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: Thermotoga neapolitana, 1,3-ß-glucanase, substrate-binding module CBM4, binding specificity
Abbreviations: CBM, carbohydrate-binding module; GHF, glycosyl hydrolase family
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INTRODUCTION |
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Soluble 1,3-ß-glucans are readily degraded by endoglucanases secreted by a number of bacteria (Schwarz et al., 1988 ; Nogi & Horikoshi, 1990
; Aida et al., 1995
; Zverlov et al., 1997
; Krah et al., 1998
). The enzymic hydrolysis of insoluble and gelatinized ß-glucans, which are protected by their highly ordered structure, is more problematic (Kanzawa et al., 1994
) and may be enhanced if hydrolytic enzyme modules are sterically correctly connected with carbohydrate-binding modules (CBMs). These non-catalytic modules were originally detected as cellulose-binding domains binding to crystalline and amorphous cellulose (Tomme et al., 1995
). The development of techniques to study the binding to soluble substrates stimulated the research of binding modules greatly (Tomme et al., 1996
). They were found to be present in many modular bacterial polysaccharide hydrolases as small, separately folding, non-catalytic protein domains or modules. Twenty-four CBM families have been defined by comparing their primary structure (Tomme et al., 1998
; Meissner et al., 2000
; Coutinho & Henrissat, 1999
). Members of the same CBM family can be connected to catalytic modules of different glycosyl hydrolase families (GHFs) and some modular enzymes contain more than one CBM and also CBMs of more than one family. The nomenclature of CBMs that has been proposed recently by Henrissat et al. (1998)
will be used here.
The three-dimensional structure of a number of CBMs has been resolved, most of them having ß-sheet topologies (reviewed by Linder & Teeri, 1997 ; Bayer et al., 1998
). CBM1, CBM2, CBM3 and CBM5 modules have a planar hydrophobic surface, on which substrate binding is mediated by interaction between exposed aromatic amino acid residues and the glucosyl-pyranose ring. These CBMs interact with multiple cellulose chains and strongly prefer insoluble microfibrils of cellulose to soluble polysaccharide molecules (Bolam et al., 1998
). In contrast, the CBM4 module CfCenC/N1 has a binding groove that recognizes one polysaccharide strand (Tomme et al., 1998
). However, the majority of CBMs are not functionally characterized as yet. The binding pattern of CBMs that have been determined can vary widely, even within one family.
The presence of these modules may influence the substrate range of their hydrolytic partner modules, especially on complex substrates like crystalline cellulose (Irwin et al., 1998 ; Gill et al., 1999
) and complex hemicellulose (Black et al., 1996
). Removal of the CBMs from a modular protein often reduces the catalytic activity on insoluble glycans without affecting the activity on soluble substrates. The activity enhancement can be explained by an increase in the effective concentration of the hydrolytic enzyme on the macromolecular substrate, either insoluble, partially soluble, gelatinized or bound to an insoluble substrate, as would be the case with hemicellulose bound to the cellulose matrix in plant cell walls (Black et al., 1996
). Moreover, it has to be pointed out that the degradation of crystalline substrates, besides the hydrolysis of the glycosidic bond, has to accomplish the dislocation of the attacked molecule from the highly ordered, crystalline surface, a (non-catalytic) activity which could be ascribed to the strongly binding CBMs. The CBMs in some enzymes have the ability to thermally stabilize the attached catalytic domain (Meissner et al., 2000
; Sunna et al., 2000
), although this seems to be a speciality of co-evolved modules, and a general thermostabilizing role can not be ascribed to them.
Thermotoga neapolitana, like its close relative Thermotoga maritima, is a saccharolytic, Gram-negative bacterium which was isolated from marine environments with volcanic activity. It is, with a maximum growth temperature of 90 °C, the most thermophilic saccharolytic bacterium isolated so far (Huber & Stetter, 1992 ) and constitutes one of the deepest branches in the phylogenetic tree of the bacteria. It hydrolyses many polysaccharides, including 1,3-ß-glucan, with a range of extracellular enzymes (Dakhova et al., 1993
; Bronnenmeier et al., 1995
; Zverlov et al., 1999
) which, due to their inherent stability, are of utmost interest for biotechnological applications. The DNA sequence of the thermostable 1,3-ß-glucanase (laminarinase) gene lamA of T. neapolitana has been determined previously (Zverlov et al., 1997
). The protein has a modular structure and consists of catalytic and non-catalytic modules. The present study investigates the role of the non-catalytic modules of Lam16A (formerly designated LamA) in the binding to soluble and insoluble substrate molecules. The primary structure of the Lam16A CBMs was compared to known CBMs and to the sequence database. Homologous sequences belong to family CBM4 and obviously fall into four subfamilies. The binding pattern of at least one member of each subfamily was determined or known. The data suggest a correlation with the hydrolytic activity of the catalytic module connected to the CBM.
