Laboratory of Molecular Enzymology, The Babraham Institute, Babraham, Cambridge CB2 4AT, UK1
Department of Animal Science, University of Western Australia, Nedlands, WA 6009, Australia2
Department of Biological and Nutritional Sciences, The University of Newcastle upon Tyne, Newcastle upon Tyne NE1 7RU, UK3
Author for correspondence: Harry J. Gilbert. Tel: +44 191 2226609. Fax: +44 191 2228684. e-mail: h.j.gilbert{at}newcastle.ac.uk
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ABSTRACT |
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Keywords: mannanase, Clostridium thermocellum, cellulosome, family 26
Abbreviations: CBD, cellulose-binding domain; CD, catalytic domain
The GenBank/EMBL accession number for the sequence reported in this paper is AJ242666.
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INTRODUCTION |
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Enzymes active in the degradation of cellulose (endoglucanases, exoglucanases and ß-glucosidases) account for the majority of cellulosomal subunits, but several highly active thermostable xylanases are also present, even though C. thermocellum is unable to utilize xylose as a source of fermentable carbon (Wiegel et al., 1985 ). One possible explanation for this is that efficient utilization of the plant cell wall as a substrate for microbial growth necessitates the hydrolysis of not only cellulose, but a range of hemicellulosic polysaccharides which contribute to the structural complexity of the cell wall and could prevent cellulases from reaching their target substrate.
To test this theory, we have examined the capacity of C. thermocellum to hydrolyse mannan, a polysaccharide that is found in significant quantity in the hemicellulose fraction from softwoods, the seeds of leguminous plants and a variety of nuts and beans. The simplest mannan is a linear homopolymer of ß-1,4-linked mannose residues (Puls & Schuseil, 1993 ). Galactomannan has a similar backbone, but is substituted with
-1,6-linked galactose side-chains, while in glucomannans, ß-1,4-glucose residues are interspersed through the backbone, which can be substituted with
-1,6-galactoses. Substitution with O-acetyl groups at the C-2 and C-3 positions of mannose is common for all mannans.
Enzymic depolymerization of the mannan backbone is effected by endo-ß-1,4-mannanases which cleave randomly within the chain, producing mannobiose and longer manno-oligosaccharides from which mannose is released by exo-acting ß-mannosidases. In addition, -galactosidase and ß-glucosidase are required for the complete breakdown of galactoglucomannan. The most intensively studied member of this enzyme consortium is endomannanase (mannanase). To date, primary sequences have been reported for 10 mannanases from bacteria and fungi. Based on sequence alignments and hydrophobic cluster analysis, all known mannanase catalytic domains (CDs) can be attributed to two distinct glycosyl hydrolase families, namely families 5 and 26. Interestingly, both enzyme families contain cellulases, although the mannanases analysed to date have narrow substrate ranges and do not exhibit significant activity against cellulose.
In this paper we show that C. thermocellum has the capacity to depolymerize mannan and we describe the biochemical properties and molecular architecture of a thermostable mannanase that is part of the cellulosome complex. Sectioning of the mannanase gene coupled with functional analysis of the truncated gene products and primary sequence comparisons reveal that the enzyme contains a family 26 CD, a domain that binds mannan weakly which is conserved in mannanases from anaerobic fungi and the ubiquitous dockerin that is characteristic of all C. thermocellum cellulosomal subunits.
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METHODS |
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Construction and screening of gene library.
C. thermocellum genomic DNA, prepared as described previously (Romaniec et al., 1987 ), was partially digested with MboI and DNA in the size range 39 kb was purified by agarose gel electrophoresis and ligated into XhoI-cleaved
ZAPII (Stratagene) using the partial fill-in technique described in the suppliers protocol. The DNA was packaged in vitro and infected into E. coli XLI-Blue to generate approximately 106 recombinant phage. To screen the library for mannanase-producing clones, phage were plated out on NZY top agarose supplemented with 0·2% azo carob galactomannan (Megazyme).
After incubation at 37 °C for 16 h, mannanase-synthesizing phage were evident by the appearance of clear haloes against a blue background. Clostridial DNA from mannanase-positive phage was excised and recircularized to form phagemid (pBluescript SK-) as described previously (Black et al., 1994 ).
General recombinant DNA methodology.
