Molecular cloning, and characterization of a modular acetyl xylan esterase from the edible straw mushroom Volvariella volvacea

A new Volvariella volvacea gene encoding an acetyl xylan esterase (designated as Vvaxe1) was cloned and expressed in Pichia pastoris. The cDNA contained an ORF of 1047 bp encoding 349 amino acids with a calculated mass of 39 990 Da. VvAXE1 is a modular enzyme consisting of an N-terminal signal peptide, a catalytic domain, and a cellulose-binding domain. The amino acid sequence of the enzyme exhibited a high degree of similarity to cinnamoyl esterase B from Penicillium funiculosum, and acetyl xylan esterases from Aspergillus oryzae, Penicillium purpurogenum, and Aspergillus ficuum. Recombinant acetyl xylan esterase released acetate from several acetylated substrates including beta-d-xylose tetraacetate and acetylated xylan. No activity was detectable on p-nitrophenyl acetate. Enzyme-catalyzed hydrolysis of 4-methylumbelliferyl acetate was maximal at pH 8.0 and 60 degrees C, and reciprocal plots revealed an apparent K(m) value of 307.7 microM and a V(max) value of 24 733 IU micromol(-1) protein. ReAXE1 also exhibited a capacity to bind to Avicel and H(3)PO(4) acid-swollen cellulose.     

Identification of the cellulose-binding domain of the cellulosome subunit S1 from Clostridium thermocellum YS

The 3' region of a gene designated cipB, which shows strong homology with cipA that encodes the cellulosome SL subunit of Clostridium thermocellum ATCC 27405, was isolated from a gene library of C. thermocellum strain YS. The truncated S1 protein encoded by the cipB derivative bound tightly to cellulose. The cellulose-binding domain in this polypeptide consisted of a C-terminal proximal 167 residue sequence which showed complete identity with residues 337-503 of mature SL from C. thermocellum strain ATCC 27405. The cellulose-binding domain interacted with both crystalline and amorphous cellulose, but not with xylan.     

Do the non-catalytic polysaccharide-binding domains and linker regions enhance the biobleaching properties of modular xylanases?

Xylanase A (XylA) from Pseudomonas fluorescens subsp. cellulosa consists of an N-terminal non-catalytic cellulose-binding domain joined to a functionally independent C-terminal catalytic domain by a sequence rich in serine residues. Xylanase D (XylD) from Cellulomonas fimi also exhibits a modular structure comprising an N-terminal catalytic domain linked to an internal non-catalytic xylan-binding domain and a C-terminal cellulose-binding domain. To determine the importance of the non-catalytic polysaccharide-binding domains and linker sequences of XylA and XylD in relation to their capacity to hydrolyse pulp xylan and enhance bleachability, purified full-length and modified derivatives of both enzymes were incubated with a hardwood kraft pulp. Deletion of the cellulose-binding domain or linker region from XylA decreased the activity of the enzyme against pulp xylan, but had no significant effect on the capacity of the enzyme to facilitate delignification and reduce pulp kappa number. While full-length and truncated forms of XylD, lacking either the cellulose-binding or the cellulose- and xylan-binding domains, were equally effective in hydrolysing pulp xylan, enzyme derivatives containing a polysaccharide-binding domain were marginally more efficient in reducing pulp kappa number. The reduction in kappa number elicited by full-length and isolated catalytic domains of XylA and XylD was reflected in an increase in the brightness of paper handsheets derived from pretreated pulps. Thus, the polysaccharide-binding domains of XylA and XylD did not appear to confer any advantage in terms of the ability of the enzymes to improve pulp bleachability. However, XylA and XylD, which belong to different glycosyl hydrolase families, differed in their ability to hydrolyse pulp xylan and facilitate the delignification of kraft pulp. Received: 21 March 1996 / Received revision: 11 July 1996 / Accepted: 19 July 1996     

Purification and characterization of five cellulases and one xylanase from Penicillium brasilianum IBT 20888