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METHODS |
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Molecular biological methods.
PCR was carried out using oligonucleotide primer pairs with chromosomal DNA as the template and the Expand High Fidelity PCR System (Boehringer Mannheim). The oligonucleotide primers were: for the amplification of LamA-H1 primer pBamI-1 (5'-TGTTTGGATCCTGGCTCAGAATATTTTAC-3') and pSalI-c3 (5'-CTTATGTCGACTCTCGATCTCCCCTCTTC-3'); for LamA-H2, pBamI-1 and pSalI-c2 (5'-TGATCGTCGACTATCGGTTCATCGAATGTG-3'); for LamA-H3, pBamI-2 (5'-ATGTGGGATCCCTGGAAGTCAGTGGTGAGG-3') and pSalI-c3; for LamA-H4, pBamI-2 and pSalI-c2; and for LamA-H5, pBamI-1 and pSalI-c1 (5'-GACAAGTCGACAGTCTTCCACCTTGTCCTC-3').
The DNA sequence of cloned PCR products was verified by sequencing supercoiled double-stranded plasmid DNA on both strands by using the Thermo Sequenase Cycle Sequencing Kit (Amersham) for extension of 5'-biotinylated primers. DNA fragments were separated with a GATC 1500 Direct-Blotting-Electrophoresis apparatus and detected using streptavidin-conjugated alkaline phosphatase and nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (Serva) as the chromogenic substrate. Sequence data were analysed with the DNASIS/PROSIS software package (Hitachi Software Engineering). Nucleotide and protein databases were screened using the FASTA and BLAST software at the EBI-server at EMBL (http://www.ebi.ac.uk). Sequence alignment was done with the CLUSTAL W software at the same server.
Purification of recombinant proteins.
Recombinant proteins were purified from 50 ml IPTG-induced E. coli cultures with Ni-NTA spin columns according to the manufacturers instructions (Qiagen). The purity of the proteins was verified by SDS-PAGE and staining with Coomassie brilliant blue G-250 (Serva).
Enzyme assays.
Enzyme aliquots in standard assays were incubated in phosphate/citrate buffer (50 mM, pH 6·2) with 0·7% (w/v) substrates at 85 °C. Reducing sugars released from 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. To get values within the linear rate of reaction, the amount of enzyme and the time of incubation were chosen to result in values well below the saturation point of the test. All tests were performed in triplicate and the coefficients of variation (standard deviation divided by the mean) were less than 8%.
Analysis of binding to soluble substrates by affinity electrophoresis.
The affinity electrophoresis method described by Tomme et al. (1996) was applied. Native polyacrylamide gels (10%), with and without binding substrates (1 mg ml-1) added prior to polymerization, were prepared separately and run in parallel at 4 °C. BSA fraction V was used as a negative non-interacting control. Proteins were visualized by staining with Coomassie brilliant blue G-250.
The retardation coefficient Kr was quantified by calculating the relative migration distances r (with substrate) and r0 (without substrate): Kr=rx(r0-r)-1x[S], where [S] is the substrate concentration (mg ml-1). The relative migration distance, r, was determined as the ratio of the migration distance of the major protein band and the migration front of the gel.
Analysis of binding to insoluble substrates.
Binding of proteins to insoluble carbohydrates was carried out in 1·5 ml polypropylene tubes at 4 °C. Purified protein (510 µg) was added to 10 mg substrate in 0·5 ml 80 mM imidazole/HCl buffer (pH 6·8) containing 0·2 M NaCl and incubated on a shaker. The samples were then centrifuged for 5 min to remove the ligand together with the bound protein. Free protein in the cleared supernatants was determined with the Protein Assay Kit (Bio-Rad). The measurements were done at least in triplicate using BSA as a negative control. The residual protein concentration (percentage) was determined as the ratio between the initial and the free protein concentration after incubation with the substrate.
Substrates.
Birchwood xylan, chitin from crab shells, avicel CF1, carboxymethylcellulose (low viscosity; degree of substitution, 0·9), lichenan and laminarin were obtained from Sigma-Aldrich, barley ß-glucan and pachyman from Megazym, curdlan from Wako Pure Chemical Industries, and pustulan from Roth. Hydroxyethyl-cellulose HEC20 was manufactured by Clarian as Tylose H20g4 (degree of substitution, 1·2; molar substitution, 1·7) and obtained through Serva. The ß-glucans used in this study are listed in Table 1.