Agarose-gel electrophoresis, Southern hybridization, transformation of E. coli and the use of restriction enzymes and T4 DNA ligase were as described by Gilbert et al. (1987) . Plasmid DNA was prepared using Qiagen columns. To sequence manA, nested deletions of appropriate restriction fragments were generated using the Exonuclease III/S1-nuclease method. DNA sequencing was carried out using an ABI 373 automated DNA sequencer, in conjunction with the PRISM dye-deoxy termination sequencing kit (Applied Biosystems). Custom-made primers were used where necessary to complete the sequences of both strands.
High-level expression and purification of mannanase A (Man26A).
The region of the manA gene encoding the CD of Man26A (Man26A') or the CD and the N-terminal 130 residues of mature Man26A (Man26A') were amplified using primers containing the appropriate restriction sites at the 5' and 3' ends, respectively. The reaction (100 µl) contained 50 mM Tris/HCl buffer, pH 9·0, 50 mM NaCl, 10 mM MgCl2, 200 µM dNTPs, 50 pmol each primer, 250 ng template DNA (pJH1) and 2 units Taq DNA polymerase (MBI Fermentas). After overlaying with mineral oil, the reaction was subjected to 20 cycles of 30 s at 94 °C, 45 s at 45 °C and 2 min at 72 °C. The primers used to amplify the truncated mannanase genes were as follows: Man26A' (pJH7), 5'-CGGGATCCCCTGCGGATGAAAGCATTG and 3'-CCGCTCGAGTTAATTGGAATCCTCATAATC; Man26A' (pJH8) 5'-CGGGATCCTCATGCATCAATGTGTTCA and 3'-CCGCTCGAGTTAATTGGAATCCTCATAATC. The amplified DNA was digested with BamHI and XhoI and ligated into BamHI/XhoI-restricted pET32a (Novagen) to generate pJH7 and pJH8.
E. coli BL21(DE3) harbouring pJH6 or pJH7 was cultured to mid-exponential phase (OD600=0·6) at 37 °C in LB broth containing ampicillin (50 µg ml-1). IPTG (1 mM) was added and after a further 4 h incubation at 37 °C, cells were harvested, disrupted by ultrasonication in buffer containing 50 mM NaH2PO4, 10 mM Tris/HCl, pH 8·0, 100 mM NaCl, and a cell-free extract was obtained by centrifugation (10000 g, 20 min). Hybrid proteins containing Man26A' or Man26A' appended to thioredoxin were purified by nickel affinity chromatography on TALON resin (Clontech) according to the manufacturers protocol. Bound fusion protein was cleared from the affinity resin during 2 h at room temperature using restriction-grade thrombin (Sigma) at a concentration of 0·5 U (mg fusion protein)-1 and the recommended buffer conditions (Novagen). Man26A' contained in the column eluant was dialysed against 10 mM Tris/HCl, pH 8·0.
Assays and substrates.
Standard enzyme assays were performed using 0·2% (final concentration) of the appropriate plant structural polysaccharide as the substrate in 0·5 ml 50 mM potassium phosphate, 12 mM citric acid buffer, pH 6·5 (PC buffer). The reaction was initiated by adding 50 µl of an appropriate dilution of enzyme and the assay was allowed to proceed for 10 min at 60 °C. The reaction was terminated by adding 0·5 ml dinitrosalicylic acid (Miller, 1959 ) and heating to 100 °C for 20 min. Measurement of A595 was used to quantify the amount of reducing sugar released from the various substrates. One unit of enzyme activity is the amount of enzyme that releases 1 µmol reducing sugar min-1 at 37 °C.
To identify the products released by Man26A, purified Man26A' was incubated with 0·66 mg manno-oligosaccharide ml-1 in PC buffer and at regular intervals the reactions were terminated by heating to 100 °C for 5 min. The reaction products were subjected to HPLC analysis using a Dionex PA1 column eluted with 100 mM NaOH containing a 075 mM gradient of sodium acetate. The sugars were detected with a pulsed amperometer and quantified by comparing the response produced by the various test samples with standard amounts of mannose and manno-oligosaccharides up to mannohexaose. There was a linear relationship between the amount of standard subjected to HPLC and the response of the pulsed amperometer. This applied for each standard evaluated.
Antiserum and Western blotting.
