1 Research Group Microbial Biotechnology, Technische Universität München, Am Hochanger 4, D-85350 Freising-Weihenstephan, Germany
2 Institute of Molecular Genetics, Russian Academy of Science, Kurchatov Sq., 123182 Moscow, Russia
3 GSF - National Research Center for Environment and Health, Institute for Ecological Chemistry, Ingolstädter Landstrasse 1, D-85764 Neuherberg, Germany
Correspondence
Wolfgang H. Schwarz
schwarz{at}mikro.biologie.tu-muenchen.de
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are AJ585344 (xghA) and AJ585345 (xynD).
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INTRODUCTION |
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The function of the non-cellulolytic enzymes is presumably the unwrapping of the cellulose crystals from the covering matrix of lignin, pectin and hemicellulose. Whereas lignin apparently cannot be degraded by the anaerobic bacteria, the heterogeneous hemicelluloses like xyloglucan, differently derivatized xylans, galactomannan and related polysaccharides, as well as pectin, are hydrolysed. Xylan has a -1,4-linked backbone of xylosyl residues which are substituted in the C-2 or C-3 position by
-arabinofuranosyl or glucuronic acid residues (Schwarz et al., 2004
). Xyloglucan is the major hemicellulose in dicotyledon type I cell walls and has a backbone of
-1,4-linked cellotetraose units, which are substituted in C-6 by
-D-xylosyl residues at two or three of the first three glucosyl residues (from the reducing end) (York et al., 1993
). Some of the xylosyl residues are further substituted by galactosyl, arabinosyl or fucosyl residues (Fry et al., 1993
). The evident cross-linking between xyloglucan chains is a major factor in the stability of the three-dimensional matrix between adjacent cellulose microfibrils and its removal is a precondition for complete lignocellulosic biomass decomposition (Whitney et al., 1995
).
Non-cellulase polysaccharide hydrolases are usually active in an endo-mode, opening the molecule strands anywhere in the molecule and producing a new reducing end with each cut. This enzymic activity reduces the local viscosity of the hemicellulose matrix. If eventually two cuts are near enough to each other, a short oligosaccharide is produced which is soluble and can diffuse away, thus removing the non-cellulosic matrix from the surface of the cellulose crystals and allowing access of the cellulases. It is therefore no surprise that the cellulosome of C. thermocellum contains a number of non-cellulolytic enzymes, namely xylanases as well as 1,3-1,4--glucanases, a mannanase and a chitinase, which make up about half of the cellulosomal protein (Hayashi et al., 1997
, 1999
; Hallstead et al., 1999
; Blum et al., 2000
; Kurokawa et al., 2001
; Zverlov et al., 1994
, 2002
, 2005
).
The exceptional efficiency of the extracellular hydrolytic machinery present in anaerobic bacteria is due to the combination of all of those activities together with the cellulases in the cellulosome (Shoham et al., 1999). About 30 genes involved in cellulosome formation have hitherto been isolated by screening of genomic libraries (Bayer et al., 2000
; Schwarz, 2001
). The following enzymes have recently been identified among the most abundant subunits in the cellulosome and therefore might have a major function in cellulolysis: CelA, CelG, CelK, CelN, CelR, CelS, CbhA, ChiA, XynC and XynZ (Zverlov et al., 2005
). In addition two new presumably hemicellulolytic subunits were identified: the xylanase XynD and the putative xyloglucanase XghA. Both were among the most prominent hemicellulolytic proteins in the cellulosome. The xylanases XynC and XynZ have been described earlier (Grépinet et al., 1988
; Hayashi et al., 1997
). A xyloglucanase has not hitherto been described as a subunit of the cellulosome.
The aim of the paper was to evaluate the gene sequences and the biochemical characteristics of the newly identified cellulosomal hemicellulolytic subunits Xyn10D and Xgh74A, which belong to the glycosyl hydrolase families GHF10 and GHF74 respectively and represent a new endoxylanase and the first xyloglucanase in the cellulosome.
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METHODS |
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Recombinant DNA techniques.
