Effect of endoglucanase and cellobiohydrolase from Trichoderma reesei on cellulose microfibril structure

Abstract Cellobiohydrolase (CBH, EC 3.2.91) was purified to homogeneity from Trichoderma reesei culture fluids by means of preparative isoelectric focussing (IEF). Its isoelectric points was 4.2. The degradation product of crystalline cellulose (Avicel and cotton) was predominantly cellobiose. The action of purified endoglucanase (EG) and CBH on cellulose microfibrils was followed by transmission electron microscopy (TEM) observations after Pt-C shadowing of the specimen. EG pretreatment of microfibrils resulted in submicrofibril formation. Addition of CBH induced the conversion of submicrofibrils into heterogeneous cellulose clusters and into homogeneous cellulose plaques. One structural effect of CBH was the increase in accessible cellulose surface area, possibly providing intermolecular entrace of water molecules between adjacent cellulose chains. Plaque formation is interpreted as a visible CBH action on crystalline cellulose to form swollen water-insoluble cellulose intermediates.     

The competitive inhibition of Trichoderma reesei C30 cellobiohydrolase I by guanidine hydrochloride

TheP-nitrophenylcellobiosidase (PNPCase) activity of Trichoderma reesei cellobiohydrolase I (CBH I) was competitively inhibited by concentrations of guanidine hydrochloride (Gdn HC1) that did not affect the tryptophan fluorescence of this enzyme. The K_m of CBH I, 3.6 mM, was increased to 45.4 mM in the presence of 0.14 M Gdn HCl, the concentration that was required to inhibit the enzyme by 50%. A similar concentration of lithium chloride and urea had little effect on the PNPCase activity of CBH I. Maximal inhibition was pH dependent, occurring in the range of pH 4.0 to 5.0, which is in the range for maximal activity. Analysis of the inhibition data indicated that 1.2 molecules of Gdn HCl combine reversibly with I molecule of CBH I. Other hydrolases and proteases were also inhibited by Gdn HCl. It is suggested that the inhibition of CBH I by Gdn HCl occurs as a result of the interaction between the positively charged guanidinium group of Gdn HCl and the carboxylate group of glutamic acid 126, postulated to be in the catalytic center of this enzyme.     

Purification of cellobiohydrolase I fromTrichoderma reesei using monoclonal antibodies in an immunomatrix

Summary The several components of the fungal cellulase system present practical problems in devising facile and efficient schemes for their purification. We report on a new single-step affinity chromatographic method for purification of cellobiohydrolase I ofTrichoderma reesei based on its selective absorption and elution using an immunomatrix constructed with CnBr-activated Sepharose 4B and monoclonal antibody specific for the enzyme. Isoenzymes of cellobiohydrolase I were purified directly from crude culture filtrate. The method is fast, simple, and of high resolution.     

Three-dimensional structures of three engineered cellulose-binding domains of cellobiohydrolase I from Trichoderma reesei.

Three-dimensional solution structures for three engineered, synthetic CBDs (Y5A, Y31A, and Y32A) of cellobiohydrolase I (CBHI) from Trichoderma reesei were studied with nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy. According to CD measurements the antiparallel beta-sheet structure of the CBD fold was preserved in all engineered peptides. The three-dimensional NMR-based structures of Y31A and Y32A revealed only small local changes due to mutations in the flat face of CBD, which is expected to bind to crystalline cellulose. Therefore, the structural roles of Y31 and Y32 are minor, but their functional importance is obvious because these mutants do not bind strongly to cellulose. In the case of Y5A, the disruption of the structural framework at the N-terminus and the complete loss of binding affinity implies that Y5 has both structural and functional significance. The number of aromatic residues and their precise spatial arrangement in the flat face of the type I CBD fold appears to be critical for specific binding. A model for the CBD binding in which the three aligned aromatic rings stack onto every other glucose ring of the cellulose polymer is discussed.     

Characterization of cellobiohydrolase I (Cel7A) glycoforms from extracts of Trichoderma reesei using capillary isoelectric focusing and electrospray mass spectrometry

