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thermocellum. CelK is composed of an N-terminal family IV classified into a new family of carbohydrate binding domains cellulose-binding domain (CBDIVCelK), ...
Protein Engineering vol.14 no.3 pp.167–172, 2001

Do domain interactions of glycosyl hydrolases from Clostridium thermocellum contribute to protein thermostability?

Irina A.Kataeva1, David L.Blum2, Xin-Liang Li and Lars G.Ljungdahl Center for Biological Resources Recovery and Department of Biochemistry and Molecular Biology, A210 Life Sciences Building, University of Georgia, Athens, GA 30602-7229, USA 2Present

address: Diversa Corporation, 4955 Directors Place, San Diego, CA 92121, USA 1To

whom correspondence should be addressed. E-mail: [email protected]

Cellulolytic and hemicellulolytic enzymes usually have a domain composition. The mutual influence of a cellulosebinding domain and a catalytic domain was investigated with cellobiohydrolase CelK and xylanase XynZ from Clostridium thermocellum. CelK is composed of an N-terminal family IV cellulose-binding domain (CBDIVCelK), a family 9 glycosyl hydrolase domain (Gh9CelK) and a dockerin domain (DD). CelK without the DD, (CBDIV–Gh9)CelK and CBDIVCelK bound cellulose. The thermostability of (CBDIV–Gh9)CelK was significantly higher than that of CBDIVCelK and Gh9CelK. The temperature optima of (CBDIV–Gh9)CelK and Gh9CelK were 65 and 45°C, respectively. XynZ consists of an N-terminal feruloyl esterase domain (FAEXynZ), a linker (L), a family VI CBD (CBDVIXynZ), a DD and a xylanase domain. FAEXynZ and (FAE–L–CBDVI)XynZ, used in the present study did not bind cellulose, but both were highly thermostable. Replacement of CBDVIXynZ with CBDIVCelK resulted in chimeras with feruloyl esterase activity and the ability to bind cellulose. CBDIVCelK–FAEXynZ bound cellulose with parameters similar to that of (CBDIV–Gh9)CelK. (FAE–L)XynZ–CBDIVCelK and FAEXynZ–CBDIVCelK had lower relative affinities and binding capacities than those of (CBDIV–Gh9)CelK. The three chimeras were much less thermostable than FAEXynZ and (FAE–L–CBDVI)XynZ. The results indicate that domains of glycosyl hydrolases are not randomly combined and that domain interactions affect properties of these domain-structured enzymes. Keywords: domain structure/glycosyl hydrolases/hybrid enzymes/interactions between domains/thermostability

Introduction Plant cell wall-degrading enzymes include glycosyl hydrolases, which are produced by a large number of microorganisms (Ljungdahl and Eriksson, 1985; Be´guin, 1990). Some hydrolases are relatively simple and consist of only a catalytic domain. However, usually they have rather complex organization with the catalytic domain combined with other domains, that may include a second catalytic domain of similar nature or with different activity (bifunctional enzymes), substratebinding domains, domains with surface-layer homology, immunoglobin similarity, as well as linkers and domains whose functions are unknown (Ohmiya et al., 1997; Bayer et al., © Oxford University Press

