Göran Pettersson

A critical review of cellobiose dehydrogenases

Cellobiose dehydrogenase (CDH) is an extracellular enzyme produced by various wood-degrading fungi. It oxidizes soluble cellodextrins, mannodextrins and lactose efficiently to their corresponding lactones by a ping-pong mechanism using a wide spectrum of electron acceptors including quinones, phenoxyradicals, Fe(3+), Cu(2+) and triiodide ion. Monosaccharides, maltose and molecular oxygen are poor substrates. CDH that adsorbs strongly and specifically to cellulose carries two prosthetic groups; namely, an FAD and a heme in two different domains that can be separated after limited proteolysis. The FAD-containing fragment carries all known catalytic and cellulose binding properties. One-electron acceptors, like ferricyanide, cytochrome c and phenoxy radicals, are, however, reduced more slowly by the FAD-fragment than by the intact enzyme, suggesting that the function of the heme group is to facilitate one-electron transfer. Non-heme forms of CDH have been found in the culture filtrate of some fungi (probably due to the action of fungal proteases) and were for a long time believed to represent a separate enzyme (cellobiose:quinone oxidoreductase, CBQ). The amino acid sequence of CDH has been determined and no significant homology with other proteins was detected for the heme domain. The FAD-domain sequence belongs to the GMC oxidoreductase family that includes, among others, Aspergillus niger glucose oxidase. The homology is most distinct in regions that correspond to the FAD-binding domain in glucose oxidase. A cellulose-binding domain of the fungal type is present in CDH from Myceliophtore thermophila (Sporotrichum thermophile), but in others an internal sequence rich in aromatic amino acid residues has been suggested to be responsible for the cellulose binding. The biological function of CDH is not fully understood, but recent results support a hydroxyl radical-generating mechanism whereby the radical can degrade and modify cellulose, hemicellulose and lignin. CDH has found technical use in highly selective amperometric biosensors and several other applications have been suggested.

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.

Limited proteolysis of the cellobiohydrolase I from Trichoderma reesei: Separation of functional domains

Limited proteolysis of the cellobiohydrolase I (CBH I, 65 kDa) from Trichoderma reesei by papain yields a core protein (56 kDa) which is fully active against small, soluble substrates such as the chromophoric glycosides derived from the cellodextrins and lactose. Activity against an insoluble substrate, such as Avicel, is however completely lost and concomitantly decreased adsorption onto this microcrystalline cellulose is observed. The peptide (10 kDa), initially split off during proteolysis, is identified as the heavily glycosylated carboxy‐terminal of the native CBH I. Depending on the experimental conditions the core protein is further nicked in between disulfide bonds, but its properties and stability do not appreciably differ from those of intact CBH I. These results lead to the proposal of a bifunctional organisation of the CBH I: one domain, corresponding to the carboxyterminal, acts as a binding site for insoluble cellulose and the other, localised in the core protein, contains the active (hydrolytic) site.

Chiral separation of β-blockers by high-performance capillary electrophoresis based on non-immobilized cellulase as enantioselective protein

Abstract Optical isomers of some basic parmaceutical drugs (β-blockers) were separated by means of high-performance capillary electrophoresis in a carrier-free solution, using the chiral recognition properties of a cellulase (cellobiohydrolase I). High resolution of the isomers and peaks with satisfactory symmetry were obtained only when the enzyme was dissolved at a high concentration (40 mg/ml; total 10 μg) in a buffer of high ionic strength (0.4 M sodium phosphate) supplemented with 2-propanol. Surprisingly, the isomer selectivity was lost when the electrophoresis was carried out in the buffers used for chromatographic separation of the isomers on a bed derivatized with cellulase. At pH 5.1 (the experimental pH), the enantiomers are positively charged and the enzyme is negatively charged. With the cathode at the detection end of the capillary the enzyme accordingly migrated away from the detection point and the enantiomers toward it. Disturbances in the UV detection of the enantiomers otherwise caused by the presence of the enzyme were thus avoided. As the runs are performed in the absence of a supporting medium the analyses can be automated easily, which also facilitates the screening of different proteins for their chiral recognition properties and studies to establish the optimum experimental conditions.

A new scaffold for binding haem in the cytochrome domain of the extracellular flavocytochrome cellobiose dehydrogenase.

BACKGROUND The fungal oxidoreductase cellobiose dehydrogenase (CDH) degrades both lignin and cellulose, and is the only known extracellular flavocytochrome. This haemoflavoenzyme has a multidomain organisation with a b-type cytochrome domain linked to a large flavodehydrogenase domain. The two domains can be separated proteolytically to yield a functional cytochrome and a flavodehydrogenase. Here, we report the crystal structure of the cytochrome domain of CDH. RESULTS The crystal structure of the b-type cytochrome domain of CDH from the wood-degrading fungus Phanerochaete chrysosporium has been determined at 1.9 A resolution using multiple isomorphous replacement including anomalous scattering information. Three models of the cytochrome have been refined: the in vitro prepared cytochrome in its redox-inactive state (pH 7.5) and redox-active state (pH 4.6), as well as the naturally occurring cytochrome fragment. CONCLUSIONS The 190-residue long cytochrome domain of CDH folds as a beta sandwich with the topology of the antibody Fab V(H) domain. The haem iron is ligated by Met65 and His163, which confirms previous results from spectroscopic studies. This is only the second example of a b-type cytochrome with this ligation, the first being cytochrome b(562). The haem-propionate groups are surface exposed and, therefore, might play a role in the association between the cytochrome and flavoprotein domain, and in interdomain electron transfer. There are no large differences in overall structure of the cytochrome at redox-active pH as compared with the inactive form, which excludes the possibility that pH-dependent redox inactivation results from partial denaturation. From the electron-density map of the naturally occurring cytochrome, we conclude that it corresponds to the proteolytically prepared cytochrome domain.