Capillary isoelectric focusing (CIEF) was used to profile the cellulase composition in complex fermentation samples of secreted proteins from Trichoderma reesei. The enzyme cellobiohydrolase I (CBH I, also referred to as Cel7A), a major component in these extracts, was purified from different strains and characterized using analytical methods such as CIEF, high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC–PAD), and capillary liquid chromatography–electrospray mass spectrometry (cLC–ESMS). ESMS was also used to monitor the extent of glycosylation in CBH I isolated from T. reesei strain RUT-C30 and two derivative mutant strains. Selective identification of tryptic N-linked glycopeptides was achieved using LC–ESMS on a quadrupole/time-of-flight instrument with a mixed scan function. The suspected glycopeptides were further analyzed by on-line tandem mass spectrometry to determine the nature of N-linked glycans and their attachment sites. This strategy enabled the identification of a high mannose glycan attached to Asn270 (predominantly Man₈GlcNAc₂) and single GlcNAc occupancy at Asn45 and Asn384 with some site heterogeneity depending on strains and fermentation conditions. The linker region of CBH I was shown to be extensively glycosylated with di-, and tri-saccharides at Thr and Ser residues as indicated by MALDI-TOF and HPAEC–PAD experiments. Additional heterogeneity was noted in the CBH I linker peptide of RUT-C30 strain with the presence of a phosphorylated di-saccharide.     

The primary structure of a 1,4-β-glucan cellobiohydrolase from the fungus Trichoderma reesei QM 9414

The sequence of the approx. 490 amino acid residues of the main 1,4-β-glucan cellobiohydrolase (CBH I) (EC 3.2.1.91) from culture filtrates of the fungus Trichoderma reesei QM 9414 has been established by automatic liquid phase Edman degradation. Peptides obtained by chemical and enzymatic cleavage of the reduced and S-carboxymethylated protein were isolated by a combination of gel filtration and high-performance liquid chromatography. The amino-terminus of the single polypeptide chain is blocked by a pyroglutamyl residue. Most of the neutral carbohydrate present in the glycoprotein is bound within a short region near the carboxyl-terminus. Three attachment sites of glucosamine residues have also been established.     

Sugar-binding sites on the surface of the carbohydrate-binding module of CBH I from Trichoderma reesei

Molecular dynamics simulations were carried out for a system consisting of the carbohydrate-binding module (CBM) of the cellulase CBH I from Trichoderma reesei (Hypocrea jecorina) in a concentrated solution of β-d-glucopyranose, to determine whether there is any tendency for the sugar molecules to bind to the CBM. In spite of the general tendency of glucose to behave as an osmolyte, a marked tendency for the sugar molecules to bind to the protein was observed. However, the glucose molecules tended to bind only to specific sites on the protein. As expected, the hydrophobic face of the sugar molecules, comprising the axial H1, H3, and H5 aliphatic protons, tended to adhere to the flat faces of the three tyrosine side chains on the planar binding surface of the CBM. However, a significant tendency to bind to a groove-like feature on the upper surface of the CBM was also observed. These results would not be inconsistent with a model of the mechanism for this globular domain in which the cellodextrin chain being removed from the surface of crystalline cellulose passes over the upper surface of the CBM, presumably then available for hydrolysis in the active site tunnel of this processive cellulase.     

Lactosylamidine-based affinity purification for cellulolytic enzymes EG I and CBH I from Hypocrea jecorina and their properties

Selective adsorption and separation of β-glucosidase, endo-acting endo-β-(1→4)-glucanase I (EG I), and exo-acting cellobiohydrolase I (CBH I) were achieved by affinity chromatography with β-lactosylamidine as ligand. A crude cellulase preparation from Hypocrea jecorina served as the source of enzyme. When crude cellulase was applied to the lactosylamidine-based affinity column, β-glucosidase appeared in the unbound fraction. By contrast, EG I and CBH I were retained on the column and then separated from each other by appropriately adjusting the elution conditions. The relative affinities of the enzymes, based on their column elution conditions, were strongly dependent on the ligand. The highly purified EG I and CBH I, obtained by affinity chromatography, were further purified by Mono P and DEAE chromatography, respectively. EG I and CBH I cleave only at the phenolic bond in p-nitrophenyl glycosides with lactose and N-acetyllactosamine (LacNAc). By contrast, both scissile bonds in p-nitrophenyl glycosides with cellobiose were subject to hydrolysis although with important differences in their kinetic parameters.     

N-glycoform diversity of cellobiohydrolase I from Penicillium decumbens and synergism of nonhydrolytic glycoform in cellulose degradation