1998). Expressed separately, catalytic domains of glycosyl hydrolases have catalytic activity and thus function independently of the other domains. The influence of the other domains on the catalytic domains has not been studied extensively and mostly focused on the effect of a CBD on the activity of the catalytic domain with insoluble substrates (Rixon et al., 1996; Black et al., 1997; Srisodsuk et al., 1997; Bolam et al., 1998). It has been also found that some xylanases and at least one cellulase from thermophilic bacteria contain domains which when deleted led to a decrease in the thermostability of the enzymes (Lee et al., 1993; Fontes et al., 1995; Hayashi et al., 1997; Riedel et al., 1998). Such domains have been termed ‘thermostability domains’ and are considered to confer thermostability on catalytic domains. Recently these domains were classified into a new family of carbohydrate binding domains based on the ability of two representatives to bind xylan (Charnock et al., 2000; Sunna et al., 2000). Several anaerobic microorganisms produce multi-protein complexes termed cellulosomes (Felix and Ljungdahl, 1993; Be´laich et al., 1997; Doi et al., 1998; Bayer et al., 1998) first discovered in Clostridium thermocellum in 1983 (Lamed et al., 1983). The C.thermocellum cellulosome is composed of at least 26 polypeptides, of which 18 possess enzymatic activity (Bayer et al., 1998). The non-catalytic cellulose-integrating protein CipA, also called scaffoldin, is important for the assembly of the cellulosome. CipA is composed of nine cohesin domains, a cellulose-binding domain (CBD) (Gerngross et al., 1993) and a type II special dockerin domain attaching the cellulosome to the cell surface (Leibovitz and Be´guin, 1996). All known catalytic subunits of the cellulosome have a type I dockerin domain, through which they are bound to the cohesins of the scaffoldin forming the cellulosomal complex (Tokatlidis et al., 1993). The catalytic subunits of the cellulosome are present in different quantities (Kohring et al., 1990). One of the most abundant subunits is a cellobiohydrolase, CelK (Choi and Ljungdahl, 1996). CelK is composed of an N-terminal family IV cellulose-binding domain (CBDIVCelK), a family 9 glycosyl hydrolase domain (Gh9CelK) and a dockerin domain (Kataeva et al., 1999a). We have reported that CelK has high thermostability and efficiently binds acid swollen cellulose (ASC), whereas a truncated form Gh9CelK missing the CBD is less thermostable and only weakly binds ASC (Kataeva et al., 1999b). Another subunit of the C.thermocellum cellulosome is XynZ. Starting with the N-terminus it consists of feruloyl esterase domain (FAEXynZ), a proline-rich linker (L), a family VI CBD (CBDVIXynZ), a dockerin and a xylanase family 10 catalytic domain (Grepinet et al., 1988; Blum et al., 2000). In the present paper we report studies of thermostability, catalytic activity and cellulose-binding capacity of (CBDIV–Gh9)CelK, Gh9CelK, CBDIVCelK and chimeric polypeptides composed of the FAE domain of XynZ and CBDIVCelK. The results indicate that interactions between domains are important for activity, thermostability and cellulose binding. They do not sustain the idea of separate thermostability domains but rather that this 167

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Table I. Nucleotide sequence of PCR primersa No.

Primer

Sequenceb

Restriction site

Location

Direction

1 2 3 4 5 6 7 8 9 10 11 12

CelKF1 CelKR1 CelKF2 CelKR2 XynZF1 XynZR1 XynZR2 XynZR3 XynZR4 CelKR3 XynZF2 XynZR5

5⬘-CTAGCTAGCTTGGAAGACAAGTCTTCAAAG-3⬘ 5⬘-TTTTCCTTTTGCGGCCGCTTATATTAAGTCAATTTCATCGAG-3⬘ 5⬘-ACGCGTCGACTTGGAAGAAGACAAGTCTTCAAAG-3⬘ 5⬘-TTTTCCTTTTGCGGCCGCTTACTAGAGAGATACATCATCAAG-3⬘ 5⬘-CGGGATCCGCTTGTCACAATAAGCAGTACA-3⬘ 5⬘-GAGGAAGCTTAAACGCCAAAAGTGAACCAGTC-3⬘ 5⬘-TTTTCCTTTTGCGGCCGCTTAGTTTCCATCCCTCGTCAA-3⬘ 5⬘-GACGTCGACACGTGTGTTTGCCGGCTTTGGACT-3⬘ 5⬘-GACGTCGACGTTTCCATCCCTCGTCAATCCGGC-3⬘ 5⬘-GACGTCGACGTAGAGAGATACATCATCAAG-3⬘ 5⬘-ACGCGTCGACCTTGTCACAATAAGCAGTACA-3⬘ 5⬘-TTTTCCTTTTGCGGCCGCTTAGTTTCCATCCCTCGTCAATCCGGC-3⬘

NheI NotI SalI NotI BamHI HindIII NotI SalI SalI SalI SalI NotI

1224–1244 3573–3593 1224–1244 1713–1733 158–178 1343–1363 941–958 977–1000 935–958 713-1733 158–178 935–958

Forward Reverse Forward Reverse Forward Reverse Reverse Reverse Reverse Reverse Forward Reverse

aRestriction

sites are underlined and stop codons are indicated by bold letters. amino acid sequences of CelK (accession af039030) and XynZ (accession m22624) were used to design primers. Numbers of primers correspond those in Figure 1.