Antiserum against purified Man26A was raised in Dutch rabbits. Initial injections of antigen (200 µg) in Freunds complete adjuvant were administered via intramuscular and subcutaneous routes; subsequent doses, mixed with incomplete adjuvant, were given subcutaneously at 21 d intervals. Western blots were carried out according to the protocol recommended for the Enhanced Chemiluminescence detection system (ECL; Amersham).
Polysaccharide-binding studies.
Binding of recombinant enzymes to mannan was evaluated as described previously (Clarke et al., 1991 ), based on the method of Gilbert et al. (1990)
. A suitable volume of recombinant purified protein was incubated with 1 mg ivory-nut mannan (made up to a volume of 1 ml with 100 mM Tris/HCl buffer, pH 8·0, containing 150 mM NaCl) for 1 h at 4 °C with constant mixing.
Gel retardation studies were performed using native gels containing a final concentration of 0·1% (v/v) soluble carob galactomannan. Gels contained 7·5% (v/v) acrylamide and were made up in 25 mM Tris/HCl buffer, pH 8·3, containing 250 mM glycine. Duplicate gels were run on the same apparatus in tandem; one gel containing carob galactomannan, the other containing no polysaccharide. Gels were then subjected to Western analysis using anti-Man26A antiserum as probe. As the two gels had migrated at exactly the same speed, a direct comparison could be made to determine the extent of retardation.
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RESULTS |
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Nucleotide sequence of C. thermocellum manA
Sequencing of pJH1 in both strands revealed an incomplete ORF adjacent to the polylinker of pBluescript SK-. Southern analysis identified a HindIII fragment, 3·0 kb in length, which would theoretically extend the ORF by 1·0 kb. A partial DNA library of C. thermocellum genomic DNA digested with HindIII was constructed in the low-copy-number vector pMTL6000. Subsequent hybridization screening of the C. thermocellum genomic DNA library with the insert from pJH5 (containing incomplete manA) resulted in the isolation of a single clone homologous to the probe DNA. This plasmid, designated pJH6, was sequenced on both strands and the manA ORF sequence was completed.
The manA ORF consisted of 1767 nt encoding a polypeptide of 589 aa with a predicted molecular mass of 66816 Da. Upstream (8 nt) of the putative ATG start codon was the sequence GGAGG, which is the most frequently observed ribosome-binding site in C. thermocellum cellulases and hemicellulases and is similar to other Gram-positive bacterial ShineDalgarno motifs. Clostridial ORFs most frequently terminate with the stop codon TAA (Ochre); however, the manA ORF terminates with the TAG (amber) stop codon. The 3' flanking region of manA did not contain any palindromic sequences resembling stemloop structures, suggesting -factor-dependent termination. The frequent use of the codons AGG (arginine), UUG (leucine) and CCG (proline) is indicative of C. thermocellum DNA and is a feature rarely found in mesophilic clostridia.
Analysis of the translated amino acid sequence of C. thermocellum Man26A
Inspection of the primary structure of Man26A from C. thermocellum revealed a modular architecture consisting of two distinct domains, the catalytic and dockerin domains, delineated by a linker region. The N-terminal region contained a typical prokaryotic signal peptide with two lysine residues followed by a stretch of small hydrophobic residues capable of -helix formation. The predicted signal peptide cleavage site was between V23 and S24 which is unusual in that prokaryotic leader peptidases most frequently cleave after alanine residues (Perlman & Halvorson, 1983
), but a terminal serine residue has also been observed in the signal peptide of C. thermocellum CelC. The region extending between residues 166 and 507 of Man26A exhibited extensive sequence identity with the CDs of other family 26 mannanases (Fig. 1
). Bolam et al. (1996)
demonstrated that two glutamate residues act as the catalytic nucleophile and acid-base residues. It is widely believed that these residues are highly conserved within members of the same family and sequence homology suggested that E339 and E438 act as the acid-base and nucleophilic residues, respectively, in C. thermocellum Man26A.
Adjacent to the catalytic region of C. thermocellum Man26A is a duplicated region of 24 residues (Fig. 1). This sequence of 71 aa exhibited strong homology with the reiterated conserved region of other C. thermocellum cellulosomal enzymes, which is termed the dockerin and mediates formation of the cellulosome by binding catalytic subunits to the cohesin domains of CipA (Tokatlidis et al., 1991
). The presence of a dockerin sequence indicated the potential of C. thermocellum Man26A to associate with the cellulosome scaffolding protein, CipA (Bayer et al., 1994
).