Four cellobiohydrolase I (CBHI) glycoforms, namely, CBHI-A, CBHI-B, CBHI-C, and CBHI-D, were purified from the cultured broth of Penicillium decumbens JU-A10. All glycoforms had the same amino acid sequence but displayed different characteristics and biological functions. The effects of the N-glycans of the glycoforms on CBH activity were analyzed using mass spectrum data. Longer N-glycan chains at the Asn-137 of CBHI increased CBH activity. After the N-glycans were removed using site-directed mutagenesis and homologous expression in P. decumbens, the specific CBH activity of the recombinant CBHI without N-glycosylation increased by 65% compared with the wild-type CBHI with the highest specific activity. However, the activity was not stable. Only the N-glycosylation at Asn-137 can improve CBH activity by 40%. rCBHI with N-glycosylation only at Asn-470 exhibited no enzymatic activity. CBH activity was affected whether or not the protein was glycosylated, together with the N-glycosylation site and N-glycan structure. N-Glycosylation not only affects CBH activity but may also bring a new feature to a nonhydrolytic CBHI glycoform (CBHI-A). By supplementing CBHI-A to different commercial cellulase preparations, the glucose yield of lignocellulose hydrolysis increased by >20%. After treatment with a low dose (5 mg/g substrate) of CBHI-A at 50 °C for 7 days, the hydrogen-bond intensity and crystalline degree of cotton fibers decreased by 17 and 34%, respectively. These results may provide new guidelines for cellulase engineering.     

The relation between solution association and surface activity of the hydrophobin HFBI from Trichoderma reesei

Hydrophobins are small fungal surface active proteins that self-assemble at interfaces into films with nanoscale structures. The hydrophobin HFBI from Trichoderma reesei has been shown to associate in solution into tetramers but the role of this association on the function of HFBI has remained unclear. We produced two HFBI variants that showed a significant shift in solution association equilibrium towards the tetramer state. However, this enhanced solution association did not alter the surface properties of the variant HFBIs. The results show that there is not a strong relationship between HFBI solution association state and surface properties such as surface activity.     

Emerging applications of small angle solution scattering in structural biology

Small angle solution X‐ray and neutron scattering recently resurfaced as powerful tools to address an array of biological problems including folding, intrinsic disorder, conformational transitions, macromolecular crowding, and self or hetero‐assembling of biomacromolecules. In addition, small angle solution scattering complements crystallography, nuclear magnetic resonance spectroscopy, and other structural methods to aid in the structure determinations of multidomain or multicomponent proteins or nucleoprotein assemblies. Neutron scattering with hydrogen/deuterium contrast variation, or X‐ray scattering with sucrose contrast variation to a certain extent, is a convenient tool for characterizing the organizations of two‐component systems such as a nucleoprotein or a lipid‐protein assembly. Time‐resolved small and wide‐angle solution scattering to study biological processes in real time, and the use of localized heavy‐atom labeling and anomalous solution scattering for applications as FRET‐like molecular rulers, are amongst promising newer developments. Despite the challenges in data analysis and interpretation, these X‐ray/neutron solution scattering based approaches hold great promise for understanding a wide variety of complex processes prevalent in the biological milieu.     

Structural characterization of the alpha-hemolysin monomer from Staphylococcus aureus

Alpha-hemolysin from Staphylococcus aureus is secreted as a water-soluble monomer and assembles on membranes to oligomerize into a homo-heptameric, water-filled pore. These pores lead to lysis and cell death. Although the structure of the heptameric pore is solved by means of X-ray crystallography, structures of intermediate states-from the soluble monomer to all potential "pre-pore" structures-are yet unknown. Here, we propose a model of the monomeric alpha-hemolysin in solution based on molecular modeling, verified by small angle X-ray scattering data. This structure reveals details of the monomeric conformation of the alpha-hemolysin, for example inherent flexibility, along with definite differences in comparison to the structures used as templates.     

Dynamic flexibility of double-stranded RNA activated PKR in solution

PKR, an interferon-induced double-stranded RNA activated serine-threonine kinase, is a component of signal transduction pathways mediating cell growth control and responses to stress and viral infection. Analysis of separate PKR functional domains by NMR and X-ray crystallography has revealed details of PKR RNA binding domains and kinase domain, respectively. Here, we report the structural characteristics, calculated from biochemical and neutron scattering data, of a native PKR fraction with a high level of autophosphorylation and constitutive kinase activity. The experiments reveal association of the protein monomer into dimers and tetramers, in the absence of double-stranded RNA or other activators. Low-resolution structures of the association states were obtained from the large angle neutron scattering data and reveal the relative orientation of all protein domains in the activated kinase dimer. Low-resolution structures were also obtained for a PKR tetramer-monoclonal antibody complex. Taken together, this information leads to a new model for the structure of the functioning unit of the enzyme, highlights the flexibility of PKR and sheds light on the mechanism of PKR activation. The results of this study emphasize the usefulness of low-resolution structural studies in solution on large flexible multiple domain proteins.     

A solution SAXS study of Borrelia burgdorferi OspA, a protein containing a single-layer beta-sheet.

