Institute of Molecular Genetics, Russian Academy of Science, Kurchatov Sq., 123182 Moscow, Russia1
Research Group Microbial Biotechnology, Technische Universität München, Am Hochanger 4, D-85350 Freising-Weihenstephan, Germany2
Author for correspondence: Wolfgang H. Schwarz. Tel: +49 8161 71 5445. Fax: +49 8161 71 5475. e-mail: schwarz{at}mikro.biologie.tu-muenchen.de
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ABSTRACT |
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Keywords: cellulase CelO, cellulosome, carbohydrate-binding module CBM3, reducing end
Abbreviations: CBM, carbohydrate-binding module; CMC, carboxymethylcellulose; GHF, glycosyl hydrolase family; PASC, phosphoric acid swollen cellulose; pNP, p-nitrophenyl
The GenBank accession number for the sequence determined in this work is AJ275975.
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INTRODUCTION |
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To hydrolyse crystalline substrates efficiently, cellulase systems depend on the simultaneous presence of non-processive endo-1,4-ß-glucanases, which produce new ends at random within a polysaccharide chain, and processive cellulases (exo-1,4-ß-glucanases), which remain attached to the substrate and split off cellobiose (cellobiohydrolase) or multimers of cellobiose (processive endocellulase) from such ends (Reverbel-Leroy et al., 1997 ; Teeri, 1997
). The catalytic domains of the cellulases have been assigned on the basis of sequence comparisons and hydrophobic cluster analysis to the glycosyl hydrolase families (GHFs) (Henrissat et al., 1998
; CAZy server: http://afmb.cnrs-mrs.fr/~pedro/CAZY). Four of these, GHF 6, 7, 9 and 48, contain cellobiohydrolases.
The endo- or exo-mode of an enzyme is determined by differences in the shape of the active-site pocket. If the hydrolytic amino acids in the active site can be freely accessed by the polysaccharide strand, i.e. if they are situated in an open, pocket-shaped cleft on the enzyme surface, the enzyme is endo-glucolytically active in a non-processive way. However, if the active site is blocked by a bulky extension of the protein, creating a tunnel-like structure covering the catalytic amino acids, the polymeric substrate can access the active site with only one of its ends and the enzyme acts processively by sliding along the substrate (Barr et al., 1996 ). The direction of this processivity, i.e. the enzyme activity on the reducing or the non-reducing end of the substrate, seems to be defined by the way that the substrate is bound and released (Parsiegla et al., 2000
).
The processive activity of cellobiohydrolases on modified cellulose, such as carboxymethylcellulose (CMC), is blocked, or at least strongly inhibited, by the occurrence of derivatized glucose residues. This leads to severe substrate limitation. Cellobiohydrolases acting from the the non-reducing end of the substrate can be assayed with chromogenic arylcellobiosides, from which the chromogenic aryl residue, attached to the reducing end of the substrate, is liberated. However, more recently, processive enzymes acting from the reducing end of the carbohydrate chain have been identified, for which chromogenic substrates are not yet commercially available (Boisset et al., 1998 ). Although only a few members of this enzyme type have been characterized, models for the hydrolysis of crystalline cellulose predict that the presence of processive cellulases with both types of chain-end-specificity is essential for productive and complete degradation of the substrate (Barr et al., 1996
; Schwarz, 2001
).
Besides the presence of different catalytic modules, the activity of cellulases on crystalline cellulose also depends on the presence of non-catalytic modules which contribute to the tight binding of the substrate (Tomme et al., 1998 ). These carbohydrate-binding modules (CBMs) have a stimulating effect by increasing enzymesubstrate proximity (Bolam et al., 1998
); they help the enzyme to overcome the liquidsolid interface (Nutt et al., 1998
) and also enhance the accessibility of the substrate surface by modifying the structure of the cellulose crystal (Pagès et al., 1997
). On the basis of sequence comparison, 26 families of substrate-binding modules from bacteria, with between 40 and 180 amino acids, have been defined (Coutinho & Henrissat, http://afmb.cnrs-mrs.fr/~pedro/DB/db.html; Tomme et al., 1998
).