bDeduced

property is attributed to the complete protein and thus to the interaction between the domains therein. Materials and methods Bacterial strains, culture conditions and plasmids C.thermocellum JW20 was used as a source of genomic DNA and cellulosomes (Wiegel and Dykstra, 1984). JW20 was grown anaerobically under a nitrogen atmosphere at 60°C in pre-reduced medium with 1% (w/v) cellobiose or 5% (w/v) Avicel PH-101 as a carbon source. Escherichia coli BL21(DE3)pLys (Stratagene Cloning Systems, La Jolla, CA), used as the cloning host for the T7 RNA polymerase expression vector pET-21b (Novagen, Madison, WI), was grown in Luria–Bertani medium supplemented with ampicillin (100 µg/ml). Genomic DNA isolation Genomic DNA of C.thermocellum was purified from a 0.5 l culture by the method of Marmur (1961) with the modifications described earlier (Kataeva et al., 1999a). Primer design, PCR and cloning Oligonucleotide primers containing restriction sites were designed according to DNA sequences of celK (Kataeva et al., 1999a) and xynZ (Grepinet et al., 1988) (Table I and Figure 1) and synthesized with an Applied Biosystems DNA synthesizer. DNA fragments were amplified by PCR using the primers in combination with purified genomic DNA as a template. PCRs were done on a Model 480 Thermal Cycler (Perkin-Elmer, Norwalk, CT). All reagents were purchased from Perkin-Elmer and used as instructed. The reactions were carried out with Vent DNA polymerase (New England Biolabs, Beverly, MA). The annealing temperature was 54°C. Extension time varied depending on the length of amplifying fragments. PCR products were separated by 1% agarose gel electrophoresis and extracted from the gel using a Geneclean II kit (Bio 101, La Jolla, CA). The extracted DNA fragments were cleaved with appropriate restriction enzymes under the conditions recommended by enzyme suppliers. After heat inactivation of the restriction enzymes, the fragments were purified using Microcon tubes (Amicon, Beverly, MA) and ligated to the pET-21b vector linearized with the corresponding restriction enzymes. To combine the cbd region of celK with the fae region of xynZ, the PCR product encoding CBDIVCelK was digested by a pair of restriction enzymes, purified as described above and ligated 168

Fig. 1. Domain structure of proteins used in the present study. All variants except Gh9CelK were cloned into pET-21b. Gh9CelK was obtained as a product of spontaneous proteolysis of (CBDIV–Gh9)CelK. Arrows indicate direction of PCR amplification. Numbers refer to primers from Table I.

to the purified pET-21 vector containing an insert encoding FAEXynZ and linearized by the same restriction enzymes. The ligation mixtures were used to transform competent E.coli BL21(DE3)pLys cells. Ampicillin-resistant colonies were isolated and their plasmid DNA was purified, cleaved with restriction enzymes, subjected to agarose gel electrophoresis and sequenced to verify the constructs. All constructs used in the present study are listed in Figure 1. Sequence analysis The Genetic Computer Group (version 10, University of Wisconsin Biotechnology Center, Madison, WI) on the VAX/ VMX system on the BioScience Computing Resource at the University of Georgia was used to analyze sequence data. Multiple sequence alignment was done using the MEME and PILEUP programs of the GCG package. Protein purifications All proteins were purified from 2 l E.coli cultures 5 h after isopropyl-β-D-thiogalactopyranoside induction. The cells were

Domains in glycosyl hydrolases interact

Fig. 2. SDS–PAGE of purified proteins. Lane 1, molecular mass standards; lane 2, (FAE–L–CBDVI)XynZ; lane 3, FAEXynZ; lane 4, CBDIVCelK; lane 5, (FAE–L)XynZ–CBDCelK; lane 6, FAEXynZ–CBDIVCelK; lane 7, CBDIVCelK– FAEXynZ.