The filamentous fungus Penicillium brasilianum IBT 20888 was cultivated on a mixture of 30 g l⁻¹ cellulose and 10 g l⁻¹ xylan for 111 h and the resulting culture filtrate was used for protein purification. From the cultivation broth, five cellulases and one xylanase were purified. Hydrolysis studies revealed that two of the cellulases were acting as cellobiohydrolases by being active on only microcrystalline cellulose (Avicel). Three of the cellulases were active on both Avicel and carboxymethyl cellulose indicating endoglucanase activity. Two of these showed furthermore mannanase activity by being able to hydrolyze galactomannan (locust bean gum). Adsorption studies revealed that the smaller of the two enzymes was not able to bind to cellulose. Similarity in molecular mass, pI and hydrolytic properties suggested that these two enzymes were identical, but the smaller one was lacking the cellulose-binding domain or an essential part of it. The basic xylanase (pI>9) was only active towards xylan. Two of the purified cellulases with endoglucanase activity were partly sequenced and based on sequence homology with known enzymes they were classified as belonging to families 5 and 12 of the glycosyl hydrolases.     

Biochemical characterization of cellulose-binding proteins (CBPA and CBPB) from the rumen cellulolytic bacterium Eubacterium cellulosolvens 5

The cellulose-binding proteins, CBPA and CBPB, of rumen cellulolytic bacterium Eubacterium cellulosolvens 5 were biochemically characterized, and their properties were compared. Recombinant CBPA and CBPB were a typical 1,4-beta-endoglucanase. Both proteins bound to insoluble polysaccharides such as Avicel cellulose, acid swollen cellulose, lichenan, chitin, and oat spelt xylan. On the other hand, only recombinant CBPB bound to agarose and starch.     

Homologous catalytic domains in a rumen fungal xylanase: evidence for gene duplication and prokaryotic origin

A cDNA (xynA), encoding xylanase A (XYLA), was isolated from a cDNA library, derived from mRNA extracted from the rumen anaerobic fungus, Neocallimastix patriciarum. Recombinant XYLA, purified from Escherichia coli harbouring xynA, had a M(r) of 53,000 and hydrolysed oat-spelt xylan to xylobiose and xylose. The enzyme did not hydrolyse any cellulosic substrates. The nucleotide sequence of xynA revealed a single open reading frame of 1821 bp coding for a protein of M(r) 66,192. The predicted primary structure of XYLA comprised an N-terminal signal peptide followed by a 225-amino-acid repeated sequence, which was separated from a tandem 40-residue C-terminal repeat by a threonine/proline linker sequence. The large N-terminal reiterated regions consisted of distinct catalytic domains which displayed similar substrate specificities to the full-length enzyme. The reiterated structure of XYLA suggests that the enzyme was derived from an ancestral gene which underwent two discrete duplications. Sequence comparison analysis revealed significant homology between XYLA and bacterial xylanases belonging to cellulase/xylanase family G. One of these homologous enzymes is derived from the rumen bacterium Ruminococcus flavefaciens. The homology observed between XYLA and a rumen prokaryote xylanase could be a consequence of the horizontal transfer of genes between rumen prokaryotes and lower eukaryotes, either when the organisms were resident in the rumen, or prior to their colonization of the ruminant. It should also be noted that Neocallimastix XYLA is the first example of a xylanase which consists of reiterated sequences. It remains to be established whether this is a common phenomenon in other rumen fungal plant cell wall hydrolases.     

Family 19 chitinases from Streptomyces thermoviolaceus OPC-520: molecular cloning and characterization