Biochemical characterization
The region of Man26A extending from residue 166 to 507, which exhibited the strongest homology with family 26 mannanase CDs, was hyperexpressed in E. coli using the pET system (Novagen). This generated a thioredoxinMan26A' fusion protein which was purified using nickel affinity chromatography. Man26A' was cleaved from the thioredoxin by thrombin cleavage.
The substrate specificity of C. thermocellum Man26A was determined by incubating purified Man26A' with a range of substrates. The enzyme was active against carob galactomannan, ivory-nut Man26A' and locust-bean galactomannan. No reducing sugar was detected when purified Man26A' was incubated with a range of other polysaccharides, including cellulose, soluble xylan, galactan, pectin, laminarin and lichenan. These data show that C. thermocellum Man26A is a mannanase with narrow substrate specificity: the enzyme does not hydrolyse other polysaccharides and specifically cleaves the ß-(1,4)-glycosidic linkages between mannopyranosyl residues. Kinetic parameters for C. thermocellum Man26A' are presented in Table 1.
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To establish whether C. thermocellum Man26A' exhibited maximum activity against polymeric mannan molecules, the capacity of the enzyme to hydrolyse a range of water-soluble manno-oligosaccharides was evaluated. The products released were analysed using Dionex HPLC. The relative time taken for 50% cleavage of the substrate of Man26A' against mannotriose, mannotetraose, mannopentaose and mannohexaose were 50, 6·7, 3·6 and 1, respectively, demonstrating that recombinant Man26A' and, by inference, C. thermocellum Man26A preferentially hydrolyses larger oligosaccharides.
Production of Man26A by C. thermocellum
To determine whether Man26A is produced by C. thermocellum as a component of the aggregated cellulosome complex, cellulosome was purified by affinity chromatography (Morag et al., 1992 ) and subjected to Western analysis, using anti-Man26A' antiserum as probe. The use of antiserum raised against Man26A' (excluding dockerin) precluded any cross-reactivity with other dockerin-containing components of the cellulosome including CipA.
A single immunoreactive band with an apparent molecular mass of 70 kDa was detected in the cellulosomal proteins purified from C. thermocellum (Fig. 2). The size of the immunoreactive protein was in good agreement with the size of Man26A calculated from the deduced amino acid sequence. The duplicated 24-residue region at the C terminus therefore appeared to be a functional dockerin domain facilitating attachment of the enzyme to CipA.
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Comparative activities of Man26A' and Man26A' against soluble and insoluble mannan demonstrated a twofold decrease in activity of Man26A' against insoluble substrates when compared to Man26A'. This suggested a role for the N-terminal domain of C. thermocellum Man26A in enhancing activity against insoluble substrates. The reduction in activity against insoluble substrates of Man26A' compared to Man26A' was attributed to the absence of the N-terminal domain in Man26A'. This property has been reported previously as characteristic of binding domains (Coutinho et al., 1993 ; Tomme et al., 1988
; McGavin & Forsberg, 1989
).
To confirm the polysaccharide-binding properties of the N-terminal domain of Man26A, binding assays were performed with insoluble mannan (Fig. 4). Purified Man26A' bound to insoluble mannan, but Man26A' did not. To further describe the binding properties of the N-terminal domain of Man26A, the capacity of the protein to bind insoluble cellulose (Avicel and acid-swollen cellulose) and xylan was also evaluated. The polypeptide bound both forms of cellulose weakly, but exhibited no affinity for insoluble xylan (data not shown). To evaluate the capacity of Man26A' to bind soluble polysaccharides, gel retardation assays were performed using soluble polysaccharides incorporated into non-denaturing polyacrylamide gels. The presence of soluble mannan caused a small retardation of Man26A' but not Man26A' (Fig. 5
). Neither protein was retarded by soluble wheat or rye arabinoxylan, barley ß-glucan, hydroxyethyl cellulose, amylose,
-1,5-arabinan, ß-1,4-galactan or polygalacturonic acid (data not shown). Although these data infer that Man26A' bound very weakly to soluble mannan (carob galactomannan), this interpretation must be viewed with some caution as the galactose substituents could reduce the affinity of the protein for the mannan backbone and thus the results may underestimate the true affinity of the polypeptide for unsubstituted mannan. In summary, these data suggest that the region of Man26A extending from residue 34 to 165 functions primarily as a weak mannan-binding domain, although it does exhibit some capacity to bind insoluble cellulose.