Table II. Properties of purified polypeptides Polypeptide

Catalytic activity (Ua µmol/protein)

PCmaxb K(l/g)c t1/2d (60°C) (µmol/g (h) cellulose)

(CBDIV–Gh9)CelK Gh9CelK CBDIVCelK (FAE–L–CBDVI)XynZ FAEXynZ (FAE–L)XynZ– CBDIVCelK FAEXynZ–CBDIVCelK CBDIVCelK–FAEXynZ

1085 818 – 558 769 716

10.8 2.2 16.4 0.6 NBf 6.9

1.4 0.4 2.6 NDe ND 0.8

590 663

6.7 10.4

0.6 1.2

62.0 7.0 0.25 97.0 140.0 0.2 0.16 1.0

of activity (U) is defined as µmoles of substrate converted in 1 min; activity of CelK and FAE derivatives was assayed with PNP-cellobioside and FAXX, respectively. bPC max ⫽ binding capacity. cK ⫽ relative equilibrium association constant. r dThe t 1/2 values were calculated by plotting log(% residual activity) versus incubation time. eNot detected. fNot bound. aUnit

collected and desintegrated using a French Press. (CBDIV– Gh9)CelK and Gh9CelK were purified as described earlier (Kataeva et al., 1998b). (FAE–L–CBDVI)XynZ was purified by heat treatment in a water-bath at 60°C for 30 min (Blum et al., 2000) and FAEXynZ by heat treatment under the same conditions, followed by centrifugation at 8000 g for 30 min to remove precipitated protein and by gel filtration on a prepacked TSK-Gel 3000SW stainless-steel column (TosoHaas, Montgomeryville, PA) (Blum et al., 2000). CBDIVCelK, (FAE–L)XynZ–CBDIVCelK, FAEXynZ–CBDIVCelK and CBDIVCelK–FAEXynZ were purified by ammonium sulfate precipitation (40% saturation) and ion-exchange chromatography on DEAESepharose CL-6B (Pharmacia Biotech, Piscataway, NJ). The purity of the proteins was monitored by SDS–PAGE. Activity assays Initial activities of (CBDIV–Gh9)CelK and Gh9CelK were determined at 65 and 45°C, respectively, in 50 mM sodium citrate buffer, pH 6.0, by measuring p-nitrophenol release from p-nitrophenylcellobioside (PNP-cellobioside) (Kataeva et al., 1999a). The concentration of PNP-cellobioside was 5 mM. Initial activities of FAE and chimeric proteins containing the FAEXynZ domain of XynZ were assayed at 60°C in 50 mM sodium citrate buffer, pH 6.0, by determining the release of ferulic acid from O-{5-O-[(E)-feruloyl]-α-Larabinofuranosyl}-(1→3)-O-β-D-xylopyranosyl-(1→4)-xylo-