Family 19 chitinase genes, chi35 and chi25 of Streptomyces thermoviolaceus OPC-520, were cloned and sequenced. The chi35 and chi25 genes were arranged in tandem and encoded deduced proteins of 39,762 and 28,734 Da, respectively. Alignment of the deduced amino acid sequences demonstrated that Chi35 has an N-terminal domain and a catalytic domain and that Chi25 is an enzyme consisting of only a catalytic domain. Amino acid sequences of the catalytic domains of both enzymes, which are highly similar to each other, suggested that these enzymes belong to the family 19 chitinases. The cloned Chi35 and Chi25 were purified from E. coli and S. lividans as a host, respectively. The optimum pH of Chi35 and Chi25 were 5-6, and the optimum temperature of Chi35 and Chi25 were 60 and 70 degrees C, respectively. Chi35 bound to chitin, Avicel, and xylan. On the other hand, Chi25 bound to these polysaccharides more weakly than did Chi35. These results indicate that the N-terminal domain of Chi35 functions as a polysaccharide-binding domain. Furthermore, Chi35 showed more efficient hydrolysis of insoluble chitin and stronger antifungal activity than Chi25. In the polysaccharide-binding domain of Chi35, there are three reiterated amino acid sequences starting from C-L-D and ending with W, and the repeats were similar to xylanase (STX-I) from the same strain. However, the repeats did not show sequence similarity to any of the known chitin-binding domains and cellulose-binding domains.     

Cloning, expression, and cell surface localization of Paenibacillus sp. strain W-61 xylanase 5, a multidomain xylanase

We have shown that a xylan-degrading bacterium, W-61, excretes multiple xylanases, including xylanase 5 with a molecular mass of 140 kDa. Here, we emend the previously used classification of the bacterium (i.e., Aeromonas caviae W-61) to Paenibacillus sp. strain W-61 on the basis of the nucleotide sequence of the 16S rRNA gene, and we clone and express the xyn5 gene encoding xylanase 5 (Xyn5) in Escherichia coli and study the subcellular localization of Xyn5. xyn5 encodes 1,326 amino acid residues, including a 27-amino-acid signal sequence. Sequence analysis indicated that Xyn5 comprises two family 22 carbohydrate-binding modules (CBM), a family 10 catalytic domain of glycosyl hydrolases, a family 9 CBM, a domain similar to the lysine-rich region of Clostridium thermocellum SdbA, and three S-layer-homologous (SLH) domains. Recombinant Xyn5 bound to a crystalline cellulose, Avicel PH-101, while an N-terminal 90-kDa fragment of Xyn5, which lacks the C-terminal half of the family 9 CBM, did not bind to Avicel PH-101. Xyn5 was cell bound, and the cell-bound protein was digested by exogenous trypsin to produce immunoreactive and xylanolytic fragments with molecular masses of 80 and 60 kDa. Xyn5 was exclusively distributed in the cell envelope fraction consisting of a peptidoglycan-containing layer and an associated S layer. Thus, Paenibacillus sp. strain W-61 Xyn5 is a cell surface-anchored modular xylanase possessing a functional cellulose-binding module and SLH domains. Possible cooperative action of multiple xylanases produced by strain W-61 is discussed on the basis of the modular structure of Xyn5.     

Nucleotide sequence and characteristics of endoglucanase gene engB from Clostridium cellulovorans

An endoglucanase gene, engB, from Clostridium cellulovorans, previously cloned into pUC19, has been further characterized and its product investigated. The enzyme, EngB, encoded by the gene was secreted into the periplasmic space of Escherichia coli. The enzyme was active against carboxymethylcellulose, xylan and lichenan but not Avicel (crystalline cellulose). The sequenced gene showed an open reading frame of 1323 base pairs and coded for a protein with a molecular mass of 48.6 kDa. The mRNA contained a typical Gram-positive ribosome-binding site sequence GGAGG and a sequence coding for a putative signal peptide. There is high amino acid and base sequence homology between the N-terminal regions of EngB and another C. cellulovorans endoglucanase, EngD, but they differ significantly in their C-termini. Deletion analyses revealed that up to 32 amino acids of the N-terminus and 52 amino acids of the C-terminus were not required for catalytic activity. The conserved reiterated domains at the C-terminus of EngB were similar to those from endoglucanases from other cellulytic bacteria. According to our deletion analyses, this region is not needed for catalytic activity.     