Preparation of chromosomal and plasmid DNA, endonuclease digestion, ligation and transformation were carried out by standard procedures or according to supplier protocols. Plasmid DNA was prepared with the QIAprep Spin Miniprep Kit (Qiagen). Restriction digests of DNA were done as recommended by the manufacturer (MBI Fermentas). E. coli cells were transformed with plasmid DNA by electroporation (Bio-Rad Gene Pulser) as suggested by the supplier.
Vectors from Invitrogen (pCR-XL-TOPO) or Qiagen (pQE32) and the E. coli host strains supplied with the kits were used for cloning. PCR was carried out using the synthetic oligonucleotide primers h14f (3'-TAGGGGAGGGGTTGTTTTAATG-5') and h14r (3'-TTAACGTTTTAAAGGCAATTCATC-5') for pCR-XL-TOPO-XynD, or h28f (3'-CGATTTGCATGCAGGCTGTAACCAGCGTG-5') and h28r (3'-TCTGAAGCTTGTTCGCCGTAAACAATACCT-5') for pQE32-XghA, with chromosomal DNA from C. thermocellum F7 as template and the Expand High Fidelity PCR System (Roche Diagnostics).
The PCR amplicons were cloned and subsequently sequenced from supercoiled double-stranded plasmid DNA on both strands using a LICOR automated sequencer (MWG Biotech) with the Thermo Sequenase fluorescent-labelling primer sequencing kit (Amersham Pharmacia Biotech). Sequence data were analysed, edited and compared with the DNASIS/PROSIS for Windows package (Hitachi Software Engineering).
Purification of enzymes.
Cellulosomes were prepared from Avicel-grown C. thermocellum F7 cultures by a modified affinity digestion method (Morag et al., 1992) as described by Zverlov et al. (2005)
. Recombinant Xyn10D protein was purified from 400 ml E. coli TOP10F' (pCR-XL-TOPO-XynD) cultures. The cells were harvested after 15 h aerated growth at 37 °C, washed with 10 mM Tris/HCl, pH 7·6, and suspended in 10 ml of the wash buffer. After sonication the extract was heated for 15 min to 60 °C and cleared by centrifugation (20 000 g, 20 min, 4 °C). The cell-free extract was applied to a HiTrapQ column (1 ml, Amersham Biosciences) and eluted with a linear NaCl gradient (00·5 M) in wash buffer. Fractions with activity on xylan were pooled and concentrated by ultrafiltration (VIVAspin 2 ml concentrator, 30 000 MWCO PES, VIVASCIENCE).
Recombinant Xgh74A proteins were purified from 400 ml E. coli(pQE32-XghA) culture according to the procedures described in the Qiagen handbook with 3 ml Ni-NTA Superflow Agarose columns (Qiagen).
Enzyme assays.
Enzyme aliquots in standard assays were incubated in MES buffer (50 mM) at the optimum pH and temperature. The substrate concentration was 1 % for soluble and 2 % (w/v) for insoluble polysaccharides. Reducing sugars released from polymeric substrates were quantitatively detected by the 3,5-dinitrosalicylic acid method (Wood & Bhat, 1988) assuming that one unit of enzyme liberates 1 µmol glucose equivalent min1 (mg protein)1. Specific activities were determined in the linear range of the reaction. All determinations were performed at least in triplicate.
The optimum pH was determined by measuring the specific activity of the enzyme at a given pH. Buffers were MES, Tris/HCl and potassium phosphate. Xylan was used as substrate for XynD and tamarind seed xyloglucan for XghA. The optimum temperature was the temperature with the highest activity of the enzyme during 30 min incubation at optimum pH. Protein concentration was determined with Coomassie brilliant blue G-250 (Protein Assay, Bio-Rad). Substrates were pachyman, tamarind seed xyloglucan and barley -glucan from Megazyme International, and birch wood glucuronoxylan (xylan) and low-viscosity carboxymethylcellulose sodium salt (CMC) from Sigma-Aldrich. Phosphoric-acid-swollen cellulose (PASC) was prepared from Avicel CF1 (Sigma-Aldrich) according to Wood & Bhat (1988)
.