pyranose (FAXX) obtained from wheat bran (Borneman et al., 1990). Ferulic acid was quantified by HPLC using a reversed-phase column (ODS microsil, 5 µm, 125⫻4 mm i.d.; Hewlett-Packard) using a mobile phase of 10 mM sodium formate, pH 3, and 30% (v/v) methanol (Borneman et al., 1990). One enzyme unit was defined as amount of the enzyme releasing 1 µmol of ferulic acid per minute. Cellulose binding assay Acid-swollen cellulose (ASC) used in adsorption experiments was prepared by treatment of Avicel PH-101 (Fluka, St. Louis, MO) with phosphoric acid (Klyosov et al., 1981). Adsorption assays were done at room temperature in 0.5 ml micro-tubes. Proteins were mixed with ASC (1 g/l) in 50 mM sodium citrate buffer, pH 6.0, in a final volume of 0.5 ml. The tube contents were continuously mixed by rotation. After equilibration for 2 h, cellulose and bound protein were removed by centrifugation at 10 000 g for 10 min, which was repeated twice to ensure removal of all cellulose from the supernatant. The concentration of unbound protein in the supernatant was then determined. The bound protein concentration was calculated as the difference between the initial protein concentration and unbound protein. Each data point is the mean of five replicates. Adsorption to cellulose was analyzed according to the model described by Gilkes et al. (1992). Protein determination During purification protein was determined with Coomassie Protein Assay Reagent (Pierce, Rockford, IL). In other experiments protein concentrations were determined on the basis of A280 values. The molecular masses of proteins were calculated from primary structures deduced from DNA sequence analysis. Molar absorption coefficients, calculated from aromatic amino acid content were as follows: for (CBDIV– Gh9)CelK, 198 800; for Gh9CelK, 158 150; for CBDIVCelK, 42 680; for (FAE–L–CBDVI)XynZ, 59 830; for FAEXynZ, 40 740; and for (FAE–L)XynZ–CBDIVCelK, FAEXynZ–CBDIVCelK and CBDIVCelK–FAEXynZ, 83 420 l/mol.cm. Thermostability assay Proteins were incubated in 50 mM Tris–HCl buffer, pH 7.5, at stated temperatures. At certain time intervals aliquots were taken, centrifuged and assayed for the residual activity. In the case of the CBDIVCelK, the residual soluble protein in the supernatant was determined. Half-life times (t1/2) were calculated using the equation for one-phase exponential decay in the GraphPad Prism program. Results Purification and activities of the protein derivatives All proteins used in this study were purified close to homogeneity as assayed with SDS–PAGE. This was shown previously for (CBDIV–Gh9)CelK and Gh9CelK (Kataeva et al., 1999b) and in Figure 2 for (FAE–L–CBDVI)XynZ, FAEXynZ, CBDIVCelK, (FAE–L)XynZ–CBDIVCelK, FAEXynZ–CBDIVCelK and CBDIVCelK–FAEXynZ. Catalytic activities of the purified enzymes are given in Table II. Deletion of the CBDIVCelK from (CBDIV– Gh9)CelK did not change the catalytic activity of Gh9CelK towards PNP-cellobioside significantly. Deletion of the linker and the CBDVI from (FAE–L–CBDVI)XynZ gave the FAEXynZ domain alone. This domain had a catalytic activity approximately one-third higher than the parent enzyme, demonstrating that the activity of this domain was not dependent on the linker or CBDVI and that actually enzymatic activity was 169

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Fig. 3. Sequence alignments of family IV CBDs and thermostabilizing domains. The number of residues located between GXTY and FYXDDV motifs is given in parentheses.

improved by removing them. Grafting the CBDIVCelK to the N- or C-terminus of FAEXynZ gave hybrid polypeptides with activity similar to that of (FAE–L–CBDVI)XynZ. The linker of XynZ retained in the (FAE–L)XynZ–CBDIVCelK construct did not affect the activity of the fused protein. Binding to acid-swollen cellulose Binding capacities (PCmax) and relative equilibrium association constants (Kr) of all polypeptides used in the present study were calculated from adsorption isotherms and are given in Table II. Kr values were used to compare the affinities of the polypeptides for a given preparation of acid-swollen cellulose (Gilkes et al., 1992). They were estimated from the limiting slope of a plot of 1/bound protein versus 1/free protein (Gilkes et al., 1992) and PCmax values were calculated by extrapolating the amount of bound protein to infinity (Table II). PCmax was the highest in the case of CBDCelK, lower in (CBDIV–Gh9)CelK and significantly lower in Gh9CelK. Truncated XynZ and FAEXynZ alone did not bind ASC, but FAEXynZ with CBDCelK attached bound to cellulose. Effect of temperature on activity and stability of protein derivatives CBDIVCelK was not thermostable, but combined with Gh9CelK giving (CBDIV–Gh9)CelK it clearly enhanced the thermostability of the latter. Thus, the highest initial activity of (CBDIV– Gh9)CelK was at 65°C and of Gh9CelK at 45°C. As a further characteristic of thermostability we used the half-life time (t1/2) at 60°C (Table II). CBDVIXynZ did not affect FAEXynZ thermostability since both (FAE–L–CBDVI)XynZ and FAEXynZ were highly thermostable. In contrast, grafting the CBDIVCelK to FAEXynZ caused a drastic decrease in the thermostability of the fused proteins (Table II). Of the hybrid proteins, CBDIVCelK–FAEXynZ was more stable than (FAE–L)XynZ–CBDIVCelK or FAEXynZ–CBDIVCelK, both of which were totally inactivated almost immediately (Table II). Comparison of family IV CBDs and thermostabilizing domains To reveal possible homology between cellulose-binding and thermostabilizing domains we aligned deduced amino acid sequences of family IV CBDs from C.thermocellum CelK (accession af039030) (Kataeva et al., 1999a), CbhA (x80993) (Zverlov et al., 1998), Cellulomonas fimi CenC (x57858) CBDN1 (Coutinho et al., 1991), Streptomyces reticuli Cel1 (x65616) (Schlochtermeier et al., 1992), Thermomonospora fusca E1 (l20094) (Lao et al., 1991), C.thermocellum LicA (direct submission, x89732) and Thermotoga neapolitana 170