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.

Separation of enantiomers using cellulase (CBH I) silica as a chiral stationary phase

A new chiral stationary phase for high-performance liquid chromatography based on a glycoprotein (celllulase, CBH I) isolated from a culture filtrate of a fungus, Trichoderma reesei, was prepared. Chiral acidic and basic drugs were resolved into their enantiomers on this phase. Compared with other similar chiral phases, high enantioselectivity was obtained for β-blocking agents despite low capacity factors. As much as 200 nmol of propranolol in a single injection could be separated into its enantiomers on an analytical column (250 × 5.0 mm I.D.) without loss of resolution. No significant decrease in enantioselectivity was observed after daily use of the phase during a period of 4 months with varying mobile phase compositions. The retention and enantioselectivity of amines increased with increasing pH of the mobile phase, whereas the opposite effect was observed for acids. Addition of organic solvents to the mobile phase both decreased the retention and increased the enantioselectivity for the analytes.

CELLOBIOSE DEHYDROGENASE (CELLOBIOSE OXIDASE) FROM PHANEROCHAETE-CHRYSOSPORIUM AS A WOOD DEGRADING ENZYME - STUDIES ON CELLULOSE, XYLAN AND SYNTHETIC LIGNIN

Degradation of carboxymethylcellulose (CMC), xylan and synthetic lignin was studied in a cellobiose dehydrogenase system, that reduced Fe(III) to Fe(II) with cellobiose as electron donor, which in the presence of hydrogen peroxide degraded all the above representatives of the main wood components, probably by forming Fentons reagent. The production of hydroxyl radicals was shown by benzoate decarboxylation. For the CMC and xylan studies viscometry was used, while lignin degradation was studied by measuring the passage of 14C-labelled synthetic lignin (DHP) through membranes of different molecular-mass cut-off. The possible participation of cellobiose dehydrogenase, Fe(III) and hydrogen peroxide in wood degradation by white-rot and brown-rot fungi is discussed.

The 1,4-β-d-glucan cellobiohydrolases from Phanerochaete chrysosporium. I. A system of synergistically acting enzymes homologous to Trichoderma reesei

A physico-chemical and structural characterization of three 1,4-beta-D-glucan cellobiohydrolases (EC. 3.2.1.91), isolated from a culture filtrate of the white-rot fungus Phanerochaete chrysosporium, reveals that the cellulolytic enzyme secretion pattern and thus the general degradation strategy for P. chrysosporium is similar to that of Trichoderma reesei. Partial sequence data show that two of the isolated enzymes, i.e., CBHI, pI 3.82 and CBH62, pI 4.85, are homologous with CBHI and EGI from T. reesei; while, the third, i.e., CBH50, pI 4.87, is homologous to T. reesei CBHII. Limited proteolysis with papain cleaved each of the three enzymes into two domains: a core protein which retained full catalytic activity against low molecular weight substrates and a peptide fragment corresponding to the cellulose binding domain, in striking similarity to the structural organization of T. reesei. CBHI and CBH62 have their binding domain located at the C-terminus, whereas in CBH50 it is located at the N-terminus. It is evident that synergistically acting cellobiohydrolases is a general requirement for efficient hydrolysis of crystalline cellulose by cellulolytic fungi.

Do the extracellular enzymes cellobiose dehydrogenase and manganese peroxidase form a pathway in lignin biodegradation

The extracellular enzyme manganese peroxidase is believed to degrade lignin by a hydrogen peroxide‐dependent oxidation of Mn(II) to the reactive species Mn(III) that attacks the lignin. However, Mn(III) is not able to directly oxidise the non‐phenolic lignin structures that predominate in native lignin. We show here that pretreatment of a non‐phenolic lignin model compound with another extracellular fungal enzyme, cellobiose dehydrogenase, allows the manganese peroxidase system to oxidise this molecule. The mechanism behind this effect is demethoxylation and/or hydroxylation, i.e. conversion of a non‐phenolic structure to a phenolic one, mediated by hydroxyl radicals generated by cellobiose dehydrogenase. This suggests that cellobiose dehydrogenase and manganese peroxidase may act in an extracellular pathway in fungal lignin biodegradation. Analytical techniques used in this paper are reverse‐phase high‐pressure liquid chromatography, gas chromatography connected to mass spectroscopy and UV‐visible spectroscopy.