Paenibacillus curdlanolyticus strain B-6 xylanolytic-cellulolytic enzyme system that degrades insoluble polysaccharides

A facultatively anaerobic bacterium, Paenibacillus curdlanolyticus B-6, isolated from an anaerobic digester produces an extracellular xylanolytic-cellulolytic enzyme system containing xylanase, beta-xylosidase, arabinofuranosidase, acetyl esterase, mannanase, carboxymethyl cellulase (CMCase), avicelase, cellobiohydrolase, beta-glucosidase, amylase, and chitinase when grown on xylan under aerobic conditions. During growth on xylan, the bacterial cells were found to adhere to xylan from the early exponential growth phase to the late stationary growth phase. Scanning electron microscopic analysis revealed the adhesion of cells to xylan. The crude enzyme preparation was found to be capable of binding to insoluble xylan and Avicel. The xylanolytic-cellulolytic enzyme system efficiently hydrolyzed insoluble xylan, Avicel, and corn hulls to soluble sugars that were exclusively xylose and glucose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of a crude enzyme preparation exhibited at least 17 proteins, and zymograms revealed multiple xylanases and cellulases containing 12 xylanases and 9 CMCases. The cellulose-binding proteins, which are mainly in a multienzyme complex, were isolated from the crude enzyme preparation by affinity purification on cellulose. This showed nine proteins by SDS-PAGE and eight xylanases and six CMCases on zymograms. Sephacryl S-300 gel filtration showed that the cellulose-binding proteins consisted of two multienzyme complexes with molecular masses of 1,450 and 400 kDa. The results indicated that the xylanolytic-cellulolytic enzyme system of this bacterium exists as multienzyme complexes.     

Family 19 Chitinases from Streptomyces thermoviolaceus OPC-520: Molecular Cloning and Characterization

Family 19 chitinase genes, chi35 and chi25 of Streptomyces thermoviolaceus OPC-520, were cloned and sequenced. The chi35 and chi25 genes were arranged in tandem and encoded deduced proteins of 39,762 and 28,734 Da, respectively. Alignment of the deduced amino acid sequences demonstrated that Chi35 has an N-terminal domain and a catalytic domain and that Chi25 is an enzyme consisting of only a catalytic domain. Amino acid sequences of the catalytic domains of both enzymes, which are highly similar to each other, suggested that these enzymes belong to the family 19 chitinases. The cloned Chi35 and Chi25 were purified from E. coli and S. lividans as a host, respectively. The optimum pH of Chi35 and Chi25 were 5-6, and the optimum temperature of Chi35 and Chi25 were 60 and 70°C, respectively. Chi35 bound to chitin, Avicel, and xylan. On the other hand, Chi25 bound to these polysaccharides more weakly than did Chi35. These results indicate that the N-terminal domain of Chi35 functions as a polysaccharide-binding domain. Furthermore, Chi35 showed more efficient hydrolysis of insoluble chitin and stronger antifungal activity than Chi25. In the polysaccharide-binding domain of Chi35, there are three reiterated amino acid sequences starting from C-L-D and ending with W, and the repeats were similar to xylanase (STX-I) from the same strain. However, the repeats did not show sequence similarity to any of the known chitin-binding domains and cellulose-binding domains.     

Paenibacillus curdlanolyticus Strain B-6 Xylanolytic-Cellulolytic Enzyme System That Degrades Insoluble Polysaccharides

A facultatively anaerobic bacterium, Paenibacillus curdlanolyticus B-6, isolated from an anaerobic digester produces an extracellular xylanolytic-cellulolytic enzyme system containing xylanase, β-xylosidase, arabinofuranosidase, acetyl esterase, mannanase, carboxymethyl cellulase (CMCase), avicelase, cellobiohydrolase, β-glucosidase, amylase, and chitinase when grown on xylan under aerobic conditions. During growth on xylan, the bacterial cells were found to adhere to xylan from the early exponential growth phase to the late stationary growth phase. Scanning electron microscopic analysis revealed the adhesion of cells to xylan. The crude enzyme preparation was found to be capable of binding to insoluble xylan and Avicel. The xylanolytic-cellulolytic enzyme system efficiently hydrolyzed insoluble xylan, Avicel, and corn hulls to soluble sugars that were exclusively xylose and glucose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of a crude enzyme preparation exhibited at least 17 proteins, and zymograms revealed multiple xylanases and cellulases containing 12 xylanases and 9 CMCases. The cellulose-binding proteins, which are mainly in a multienzyme complex, were isolated from the crude enzyme preparation by affinity purification on cellulose. This showed nine proteins by SDS-PAGE and eight xylanases and six CMCases on zymograms. Sephacryl S-300 gel filtration showed that the cellulose-binding proteins consisted of two multienzyme complexes with molecular masses of 1,450 and 400 kDa. The results indicated that the xylanolytic-cellulolytic enzyme system of this bacterium exists as multienzyme complexes.     