In this study we report the structure of the celO gene and the corresponding cellulase, CelO, from Clostridium thermocellum F7. CelO was shown to consist of a leader peptide, a substrate-binding domain CBM3b (which bound to cellulose), a short proline-rich linker, a catalytic domain of GHF5 and a dockerin domain. We present evidence which suggests that the enzyme is a cellobiohydrolase which hydrolyses cellodextrins from the reducing end.
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METHODS |
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Molecular biological methods.
The DNA sequence of clone pCU107 was determined from supercoiled, double-stranded plasmid DNA from both strands (Thermosequenase Cycle Sequencing Kit, Amersham) with 5'-biotinylated oligonucleotide primers. The DNA sequence was determined with a GATC 1500 Direct-Blotting Electrophoresis sequencer (GATC, Konstanz) using streptavidin-conjugated alkaline phosphatase and the chromogenic substrate NBT-BCIP (Promega). Sequence data were analysed with the DNASIS/PROSIS software package (Hitachi Software Engineering). Nucleotide and protein sequence databases were screened using FASTA and BLAST (http://www.ebi.ac.uk).
The CLUSTAL W program (http://www.ebi.ac.uk/clustalw) was applied for progressive multiple sequence alignment by calculating maximum-parsimony, maximum-likelihood and position-specific gap-penalties (Thompson et al., 1994 ), and by using significant sequence similarity based on the amino acid substitution matrix of Henikoff & Henikoff (1992)
.
PCR was carried out using chromosomal DNA as a template and the Expand High Fidelity PCR System (Boehringer Mannheim). The PCR oligonucleotide primers were designed to truncate the gene for the leader peptide and the dockerin domain (rCelO, primers cel4-1-Bam and cel4-2-Sal), or the leader peptide, the CBM and the dockerin domain (rCelO-Cat, primers cel4-2-Bam and cel4-2-Sal). The primers were also designed to introduce recognition sites for the restriction endonucleases SalI (GTCGAC) or BamHI (GGATCC), allowing for cloning into the expression vectors pQE30 and pQE31, respectively. The primers used were: cel4-1-bam (3'-CCCTGATTGGATCCTGCCTTTTCAG-5'); cel4-2-bam (3'-AACCGACGGATCCGCCAAACAACG-5'); and, cel4-2-sal (3'-GTTAACGTCGACATATTTAAAGGTATCC-5').
Purification of recombinant proteins.
Recombinant proteins were purified from 400 ml E. coli cultures according to the manufacturers instructions (Qiagen) using 3 ml Ni-NTA superflow columns (Qiagen). The purity of the proteins was verified by SDS-PAGE and by staining with Coomassie brilliant blue G-250 dye (Serva).
Enzyme assays.
Enzyme aliquots for standard assays were incubated in sodium phosphate buffer (50 mM, pH 6·6) at 65 °C. The concentration of the substrates used was 1% (w/v) for soluble polysaccharides and 2% (w/v) for insoluble polysaccharides. Reducing sugars released from the polymeric substrates were detected by the 3,5-dinitrosalicylic acid method (Wood & Bhat, 1988 ), assuming that 1 U enzyme liberates 1 µmol glucose equivalent min-1 (mg protein)-1. Specific activities were determined in the linear range of the reaction. The protein concentration was determined with Coomassie brilliant blue as described by Sedmak & Grossberg (1977)
. p-Nitrophenol liberation from p-nitrophenyl(pNP)-glycosides was measured by its absorbance in alkaline solution (0·6 M Na2CO3) at 395 nm. One unit of activity was defined as the amount of enzyme producing 1 µmol p-nitrophenol min-1 (0·013
OD395=1 nmol).
Analysis of protein binding to insoluble cellulose.
Binding of the proteins to insoluble carbohydrates was carried out in 1·5 ml polypropylene caps at 4 °C with shaking for 2 h. The purified protein was added to 1 mg substrate in 0·5 ml 50 mM sodium phosphate buffer, pH 6·6, containing 0·2 M NaCl. The samples were then centrifuged twice at 16000 g for 5 min to remove the ligand and the bound protein. Free protein in the cleared supernatant was determined in a 1 cm cell by UV absorption at 280 nm, assuming a molar absorption coefficient of 163480 M-1 cm-1 for rCelO and 132161 M-1 cm-1 for rCelO-Cat as calculated from the amino acid sequence with the biopolymer calculator (http://paris.chem.yale.edu/cgi-bin/extinct.p). The measurements were done at least in triplicate.
