a gene (lpo) coding for lignin peroxidase (LPO), the coding region of which is identical to a lpo cDNA sequence which had previously been described [M. Tien and C.-P.D. Tu, Nature 326 (1987) 520-523]. The deduced amino acid (aa) sequence corresponds to 372 aa residues and the coding region is interrupted by eight short introns that range in size from 50 to 62 nt. Southern blot experiments using the cloned lpo gene as hybridization probe revealed a complex restriction fragment pattern, indicating that there are a number of lpo-related nucleotide sequences present in P. chrysosporium DNA which cross-hybridize. We also present data on the in vivo expression of the lpo genes and show that they are regulated at the RNA level and that the structure of the transcripts as judged from S1 experiments is complex. These data are consistent with the idea that there are a number of related lpo genes in P. chrysosporium which constitute a gene family.

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.

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.

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.

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.

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.

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.

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.