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DISCUSSION |
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The identification of a domain that binds weakly to mannan in the cellulosomal enzyme Man26A is intriguing. Although the domain appears to cause a modest increase in the catalytic activity of the mannanase against recalcitrant insoluble substrates, it may also play a general role in providing the cellulosome with a receptor for mannan. Cellulosome attachment to cellulose microfibrils is mediated via the family IIIa CBD of CipA (Morag et al., 1995 ). Therefore, cellulosomal enzymes containing discrete CBDs would at first glance appear superfluous. However, several C. thermocellum cellulases and xylanases which are components of the cellulosome, also contain discrete CBDs (reviewed by Bayer et al., 1998
; Hazlewood & Gilbert, 1997
). It has been proposed that they may serve to increase further the affinity of the cellulosome for cellulose by the accumulative effect of these CBDs and CipACBD, or play a more direct role in the action of specific catalytic subunits within the complex. The cellulosome displays low affinity for ivory-nut mannan (results not shown). Whether this is attributable to Man26A, CipACBD or both remains unclear at this time (Tomme et al., 1995
).
Interestingly, the lack of a linker region delineating the weak mannan-binding region and the CD of Man26A would appear to disagree with the accepted dogma for the evolution of microbial glycosyl hydrolase architecture. The majority of functional domains of modular glycosyl hydrolases are separated by linker regions rich in hydroxy amino acids, including serine, threonine and proline (Ferreira et al., 1990 ). It has been suggested that recombination between the DNA encoding these linker sequences has led to the evolution of multidomain glycosyl hydrolases (Ferreira et al., 1990
). The lack of a linker region delineating the binding and CDs of Man26A is unusual. It is possible that the mannan-binding domain was acquired by C. thermocellum through horizontal gene transfer and was fused to a bacterial mannanase gene in a subsequent recombination event. Alternatively, the mannan-binding domain/mannanase CD may be more closely related to the anaerobic fungal mannanases and a single gene encoding these two domains may have been imported directly into C. thermocellum. The absence of a linker sequence between the mannan-binding domain and CD of Man26A questions the importance of these regions in modular glycosyl hydrolases. In previous studies, linker-less derivatives of P. fluorescens subsp. cellulosa XYLA and Trichoderma reesei cellobiohydrolase I retained the ability to hydrolyse soluble substrates and to bind to cellulose when compared to their respective full-length enzymes (Ferreira et al., 1990
; Srisodsuk et al., 1993
). These results inferred that the CD and CBD of these enzymes do not need to be separated by linker sequences to retain function. In contrast, removal of linker sequences from Cellulomonas fimi endoglucanase A reduced activity of the enzyme against soluble and highly crystalline substrates and affected its desorption from cellulose (Shen et al., 1991
). This was attributed to the steric hindrance generated by the close proximity of the CBD to the CD. The identification of a glycosyl hydrolase not delineated by a linker region, but which still comprises discrete functional domains, demonstrates that linker regions are not always essential for the discrete domains in glycosyl hydrolases to retain their proper function. In nature, two glycosyl hydrolases, endoglucanase E4 and CelG from Thermomonospora fusca and Clostridium cellulolyticum, respectively, were shown to lack a linker sequence between the CD and the type IIIc CBD, which exhibited very weak affinity for cellulose (Irwin et al., 1998
; Gal et al., 1997
). Resolution of the three-dimensional structure of a truncated form of E4 showed that the type IIIc CBD extended the substrate-binding cleft of the CD such that the enzyme could accommodate 12 glucose residues. It was proposed that the type IIIc CBD fed cellulose chains into the active site of the enzyme and that this played a pivotal role in the hydrolysis of recalcitrant crystalline substrates (Sakon et al., 1997
), and explained the processive mode of action of the enzyme. Although it is tempting to speculate that the mannan-binding domain in Man26A plays a similar role to the type IIIc CBD in endoglucanases E4 and CelG, we believe that this is unlikely as there is no apparent loss in activity against soluble substrates when this domain is removed and only a twofold reduction in catalytic rate against crystalline mannan. In addition, the hydrolysis patterns of Man26A' and Man26A' are not substantially different.
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Received 21 April 1999;
revised 6 July 1999;
accepted 22 July 1999.