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.

Binding of 4-methylumbelliferyl-beta-D-ribopyranoside to beta-D-xylosidase from Bacillus pumilus.

The determination of the binding parameters of 4-methylumbelliferyl-beta-D-ribopyranoside, a fluorescent ligand of beta-D-xylosidase (exo-1,4-beta-xylosidase, EC 3.2.1.37) from Bacillus pumilus, is described. A single binding site per polypeptide chain (60 000 daltons) was found and the homogeneity of the binding sites in the dimeric or tetrameric forms of the enzyme were shown. The association constants, as a function of temperature and ionic strength, were obtained from equilibrium binding experiments and compared to the kinetically determined inhibition constants. The apparent discrepancies are attributed to a temperature, ionic strength and concentration dependent shift in the dimer-tetramer equilibrium of the enzyme and different affinities of the ligand for both oligomeric forms. Sedimentation velocity experiments seem to corroborate this hypothesis.

Family A cellulases: two essential tryptophan residues in endoglucanase III from Trichoderma reesei.

Three tryptophan residues are readily oxidised by N-bromosuccinimide in endoglucanase III from Trichoderma reesei. Evidence was obtained that the residue first modified is situated in the cellulose-binding domain and the second in the enzyme's catalytic site. The latter influences the binding and hydrolysis of soluble substrates. The modification of a third residue does not further affect the catalytic properties. The present results complement published data concerning other identified catalytic residues, and help to clarify the active site structure of family A cellulases.

Beta-D-xylosidase from Bacillus pumilus. Molecular properties and oligomeric structure.

Bacillus pumilus beta-D-xylosidase, purified by affinity chromatography, seems to be homogeneous, as judged by disc electrophoresis and gel filtration. The absorption coefficient at 280 nm, Ao, 1% 1cm, determined by the dry weight method, is 1.78. The complete amino acid composition is determined. Sedimentation velocity studies show the presence of two components with S20, W values of 10.0 S and 6.6 S. After glutaraldehyde cross-linking two, enzymically active, components, with apparent molecular weights 126 000 and 243 000, can be isolated by preparative sucrose gradient ultracentrifugation. These values are confirmed by analytical disc electrophoresis at different acrylamide concentrations. The subunit molecular weight is 60 000. L-Methionine is the only N-terminal amino acid detectable. The possible presence of both dimeric and tetrameric forms of the beta-D-xylosidase in solution has to be envisaged.

Purification and properties of an induced beta-D-glucosidase from stachybotrys atra.

We have purified an induced beta-D-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) from Stachybotrys atra to apparent homogeneity. The induced enzyme was clearly different from the constitutive beta-D-glucosidase. The molecular weight was 65 500-69 000, the pH optimum was at 6.7 and the isoelectric point at 4.8. Carbohydrate content (related to glucose) was 14.4%. The enzyme showed beta-D-glucosidase, beta-D-xylosidase and beta-D-thioglucosidase activity. These three activities sppear to be due to the same enzyme. The enzyme was strongly inhibited by D-glucono-(1 leads to 5)-lactone and nojirimycin and was very sensitive to sulfhydryl reagents.

Monoclonal antibodies against different domains of cellobiohydrolase I and II from Trichoderma reesei.

Monoclonal antibodies have been produced against two functionally different domains present in two cellobiohydrolases from Trichoderma reesei (CBH I and CBH II). Four groups of antibodies were obtained, which specifically recognized (Western blotting, ELISA) (a) the core protein within CBH I, (b) the core protein within CBH II, (c) the BA region of CBH I, and (d) the ABB' region of CBH II. No cross-reactivities within these four groups were observed. The antibodies reacted also specifically with proteins of similar size to CBH I and CBH II (SDS-PAGE) from other Trichoderma strains (Western blotting), whereas no reaction was observed with cellulases from other fungal sources. Analysis of culture filtrates of T. reesei QM 9414 harvested at various times of growth on cellulose under buffered conditions (pH 5-6) indicated the presence of only single bands of CBH I and CBH II, even after prolonged cultivation (160 h). Cultivation on cellulose in unbuffered media, however, showed the appearance (Western blotting) of additional lower molecular weight proteins, which reacted with the monoclonal antibodies directed against the cores of CBH I and II, but not with those recognizing the respective BA and ABB' regions. The appearance of these lower molecular weight bands was most pronounced in unbuffered media, supplemented with a 3-fold (w/w) amount of organic nitrogen (peptone). Analysis of some commercial cellulase preparations from T. harzianum revealed the same pattern of lower molecular weight proteins, in contrast to samples from other fungal cellulases. Those samples or preparations, showing a multiple pattern of CBH I and CBH II, exhibited higher activities of an acid proteinase. These results imply that the use of unbuffered, high nitrogen-supplemented culture conditions for production of cellulases may lead to considerable proteolytic modification of the secreted cellobiohydrolases.