The equilibrium binding constant (Ka) was calculated from the depletion isotherm with the SigmaPlot program package for PCs (SPSS Science Software) by non-linear regression of the raw data to a Langmuir-type adsorption model:
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where [B] is the concentration of bound CBM [mol (g cellulose)-1], [F] is the concentration of free CBM (M), [N0] is the total concentration of available binding sites on the cellulose surface, also called the capacity value for cellulose [mol (g cellulose)-1], and Ka is the equilibrium association constant (M-1). The concentration of bound protein was determined as the difference between the initial protein concentration and the free protein concentration after incubation with the substrate. The measurements were done at least in triplicate and the standard error was calculated.
TLC.
Polymeric and oligomeric substrates were hydrolysed to completion under the conditions stated above. Hydrolysis products were separated on 0·2 mm aluminium sheet silica gel 60 plates (Merck) with acetronitrile/water (80:20, v/v) as eluent. Sugars were detected by spraying the plates with a freshly prepared mixture of 10 ml stock solution and 1 ml orthophosphoric acid, followed by heating the plates at 120 °C until colour developed. The stock solution consisted of 1 g diphenylamine and 1 ml aniline dissolved in 100 ml acetone.
Viscometry.
A 1% (w/v) solution of CMC in 0·1 M phosphate buffer of the appropriate pH, according to the optimal activity profile of the enzyme, was hydrolysed at 50 °C. The flow-time of the reaction mixture was determined at intervals in an Oswald viscometer at 50 °C, together with the reducing sugar content (3,5-dinitrosalicylic acid method). The relative fluidity was calculated as:
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where T0 is the flow-time measured for the buffer, the flow-time of the CMC solution without enzyme, and T is the flow-time of the reaction mixture with the enzyme.
Substrates.
Birchwood xylan, Avicel CF1, CMC (low viscosity), and pNP-glycosides were obtained from Sigma, cellodextrins from Merck, barley ß-glucan from Megazym, and pustulan from Roth. Phosphoric acid swollen cellulose (PASC) was prepared from Avicel CF1 by the method of Wood (1988) . NaBH4-reduced cellodextrins were prepared from cellobiose, cellotriose and cellopentaose as described by Barr et al. (1996)
.
Sequence accession number.
The GenBank accession number for the nucleotide sequence referred to in this study is AJ275975. For comparison with other GHF5 sequences, the nucleotide and protein sequences can be found at http://afmb.cnrs-mrs.fr/~pedro/CAZY
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RESULTS |
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To characterize CelO, and the role of its modules, two truncated proteins were constructed by PCR: rCelO, with the leader peptide and the dockerin module deleted (587 aa, 67272 Da), and rCelO-Cat, containing only the catalytic domain devoid of the CBM of CelO (415 aa, 47946 Da). The resulting recombinant enzymes were purified to homogeneity by affinity chromatography of the hexahistidyl-tagged recombinant proteins and showed a molecular mass consistent with that calculated from the sequence.
The hydrolytic activities of rCelO and rCelO-Cat were identical within the error limits of the assay if molar activities were calculated (Table 1). rCelO hydrolysed soluble and insoluble 1,4-ß-linked substrates, with the highest activity shown for the soluble barley ß-glucan, containing alternating 1,3-ß- and 1,4-ß-linkages in a ratio of 1:3, whereas no activity could be observed with laminarin, a 1,3-ß-glucan. rCelO released reducing sugar from soluble CMC and from PASC, an amorphous cellulose preparation. Only minor activity near the detection limit was observed with xylan (1,4-ß-D-xylan) or pustulan (1,6-ß-D-glucan) and this could be the result of substrate contamination. The pNP compounds of ß-glucoside, ß-xyloside, ß-mannoside, lactoside and ß-galactoside were not hydrolysed. With an optimum temperature of 65 °C at its optimal pH of 6·6 for hydrolysis of barley ß-glucan, CelO is a relatively thermostable enzyme. These values are comparable to those found for other exoenzymes from the thermophilic bacteria C. thermocellum and Clostridium stercorarium.