Sequence of xynC and properties of XynC, a major component of the Clostridium thermocellum cellulosome.

The nucleotide sequence of the Clostridium thermocellum F1 xynC gene, which encodes the xylanase XynC, consists of 1,857 bp and encodes a protein of 619 amino acids with a molecular weight of 69,517. XynC contains a typical N-terminal signal peptide of 32 amino acid residues, followed by a 165-amino-acid sequence which is homologous to the thermostabilizing domain. Downstream of this domain was a family 10 catalytic domain of glycosyl hydrolase. The C terminus separated from the catalytic domain by a short linker sequence contains a dockerin domain responsible for cellulosome assembly. The N-terminal amino acid sequence of XynC-II, the enzyme purified from a recombinant Escherichia coli strain, was in agreement with that deduced from the nucleotide sequence although XynC-II suffered from proteolytic truncation by a host protease(s) at the C-terminal region. Immunological and N-terminal amino acid sequence analyses disclosed that the full-length XynC is one of the major components of the C. thermocellum cellulosome. XynC-II was highly active toward xylan and slightly active toward p-nitrophenyl-beta-D-xylopyranoside, p-nitrophenyl-beta-D-cellobioside, p-nitrophenyl-beta-D-glucopyranoside, and carboxymethyl cellulose. The Km and Vmax values for xylan were 3.9 mg/ml and 611 micromol/min/mg of protein, respectively. This enzyme was optimally active at 80 degrees C and was stable up to 70 degrees C at neutral pHs and over the pH range of 4 to 11 at 25 degrees C.     

The binding of Cellulomonas fimi endoglucanase C (CenC) to cellulose and Sephadex is mediated by the N-terminal repeats

Endoglucanase C (CenC) from Cellulomonas fimi binds to cellulose and to Sephadex. The enzyme has two contiguous 150-amino-acid repeats (N1 and N2) at its N-terminus and two unrelated contiguous 100-amino-acid repeats (C1 and C2) at its C-terminus. Polypeptides corresponding to N1, N1N2, C1, and C1C2 were produced by expression of appropriate cenC gene fragments in Escherichia coli. N1N2, but not N1 alone, binds to Sephadex; both polypeptides bind to Avicel, (a heterogeneous cellulose preparation containing both crystalline and non-crystalline components). Neither C1 nor C1C2 binds to Avicel or Sephadex. N1N2 and N1 bind to regenerated ('amorphous') cellulose but not to bacterial crystalline cellulose; the cellulose-binding domain of C. fimi exoglucanase Cex binds to both of these forms of cellulose. Amino acid sequence comparison reveals that N1 and N2 are distantly related to the cellulose-binding domains of Cex and C. fimi endoglucanases A and B.     

Importance of the carbohydrate-binding module of Clostridium stercorarium Xyn10B to xylan hydrolysis

The Clostridium stercorarium xylanase Xyn10B is a modular enzyme comprising two thermostabilizing domains, a family 10 catalytic domain of glycosyl hydrolases, a family 9 carbohydrate-binding module (CBM), and two S-layer homologous (SLH) domains [Biosci. Biotechnol. Biochem., 63, 1596-1604 (1999)]. To investigate the role of this CBM, we constructed two derivatives of Xyn10B and compared their hydrolytic activity toward xylan and some preparations of plant cell walls; Xyn10BdeltaCBM consists of a catalytic domain only, and Xyn10B-CBM comprises a catalytic domain and a CBM. Xyn10B-CBM bound to various insoluble polysaccharides including Avicel, acid-swollen cellulose, ball-milled chitin, Sephadex G-25, and amylose-resin. A cellulose binding assay in the presence of soluble saccharides suggested that the CBM of Xyn10B had an affinity for even monosaccharides such as glucose, galactose, xylose, mannose and ribose. Removal of the CBM from the enzyme negated its cellulose- and xylan-binding abilities and severely reduced its enzyme activity toward insoluble xylan and plant cell walls but not soluble xylan. These findings clearly indicated that the CBM of Xyn10B is important in the hydrolysis of insoluble xylan. This is the first report of a family 9 CBM with an affinity for insoluble xylan in addition to crystalline cellulose and the ability to increase hydrolytic activity toward insoluble xylan.     