An elaboration on the syn-anti proton donor concept of glycoside hydrolases: electrostatic stabilisation of the transition state as a general strategy.

An in silico survey of all known 3D-structures of glycoside hydrolases that contain a ligand in the -1 subsite is presented. A recurrent crucial positioning of active site residues indicates a common general strategy for electrostatic stabilisation directed to the carbohydrate's ring-oxygen at the transition state. This is substantially different depending on whether the enzyme's proton donor is syn or anti positioned versus the substrate. A comprehensive list of enzymes belonging to 42 different families is given and selected examples are described. An implication for an early evolution scenario of glycoside hydrolases is discussed.

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 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.

Specificity mapping of cellulolytic enzymes: classification into families of structurally related proteins confirmed by biochemical analysis.

The specificities of 15 cellulolytic enzymes have been examined using chromophoric glycosides derived from D-glucose, cellobiose, higher cellooligosaccharides, lactose, D-xylose, and beta-(1,4)-xylobiose. Coinciding with a classification based on hydrophobic cluster analysis of amino acid sequences, six families each showing a characteristic specificity pattern were observed. Furthermore, in these cases where the anomeric forms of reaction products were determined, results seem to indicate conservation of intrinsic reaction mechanism (single or double displacement) within each family. On the other hand, the low molecular weight substrates do not discriminate exo- from endocellulases. This functional differentiation is speculated to originate from the presence, in exoenzymes, of a tunnel-shaped active site formed by extra loops in their structure.

Cellulase families revealed by hydrophobic cluster analysis.

The amino acid sequences of 21 beta-glycanases have been compared by hydrophobic cluster analysis. Six families of cellulases have been identified on the basis of primary structure homology: (A) endoglucanases B, C and E of Clostridium thermocellum; endoglucanases of Erwinia chrysanthemi and Bacillus sp.; endoglucanase III of Trichoderma reesei; endoglucanase I of Schizophyllum commune; (B) cellobiohydrolase II of T. reesei; endoglucanases of Cellulomonas fimi and Streptomyces sp; (C) cellobiohydrolases I of T. reesei and of Phanerochaete chrysosporium; endoglucanase I of T. reesei; (D) endoglucanase A of C. thermocellum and an endoglucanase from Ce. uda; (E) endoglucanase D of C. thermocellum and an endoglucanase from Pseudomonas fluorescens; (F) xylanases of C. thermocellum and of Cryptococcus albidus and the cellobio-hydrolase of Ce. fimi. For each family, conserved potentially catalytic residues have have been listed and previous allocations of the active-site residues are evaluated in the light of the alignment of the amino acid sequences. A strong homology is also reported for the putative cellulose-binding domains of cellulases of Ce. fimi and of P. fluorescens.

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.

A hydrophobic platform as a mechanistically relevant transition state stabilising factor appears to be present in the active centre of all glycoside hydrolases.

An in silico survey of the -1 subsite of all known 3D-structures of O-glycoside hydrolases containing a suitably positioned ligand has led to the recognition -- apparently without exceptions -- of a transition state stabilising hydrophobic platform which is complementary to a crucial hydrophobic patch of the ligand. This platform is family-specific and highly conserved. A comprehensive list is given with examples of enzymes belonging to 33 different families. Several typical constellations of platform - protein residues are described.

Stereochemical course of hydrolysis and hydration reactions catalysed by cellobiohydrolases I and II from Trichoderma reesei.

Cellobiohydrolase I from Trichoderma reesei catalyzes the hydrolysis of methyl beta-D-cellotrioside (Km = 48 microM, kcat = 0.7 min-1) with release of the beta-cellobiose (retention of configuration). The same enzyme catalyzes the trans-hydration of cellobial (Km = 116 microM, kcat = 1.16 min-1) and lactal (Km = 135 microM, kcat = 1.35 min-1), presumably with glycosyl oxo-carbonium ion mediation. Protonation of the double bond is from the direction opposite that assumed for methyl beta-cellotrioside, but products formed from these prochiral substrates are again of beta configuration. Cellobiohydrolase II from the same microorganism hydrolyzes methyl beta-D-cellotetraoside (Km = 4 microM, kcat = 112 min-1) with inversion of configuration to produce alpha-cellobiose. The other reaction product, methyl beta-cellobioside, is in turn partly hydrolysed by cellobiohydrolase II to form methyl beta-D-glucoside and D-glucose, presumably the alpha-anomer. Reaction with cellobial is too slow to permit unequivocal determination of product configuration, but clear evidence is obtained that protonation occurs from the si-direction, again opposite that assumed for protonating glycosidic substrates. These results add substantially to the growing evidence that individual glycosidases create the anomeric configuration of their reaction products by means that are independent of substrate configuration.