Zymograms of denatured cellulosomal proteins were prepared by running denaturing SDS-PAGE, renaturing the enzymes within the gel slabs by three successive washes in 50 mM phosphate/citrate buffer (pH 6·5) with and without 2-propanol as described previously (Schwarz et al., 1987) and soaking the gel slabs for 1 h with 0·05 % tamarind xyloglucan solution. After incubation at 60 °C in enzyme assay buffer, the gels were stained with Congo red and destained with 1 M NaCl to make bands of hydrolytic activity visible against a red background.
Thin-layer chromatography (TLC).
Hydrolysis products from oligo- and polysaccharides were separated on 0·2 mm silica gel 60 aluminium plates (VWR) with acetonitrile/water (80 : 20, v/v) as eluent. Sugars were detected by spraying the plates with a freshly prepared mixture of 10 ml stock solution (1 g diphenylamine + 1 ml aniline dissolved in 100 ml acetone) with 1 ml orthophosphoric acid, followed by heating the plates at 120 °C until colour developed.
Identification of glucan hydrolysis products by ESI/MS.
Infusion experiments were conducted using a homemade coaxial microelectrospray ionization interface coupled to a Finnigan LCQ Duo ion-trap mass spectrometer (ThermoQuest) as described by Frommberger et al. (2004). The sample flux was set to 0·35 µl min1 and the sheath liquid was methanol/water/acetic acid (90 : 10 : 1, by vol.) pumped at 5 µl min1; electrospray ionization was conducted in the positive and the negative ionization mode and the heated capillary was set to 150 °C for optimum signals (Schmitt-Kopplin & Kettrup, 2003
).
Nucleotide sequence accession numbers.
Nucleotide sequences are deposited under GenBank/EMBL/DDBJ accesssion no. AJ585344 (xghA) and AJ585345 (xynD). The draft genomic sequence from C. thermocellum ATCC 27405 with GenBank ID AABG00000000 was used for primer design (Whole Genome Shotgun project WGS by DOE).
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RESULTS |
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Analysis of the sequences revealed complex module structures similar to those found in other cellulosomal genes. In both genes the dockerin type I module for binding to the cohesin modules of the cellulosomal scaffoldin is located C-terminally. The modules are separated by short linker sequences rich in hydroxy amino acids (PTS boxes). Xyn10D contained, in addition to a central catalytic GHF10 module, an N-terminal substrate-binding module of family CBM22 known to bind xylan (Charnock et al., 2000). Xgh74A consists of an N-terminal catalytic module of family GHF74 and the dockerin module.
To characterize the proteins biochemically, the genes were expressed in E. coli. Both proteins had the expected mass in denaturing SDS-PAGE (not shown) as was calculated from the sequence, and were enzymically active (Table 1). This corroborated the correct cloning procedure and the predicted extent of the reading frames.
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The enzyme depolymerized xyloglucan completely and released reducing sugars from tamarind xyloglucan quickly. Large oligosaccharides with more than six sugar residues were produced which could not be separated sufficiently by TLC (Fig. 1b, AE). No smaller molecules were visible as hydrolysis products even after extended incubation with excess enzyme, indicating an extremely low or no activity on the oligosaccharides (Fig. 1b
, E).
The oligosaccharides produced from the xyloglucan were further investigated by infusion ESI/MS. The MS data fit well with the occurrence of the hepta- to nonaglycosides XXXG, XLXG (or XXLG) and XLLG as major products according to the nomenclature of Fry et al. (1993) with m/z values of 1080, 1242 and 1404 respectively (Fig. 2
). Minor products can be identified as XXG, XXX and XXGG. Each oligosaccharide appears as a cluster of peaks from protonated molecules as well as different salt adducts. Acetyl esters from XXXG as well as from other oligosaccharides (m/z+42) were found in traces. The majority of the products were substituted cellotetraose units of xyloglucan.