LamA (z47974) (Dakhova et al., 1993) with thermostabilizing domains from C.fimi XynC (z50866) (Clarke et al., 1996), C.thermocellum XynC (d84188) (Hayashi et al., 1997), Ruminococcus flavefaciens XynD (s61204) (Flint et al., 1993), C.thermocellum XynY (x83269) (Fontes et al., 1995) and Thermotoga maritima XynA (z46264) (Winterhalter et al., 1995). The alignment revealed no significant homology. However, two motifs were highly conserved in both domains (Figure 3). The first motif was a GXTY sequence with tyrosine conserved in all CBDs and thermostabilizing domains. The second motif was an FYXDDV sequence where the first aspartic amino acid residue was also conserved in both domains. The motifs were separated by an insert of ~75 amino acid residues. Exceptions are the T.neapolitana LamA CBD where the two motifs were separated by only 30 amino acid residues and the C.thermocellum LicA CBD in which the FYXDDV motif preceded and was closely located to GXTY (Figure 3). Discussion It is now well recognized that many proteins consist of multiple domains or motifs. It has been suggested that the domains interact and affect properties of each other (Vita et al., 1989; Palme et al., 1997, 1998; Wenk et al., 1998; Jaenicke, 1999; Wenk and Jaenicke, 1999). Glycosyl hydrolases from microorganisms that hydrolyze plant constituents such as cellulose and hemicelluloses generally have several domains with different functions. On the basis of amino acid sequence homology, catalytic domains as well as cellulose-binding domains of these enzymes are classified into 60 and 13 families, respectively (Gilkes et al., 1991; Henrissat and Bairoch, 1993; Tomme et al., 1995). In many cases these domains are separated by flexible hydroxyamino acid-rich linker sequences of different length (Gilkes et al., 1991). Numerous catalytic and substrate-binding domains have separately been expressed in E.coli, purified and characterized (Tomme et al., 1995; Be´ guin and Lemaire, 1996). The recombinant domains usually retain their functions. It has been concluded that the domains fold independently. The domains are not randomly combined (Tomme et al., 1995). Thus, family IV CBDs are attached (with only one exception) to the N-terminus of catalytic domains belonging to family 9 glycosyl hydrolases (Coutinho et al., 1991; Tomme et al., 1995; Zverlov et al., 1998; Kataeva et al., 1999a). Subfamily IIIc CBDs have been found only at the C-terminus of family 9 catalytic domains (Tormo et al., 1996). Domains