The crystal structure of a soluble form of Borrelia burgdorferi outer surface protein A (OspA) complexed with the Fab fragment of a monoclonal antibody has revealed an unusual structure that has a repetitive antiparallel beta topology with a nonglobular, single layer beta-sheet connecting the globular N- and C-terminal domains. Earlier NMR studies have shown that the local structure of OspA including the single layer beta-sheet is similar to the crystal structure. Here we report a small angle X-ray scattering (SAXS) study of the global conformation of OspA in solution. The radius of gyration (Rg) and the length distribution function (P(r)) of OspA measured by SAXS in solution are nearly identical to the calculated ones from the crystal structure, respectively. The NMR and SAXS experiments complement each other to show that OspA including the central single-layer beta-sheet is a stable structure in solution, and that the OspA crystal structure represents the predominant solution conformation of the protein.     

Structural and functional organization of the peripheral light-harvesting system in Photosystem I

This review centers on the structural and functional organization of the light-harvesting system in the peripheral antenna of Photosystem I (LHC I) and its energy coupling to the Photosystem I (PS I) core antenna network in view of recently available structural models of the eukaryotic Photosystem I–LHC I complex, eukaryotic LHC II complexes and the cyanobacterial Photosystem I core. A structural model based on the 3D homology of Lhca4 with LHC II is used for analysis of the principles of pigment arrangement in the LHC I peripheral antenna, for prediction of the protein ligands for the pigments that are unique for LHC I and for estimates of the excitonic coupling in strongly interacting pigment dimers. The presence of chlorophyll clusters with strong pigment–pigment interactions is a structural feature of PS I, resulting in the characteristic red-shifted fluorescence. Analysis of the interactions between the PS I core antenna and the peripheral antenna leads to the suggestion that the specific function of the red pigments is likely to be determined by their localization with respect to the reaction center. In the PS I core antenna, the Chl clusters with a different magnitude of low energy shift contribute to better spectral overlap of Chls in the reaction center and the Chls of the antenna network, concentrate the excitation around the reaction center and participate in downhill enhancement of energy transfer from LHC II to the PS I core. Chlorophyll clusters forming terminal emitters in LHC I are likely to be involved in photoprotection against excess energy.     

Structural/functional properties of the Glu1-HSer57 N-terminal fragment of human plasminogen: conformational characterization and interaction with kringle domains.

The Glu1-Val79 N-terminal peptide (NTP) domain of human plasminogen (Pgn) is followed by a tandem array of five kringle (K) structures of approximately 9 kDa each. K1, K2, K4, and K5 contain each a lysine-binding site (LBS). Pgn was cleaved with CNBr and the Glul-HSer57 N-terminal fragment (CB-NTP) isolated. In addition, the Ile27-Ile56 peptide (L-NTP) that spans the doubly S-S bridged loop segment of NTP was synthesized. Pgn kringles were generated either by proteolytic fragmentation of Pgn (K4, K5) or via recombinant gene expression (rK1, rK2, and rK3). Interactions of CB-NTP with each of the Pgn kringles were monitored by 1H-NMR at 500 MHz and values for the equilibrium association constants (Ka) determined: rK1, Ka approximately 4.6 mM(-1); rK2, Ka approximately 3.3 mM(-1); K4, Ka approximately 6.2 mM-'; K5, K, 2.3 mM(-1). Thus, the lysine-binding kringles interact with CB-NTP more strongly than with Nalpha-acetyl-L-lysine methyl ester (Ka < 0.6 mM(-l), which reveals specificity for the NTP. In contrast, CB-NTP does not measurably interact with rK3. which is devoid of a LBS. CB-NTP and L-NTP 1H-NMR spectra were assigned and interproton distances estimated from 1H-1H Overhauser (NOESY) experiments. Structures of L-NTP and the Glul-Ile27 segment of CB-NTP were computed via restrained dynamic simulated annealing/energy minimization (SA/EM) protocols. Conformational models of CB-NTP were generated by joining the two (sub)structures followed by a round of constrained SA/EM. Helical turns are indicated for segments 6-9, 12-16, 28-30, and 45-48. Within the Cys34-Cys42 loop of L-NTP, the structure of the Glu-Glu-Asp-Glu-Glu39 segment appears to be relatively less defined, as is the case for the stretch containing Lys5O within the Cys42-Cys54 segment, consistent with the latter possibly interacting with kringle domains in intact Glul-Pgn. Overall, the CB-NTP and L-NTP fragments are of low regular secondary structure content-as indicated by UV-CD spectra- and exhibit fast amide 1H-2H exchange in 2H2O, suggestive of high flexibility.