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RESULTS |
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Binding to insoluble polysaccharides was assayed by measuring the depletion of protein from cleared supernatant. The ratio of unbound to bound enzyme was plotted against the time of incubation (Fig. 4). No binding was observed to the 1,4-ß-linked polysaccharides Avicel (micro-crystalline cellulose), xylan and chitin. The constructs LamA-H1 and LamA-H3 (
CBM4-1) bound to the insoluble yeast cell wall ß-1,3-glucan, whereas LamA-H2 (
CBM4-2) and -H4 (
CBM4-1
CBM4-2) did not. The binding kinetics were fast: maximum binding was reached after 510 min. Only LamA-H1 and -H3 contained CBM4-2 (Fig. 2
). This clearly indicates that the CBM4-2 module is responsible for the specific physical attachment to insoluble 1,3-ß-glucan.
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DISCUSSION |
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Deletion of the non-catalytic modules diminished the hydrolytic activity of Lam16A. The greatest effect for the hydrolytic activity on soluble 1,3-ß-glucan was observed by deletion of CBM4-1, whereas on insoluble substrates and mixed-linkage glucan deletion of CBM4-2 had the greatest effect. This indicates a co-operativity of the substrate binding with the catalytic activity of the GHF16 module, supporting the model that binding modules enhance the catalytic activity of an enzyme by increasing its concentration on the substrate. This argument holds true at least for soluble substrates. For insoluble ß-glucans it cannot be ruled out that CBM4-2 interacts with the substrate surface and disrupts the ordered surface structure. This would allow the catalytic domain of the enzyme to access the substrate molecule. However, this argument cannot apply for gelatinized ß-glucans. It should also be noted that a thermostabilizing effect, as was described for non-catalytic modules homologous to CBMs in other bacterial exo-enzymes (Meissner et al., 2000 ; Sunna et al., 2000
), was not observed with the presence of the CBM4-1 and CBM4-2 modules in Lam16A. Therefore it seems unlikely that the loss of activity is due to diminished stability.
Protein databases were screened for sequences similar to the two TnLam16A binding modules: 22 sequences belonging to module family CBM4 were identified, all occurring in secreted glycosyl hydrolases of bacteria, both mesophilic and thermophilic. The sequences were aligned using the neighbour-joining method in progressive multiple sequence alignment (CLUSTAL W; Thompson et al., 1994 ) (Fig. 1
). A similarity tree was constructed (Fig. 5
) which showed a clear subdivision of the CBM4 family into four subfamilies.
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The binding pattern of seven CBMs connected with four different hydrolytic enzymes has been determined, at least one for each subfamily (Table 4). It is obvious that these binding modules bind exactly the substrates their hydrolytic partners in the modular enzymes are able to degrade. The deep branches in the dendrogram of the CBMs may thus reflect the specificity of the catalytic domains connected with them, suggesting that they are in accordance with the sequence diversity between the domains.
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The sequence of the Lam16A binding modules was compared to the secondary structure of the homologous CfCenC/CBM4-1. This module is comprised of two five-stranded ß-sheets that fold into a jellyroll-type ß-sandwich (Johnson et al., 1996 ). It is so far the only structure of a CBM binding to soluble substrates: an oligosaccharide molecule binds into a cleft that runs across one ß-sheet face of the protein, whereas the modules binding to insoluble substrates have flat binding surfaces (Bayer et al., 1998
). The 10 ß-sheet regions in CfCenC/CBM4-1 are generally well conserved in family CBM4 (Fig. 1
). It can therefore be expected that all CBM4 members have a similar tertiary structure to CfCenC/CBM4-1. Yet the non-polar and hydrophilic amino acid residues proposed to be involved in substrate binding within the binding cleft of CfCenC/CBM4-1 are not very well conserved between the subfamilies, as could be expected from the differences in binding specificity. The calcium-binding amino acid residues in ß-sheets B2 and B3 of CfCenC(1) are involved in stabilizing the protein structure (Johnson et al., 1998
) and are highly conserved in almost all CBMs of family 4.
In summary, the present work demonstrates that two 1,3-ß-glucan binding modules bound differently to various types of binding substrates: CBM4-1 was more specific and bound only soluble 1,3-ß-glucans; CBM4-2 was rather non-specific and bound more tightly to soluble ß-glucans, in addition it also bound to insoluble 1,3-ß-glucans. By sequence similarity the two modules were assigned to one of four newly defined subfamilies of family CBM4, members of which differ greatly in binding specificity to a variety of glycans, either soluble or insoluble, consisting of glucose or xylose monomers and connected by 1,3-ß-, 1,4-ß- or 1,6-ß-glycosidic bonds. The data presented above corroborate the hypothesis that the similarity tree reflects an evolutionary development within the CBM4 family, adapting the subfamilies to bind different substrates. A co-evolution of the non-catalytic modules with their attached catalytic domains can be assumed.
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ACKNOWLEDGEMENTS |
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Received 22 September 2000;
revised 20 November 2000;
accepted 23 November 2000.