Cellulosomes were purified from a cellulose-grown C. thermocellum culture and incubated with tamarind xyloglucan. The specific activity of the cellulosomal preparation on tamarind xyloglucan was 4·7 U mg1 (±0·4). The degradation pattern was indistinguishable in TLC from that obtained with purified recombinant Xgh74A (data not shown). This indicates (1) that xyloglucanase is active in native cellulosomes, and (2) that the decorated cellotetraose units produced are not further degraded by either Xgh74A, or any -glucosidase or other glycosidase eventually present in the cellulosome.
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DISCUSSION |
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The cellulosomal subunit Xyn10D cleaves xylan in endo-mode and with a hydrolysis pattern known from other endo-xylanases of GHF10. It is at present not clear why the bacterium produces at least three different GHF10 endoxylanases for xylan hydrolysis: Xyn10C, Xyn10D and Xyn10Z. Xyn10D is the third most prevalent xylanase in cellulose-grown cellulosomes, with a molar abundance of 0·3 (about one Xyn10D in each third cellulosomal particle) (Zverlov et al., 2005). The similarities in product formation between Xyn10D and Xyn10C (Hayashi et al., 1997
) were conspicuous. But nevertheless and despite an identical module architecture the sequence homology between Xyn10C and Xyn10D is low (21 % identity in the catalytic module), and the presence of these two similar genes in the genome does not seem to be the result of a gene duplication. However, another non-cellulosomal xylanase gene with much higher homology (56 % aa identity) was identified in the genome of C. thermocellum: ORF Chte02002923 (zp_00311867), which has an additional N-terminal module of unknown function and lacks a binding and a dockerin module. This ORF has not yet been investigated. It is not known if it is expressed or functional. The characterized xylanase with the highest homology to Xyn10D is XynA of Thermoanaerobacterium thermosulfurigenes, with 34 % identity (Matuschek et al., 1996
).
An explanation for the presence of multiple xylanases could be the resulting improvement in substrate decomposition, e.g. (1) in the competition for binding to different cohesin sites on CipA (and thus occupying more of the limited cohesin positions with xylanases), (2) in differences in substrate binding or (3) in the cleavage of different sites in the heterogeneous substrate, which is difficult to test. An example is Xyn10Z, which has an additional carbohydrate esterase that helps to make native xylan accessible for degradation, and another type of binding module (CBM6), which is described to bind to cellulose.
One type of hemicellulolytic enzyme which has not previously been addressed in the context of the cellulosome is the xyloglucanase hydrolysing the plant cell wall polysaccharide xyloglucan. This 1,4--glucan consists of heavily decorated cellotetraose subunits. Its hydrolysis greatly stimulates the cellulase activity in xyloglucan-cellulose copolymers (Irwin et al., 2003
) and may play a crucial role in the degradation of biomass at least from dicotyledonous plants. Xyloglucanase Xgh74A represents the first xyloglucanase identified in C. thermocellum and the first active xyloglucanase in a cellulosome. Only one ORF for a cellulosomal GHF74 protein has been identified so far: in the genome of C. acetobutylicum ATCC 824. It was annotated as sialidase, but was not characterized further and is probably a xyloglucanase (accession no. AE007608; Nolling et al., 2001
; Doi & Kosugi, 2004
). This gene encodes the GHF74 protein in the databases that is the most homologous to Xgh74A (55 % identity).
The hydrolysis pattern of Xgh74A as one of the prominent subunits in the C. thermocellum cellulosome was investigated (Fig. 2). Few enzymes of GHF74 have been characterized to date; most of them are fungal
-glucanases (Yaoi & Mitsuishi, 2002
; Hasper et al., 2002
; Irwin et al., 2003
). The GHF74 xyloglucanases show a variety of substrate specificities, mostly on the
-glucan backbone substituted with xylose. Xgh74A hydrolysed xyloglucan primarily into hepta- to nonasaccharides based on cellotetraose, seemingly XXXG, XLXG (or XXLG) and XLLG using the known composition of the substrate (Fry et al., 1993
). The Thermobifida fusca enzyme Xeg74 shows a similar product pattern and size distribution (Irwin et al., 2003
). This suggests that Xgh74A is a depolymerizing 1,4-
-glucanase like Xeg74 from T. fusca and cuts the xyloglucan backbone at the 1'-end of the unsubstituted glucose residue. Xeg74 was also reported to be an inverting enzyme (Irwin et al., 2003
), which implies that Xgh74A also inverts the anomeric carbon on hydrolysis.