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DISCUSSION |
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No negative effect on the catalytic activity or on the thermal stability of the enzyme was observed on deletion of the CBM3b in rCelO-Cat. This might be explained by the fact that CelO, unlike the non-cellulosomal enzymes, is tightly integrated in the cellulosome complex, which already contains the very strongly binding CBM3a of the scaffoldin Ct-CipA. It can be assumed that the cellulosomal enzyme components in C. thermocellum would not be dependent on additional CBMs. However, their presence could stabilize the whole complex on the substrate surface, having a back-up-function and supporting the overall activity of the cellulosome.
The catalytic GHF5 module of CelO belongs to a heterogeneous family of glucanases, subdivided into five subfamilies with amino acid sequence similarity above 25%. CelO showed a narrow substrate specificity, restricted to 1,4-ß-linked substrates like CMC, cellodextrins and amorphous and microcrystalline cellulose. The CelO sequence grouped with subfamily 1 (formerly called family A1), all members of which are hitherto described as endoglucanases, mostly based on the fact of CMC hydrolysis (Lemaire & Béguin, 1993 ; Meinke et al., 1993
; Sakon et al., 1996
). Only the hydrolytic mode of cellodextrinase CelC from Pseudomonas fluorescens was definitely characterized as an endoglucanase, hydrolysing cellohexaose to a mixture of cellobiose and cellotriose (Ferreira et al., 1991
). Despite hydrolysing CMC with a high initial reaction velocity, CelO released exclusively cellobiose from Avicel, PASC and cellodextrins. The CelO activity on CMC showed pronounced substrate limitation and a relatively small decrease in viscosity, typical for a processive mode of hydrolysis. CelO behaved as an exo-glucanolytic cellobiohydrolase (EC 3 . 2 . 1 . 91), although the decrease in viscosity and the amount of reducing sugars released from CMC does not exclude a limited ability for random attack.
The high activity on mixed-linkage ß-glucan is a trait CelO has in common with most of the cellobiohydrolases described so far. The mechanism by which sequentially active cellobiohydrolases overcome the potential block of 1,3-ß-linkages in the linear substrate molecule deserves further analysis. The apparently low activity of CelO on Avicel, only one-tenth of the value of the main cellulosomal component Ct-CelS (Kruus et al., 1995 ), may be drastically enhanced if CelO is correctly integrated into the synergistically acting cellulosome. On the other hand, Avicel might not be its natural substrate.
The lack of chromogenic activity on pNP-cellodextrins (pNP-cellobioside, -trioside, -tetraoside and -pentaoside) was at first glance interpreted as an absence of activity, because cellotriose was also not hydrolysed by CelO. However, TLC analysis of the reaction products detected the hydrolysis of pNP-cellotetraoside to pNP-glucoside and cellotriose, and could be explained by hydrolysis from the reducing end: if the substrate were hydrolysed from the non-reducing end, the expected products would be cellobiose and pNP-cellobioside. It cannot be ruled out that interactions with the aromatic aglycone may affect the pattern of degradation of pNP-cellooligodextrins. However, the model was further supported by another experiment using reduced cellopentaose as a substrate. Whereas cellobiohydrolase Ct-CbhA hydrolysed the substrate from the unmodified non-reducing end, CelO degraded only the unmodified substrates, while activity on the reduced substrates was blocked by modification at the reducing end. Fig. 7 explains the proposed model of the hydrolytic mode schematically.
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The data show that GHF5 is a cellulase family containing both endo- and exoglucanases. CelO is the first cellobiohydrolase in the cellulosome of C. thermocellum for which evidence of reducing-end specificity has been presented. As far as the cellulosome of C. thermocellum is concerned, two exoglucanases each seem to be active from either side of the polymeric substrate: CelK and CbhA from the non-reducing side (Kataeva et al., 1999 ; Zverlov et al., 1998
), and CelO and probably CelS (Teeri, 1997
) from the reducing end. This, together with the presence of binding domains for crystalline cellulose, would enable the cellulosome to widen a gap in a cellulose molecule on the crystal surface in both directions.
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ACKNOWLEDGEMENTS |
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Received 19 July 2001;
revised 28 August 2001;
accepted 31 August 2001.