Identification of an essential glutamate residue in the active site of endoglucanase III from Trichoderma reesei.

n-Propyl, n-butyl and n-pentyl beta-cellobiosides with a reactive omega-epoxide in their aglycon completely and irreversibly inactivate endoglucanase III from Trichoderma reesei. The pentyl derivative was found to be most effective. From these affinity labeling experiments evidence was found for the implication of Glu329 in the reaction mechanism. This is discussed in relation to other structural/functional data known for endoglucanase III and several other family A glycanases.

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.

The Q-Trap mass spectrometer, a novel tool in the study of protein glycosylation.

The use of the recently introduced Q-Trap mass spectrometer in the study of protein glycosylation is described. The combined ion trap and triple quadrupole scan functions make it a powerful system in both oligosaccharide and glycopeptide analysis. Several oligosaccharides, both linear and branched, were analyzed to obtain information on sequence, linkage, and branching. Quadrupole like MS/MS spectra with ion trap sensitivity but without the typical ion trap low mass cut-off were obtained. To determine the origin of fragments and to reveal the existence of new ions, the MS(3) capabilities of the system proved to be useful. Glycopeptides were selectively detected in peptide mixtures using the triple quadrupole precursor ion scan function, either in off-line experiments or during LC/MS using information dependent acquisition (IDA).

Purification and characterization of two low molecular mass alkaline xylanases from Fusarium oxysporum F3.

Two low molecular mass endo-1,4-beta-D-xylanases from Fusarium oxysporum were purified to homogeneity by gel-filtration and ion-exchange chromatography. They exhibit molecular masses of 20.8 (xylanase I) and 23.5 (xylanase II) kDa, and isoelectric points of 9.5 and 8.45-8.70, respectively. Both xylanases display remarkable pH (9.0) stability. At 40 to 55 degrees C xylanase II is more thermostable than xylanase I but less active on xylan. In contrast to xylanase I, xylanase II is able to hydrolyze 1-O-4-methylumbelliferyl-beta-D-glucopyranosyl)-beta-D-xylopyranoside (muxg). Neither of these enzymes hydrolyze xylotriose. They bind on crystalline cellulose but not on insoluble xylan. Analysis of reaction mixtures by high pressure liquid chromatography revealed that both enzymes cleave preferentially the internal glycosidic bonds of xylopentaose and oat spelts xylan. Thus the purified enzymes appeared to be true endo-beta-1,4-xylanases. The amino terminal sequences of xylanases I and II show to homology. Xylanase I shows high similarity with alkaline low molecular mass xylanases of family G/11.

Molecular cloning and enzymatic characterization of a Trichoderma reesei 1,2-alpha-D-mannosidase.

A cDNA encoding 1,2-alpha-D-mannosidase mds 1 from Trichoderma reesei was cloned. The largest open reading frame occupied 1571 bp. The predicted sequence contains 523 amino acid residues for a calculated molecular mass of 56,266 Da and shows high similarity to the amino acid sequences of 1,2-alpha-D-mannosidases from Aspergillus saitoi and Penicillium citrinum (51.6 and 51.0% identity, respectively). T. reesei mannosidase was produced as a recombinant enzyme in the yeast Pichia pastoris. Replacement of the N-terminal part with the prepro-signal peptide of the Saccharomyces cerevisiae alpha-mating factor resulted in high amounts of secreted enzyme. A three-step purification protocol was designed and the enzymatic properties were analyzed. The enzyme was characterized as a class-I mannosidase.

Sudan-ß-d-glucuronides and their use for the histochemical localization of ß-glucuronidase activity in transgenic plants.

Synthesis of five different Sudan-ß-D-glucuronides (I, II, III, IV, and RedB) was performed by condensation of a set of red Sudan diazo dyes with methyl (1-deoxy-2,3,4-tri-O-acetyl-1-trichloroacetimidoyl-α-D-glucopyran)uronate. After the acid and alcohol groups had been deprotected, the resulting compounds were used for histochemical localization of ß-glucuronidase (GUS) activity in transgenic plants (Petunia hybrida, Arabidopsis thaliana, and Nicotiana tabacum) that contained the GUS reporter system. Because the cleavage of the ß-glucuronide results in the liberation of an insoluble Sudan dye, Sudan substrates gave no diffusion artifacts as described for the commonly used 5-bromo-4-chloro-3-indolyl-ß-D-glucuronide (X-gluc). A comparison of assays with different Sudan glucuronides and X-gluc demonstrated that the SudanIV variant is a valuable glucuronide substrate for the precise histochemical localization of GUS activity in transgenic plants.