Domains in glycosyl hydrolases interact

designated as thermostabilizing are both N-terminally or internally located and associated (with just one exception) with family 10 glycosyl hydrolases catalytic domains, etc. (Hayashi et al., 1997; Sunna et al., 2000). Linkers seemed to mediate proper interaction between domains. In the case of CelK there is no visible linker between the Gh9CelK and CBDIVCelK, suggesting tight interaction between the domains. The domains expressed separately were biologically active: the Gh9CelK hydrolyzed soluble substrates, although at a lower optimal temperature than when associated with CBDIVCelK, whereas the CBDIVCelK bound ASC (Kataeva et al., 1999b). Separately, Gh9CelK and CBDIVCelK have low thermostability whereas the combination (CBDIV–Gh9)CelK has high thermostability. Lack of thermostability of the CBDIVCelK expressed alone was not expected, but this observation is consistent with data obtained with CBDN1 belonging to family IV from mesophilic C.fimi CenC (Creagh et al., 1998). Grafting of the CBDIVCelK to the C-terminal side of the FAEXynZ domain with or without linker did not affect the domain functions much: all the chimeric polypeptides bound ASC, although less efficiently and the FAE domain efficiently hydrolyzed FAXX. However, attachment of the CBDIVCelK to its ‘natural position’ at the N-terminus of the FAEXynZ resulted in a more efficient binding than attachment to the ‘wrong’ C-terminus side. While the combination of the catalytic domain and the CBD enhanced thermostability of (Gh9–CBDIV)CelK, the replacement of the CBDVIXynZ with the CBDIVCelK strongly decreased the thermostability of the hybrid enzymes regardless of the N- or C-terminal location and presence of linker. Thus, although CBDIVCelK in combination (CBDIV– Gh9)CelK positively affects thermostability, this property is not transferable. The idea that protein stability is an intrinsic property of the whole molecule was discussed about 20 years ago (Ljungdahl and Sherod, 1976; Ljungdahl, 1982). However, the finding that deletion of domains of unknown function present in some xylanases from thermophilic microorganisms resulted in a dramatic decrease in the thermostability of the residual polypeptides brought the concept of ‘thermostabilizing domains’ (Lee et al., 1993; Fontes et al., 1995; Hayashi et al., 1997). Until now no-one has reported the improved stability of a mesophilic protein by grafting it to the above ‘thermostabilizing domain’. Other examples reveal that domains in native glycosyl hydrolases interact. CelZ from C.stercorarium contained an internal C⬘ domain identified on a structural basis as a CBD of family IIIc. The C⬘ did not bind cellulose, but it was required to maintain the thermostability of CelZ (Riedel et al., 1998). Another cellulase, E4, from Thermomonospora fusca, contained two CBDs belonging to families IIIc and II. The family IIIc CBD also did not bind cellulose, but was responsible for processivity of the catalytic domain (Irwin et al., 1998). The crystal structure obtained for truncated E4 composed of a family 9 catalytic domain and a subfamily IIIc CBD was the first demonstration of a direct interaction between the two domains (Sakon et al., 1997). The interdependence observed between the Gh9CelK and the CBDIVCelK in CelK with regard to thermostability of the enzyme is in line with these structural data. All these observations imply that thermostability is a cooperative property of the entire molecule based on the interaction between different domains rather than the result of the presence of a special ‘thermostabilizing’ domain. In this regard, stabilization of the protein seemed not to be a biological role of the so-called ‘thermostabilizing domains’ (TSD).

Indeed, recent findings indicated that at least two of such domains, one from Caldibacillus cellulovorans XynA and an internal domain X6b from C.thermocellum XynY, bound polysaccharides (Charnock et al., 2000; Sunna et al., 2000). These observations gave a reason to classify thermostabilizing domains into a new family of carbohydrate-binding domains (Charnock et al., 2000). However, N-terminal TSD from XynY X6a does not bind polysaccharides, keeping the concept of thermostabilization of a catalytic domain alive (Charnock et al., 2000). The crystal structure of X6b revealed typical features found in structures of CBDs (Johnson et al., 1996; Tormo et al., 1996; Brun et al., 2000). This domain binds soluble and insoluble xylans lacking crystal structure, has binding surface as a cleft and contains one calcium ion (Charnock et al., 2000). In this regard X6b is reminiscent of CBDs belonging to family IV with affinity to amorphous but not crystalline cellulose. It is notable that family IV CBDs and former TSDs lacking sequence homology incorporate two short conservative motifs separated by about 75 amino acids (Figure 3). The GXTY motif includes Tyr111, which probably supports the biological fold of CBDCelK (Kataeva et al., 2001). The FYXDDV motif contains an aspartic residue shown to bind calcium in CBDN1, X6b (Charnock et al., 2000) and CelK (Kataeva et al., 2001). In CBDN2 this motif is missed and the domain does not bind calcium. In the alignment of 11 TSDs (Hayashi et al., 1997) and nine representatives of family IV CBDs (Kataeva et al., 1999a), both motifs are much conserved. Probably these motifs found in both family IV CBDs and TSDs separated by the same length fragment are important for the specific fold of these domains affecting the structure of the substrate binding surface and binding specificity. CBDs are ideal affinity tags. They might be attached to the N- or C-terminus of tag proteins without interference with their activity and they show specific interactions with numerous cellulosic matrices (Tomme et al., 1998). Data on domain interactions affecting the final properties of a polypeptide reported here might be of interest for protein engineering of chimeric multidomain polypeptides with desirable properties. Acknowledgement This work was supported by grant DE-FG02-93ER20127 from the US Department of Energy and Aureozyme, Atlanta, GA.

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