The high activity of Xgh74A on mixed-linkage barley -glucan is unusual amongst xyloglucanases. However, the Thermotoga maritima xyloglucanase Cel74 has its highest activity on barley
-glucan (Chhabra & Kelly, 2002
). It shows 38 % sequence identity in the catalytic module. The only 3-D structure of a GHF74 enzyme has been obtained from the OXG-RCBH xyloglucanase of Geotrichum sp. M128 (Yaoi et al., 2004
). Although this fungal enzyme is rather an oligoxyloglucan reducing-end-specific cellobiohydrolase (Yaoi & Mitsuishi, 2002
, 2004
), its basic structure as a tandem repeat of two similar seven-bladed
-propeller subunits should be preserved in the homologous Xgh74A protein (Yaoi et al., 2004
). The proposed catalytic amino acids Asp-35 and Asp-465 in the active centre of OXG-RCBH are located in highly conserved regions and are preserved in Xgh74A (D70 and D480, respectively). The extra loop aa 375485 in OXG-RCBH is missing in Xgh74A as it is in all other xyloglucanases (Yaoi et al., 2004
).
The removal of the xyloglucan in plant cell walls is obviously a precondition for efficient cellulose hydrolysis, and Xgh74A may be able to perform this task in C. thermocellum, as was demonstrated by incubating xyloglucan with purified cellulosomes. The reading frame of xghA is the only cellulosomal gene in the draft genome of C. thermocellum to which a xyloglucanase activity can be ascribed, and the only ORF with homology to GHF74 modules in the genome.
Zymogram staining of denaturing gel electrophoresis slabs from purified cellulosomes show only one band of activity (data not shown), indicating that no other -glucanase is able to degrade tamarind xyloglucan sufficiently to be detected with the Congo red staining assay. Xgh74A is thus the only xyloglucanase in the cellulosome, opening the possibility to calculate the prevalence of Xgh74A in the cellulosome. Assuming an average mass of 900 kDa for a single cellulosome unit (containing one scaffoldin molecule and nine of the major catalytic components) and applying the formula (SpAXgh74A/SpAcellulosome)x(MXgh74A/Mcellulosome), where SpA is the specific activity and M is the molecular mass, it can be concluded that one Xgh74A molecule is present in
5·7 (±0·7) cellulosomal units (Zverlov et al., 2002
). This calculated prevalence is in good agreement with the abundance of 0·2 per particle obtained from protein determinations for XghA in 2-D electrophoresis gels (Zverlov et al., 2005
).
Xgh74A can be ascribed a key role in the degradation of natural cellulosic material by the cellulosome of C. thermocellum, considering the protective role that xyloglucans have on cellulose fibres. This was shown by Irwin et al. (2003) with copolymers of bacterial cellulose and xyloglucan: cellulose was completely protected from T. fusca cellulase degradation as long as the xyloglucan was present. Only removal of the xyloglucan by T. fusca xyloglucanase Xeg74 brought about the complete digestion of the cellulose fibres. Xgh74A should have a similar function in dismantling the cellulose fibres in biomass. It is thus no surprise that it is one of the most prominent hemicelluloytic enzymes in the extracellular cellulose-degrading enzyme complex of C. thermocellum (Zverlov et al., 2005
).
We have described two hemicellulolytic subunits in the cellulosome of C. thermocellum, XghA and Xyn10D, representing the only xyloglucanase in the cellulosome and a typical endo--xylanase, respectively. Both enzymes degrade hemicellulose components protecting cellulose in plant fibres from enzymic degradation. They thus contribute to the cellulose degradation by the cellulosomes of C. thermocellum.
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ACKNOWLEDGEMENTS |
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Received 20 May 2005;
revised 23 June 2005;
accepted 30 June 2005.
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