Xylanase B and an arabinofuranosidase from Pseudomonas fluorescens subsp. cellulosa contain identical cellulose-binding domains and are encoded by adjacent genes.

The complete nucleotide sequence of the Pseudomonas fluorescens subsp. cellulosa xynB gene, encoding an endo-beta-1,4-xylanase (xylanase B; XYLB) has been determined. The structural gene consists of an open reading frame (ORF) of 1775 bp coding for a protein of Mr 61,000. A second ORF (xynC) of 1712 bp, which starts 148 bp downstream of xynB, encodes a protein, designated xylanase C (XYLC), of Mr 59,000. XYLB hydrolyses oat spelt xylan to xylobiose and xylose, whereas XYLC releases only arabinose from the same substrate. Thus XYLB is a typical xylanase and XYLC is an arabinofuranosidase. Both enzymes bind to crystalline cellulose (Avicel), but not to xylan. The nucleotide sequences between residues 114 and 931 of xynB and xynC were identical, as were amino acid residues 39-311 of XYLB and XYLC. This conserved sequence is reiterated elsewhere in the P. fluorescens subsp. cellulosa genome. Truncated derivatives of XYLB and XYLC, in which the conserved sequence had been deleted, retained catalytic activity, but did not exhibit cellulose binding. A hybrid gene in which the 5' end of xynC, encoding residues 1-110 of XYLC, was fused to the Escherichia coli pho A' gene (encodes mature alkaline phosphatase) directed the synthesis of a fusion protein which exhibited alkaline phosphatase activity and bound to cellulose.     

Cloning and sequence analysis of genes encoding xylanases and acetyl xylan esterase from Streptomyces thermoviolaceus OPC-520.

Three genes encoding two types of xylanases (STX-I and STX-II) and an acetyl xylan esterase (STX-III) from Streptomyces thermoviolaceus OPC-520 were cloned, and their DNA sequences were determined. The nucleotide sequences showed that genes stx-II and stx-III were clustered on the genome. The stx-I, stx-II, and stx-III genes encoded deduced proteins of 51, 35.2, and 34.3 kDa, respectively. STX-I and STX-II bound to both insoluble xylan and crystalline cellulose (Avicel). Alignment of the deduced amino acid sequences encoded by stx-I, stx-II, and stx-III demonstrated that the three enzymes contain two functional domains, a catalytic domain and a substrate-binding domain. The catalytic domains of STX-I and STX-II showed high sequence homology to several xylanases which belong to families F and G, respectively, and that of STX-III showed striking homology with an acetyl xylan esterase from S. lividans, nodulation proteins of Rhizobium sp., and chitin deacetylase of Mucor rouxii. In the C-terminal region of STX-I, there were three reiterated amino acid sequences starting from C-L-D, and the repeats were homologous to those found in xylanase A from S. lividans, coagulation factor G subunit alpha from the horseshoe crab, Rarobacter faecitabidus protease I, beta-1,3-glucanase from Oerskovia xanthineolytica, and the ricin B chain. However, the repeats did not show sequence similarity to any of the nine known families of cellulose-binding domains (CBDs). On the other hand, STX-II and STX-III contained identical family II CBDs in their C-terminal regions.     

A bifunctional xylanase encoded by the xynA gene of the rumen cellulolytic bacterium Ruminococcus flavefaciens 17 comprises two dissimilar domains linked by an asparagine/glutamine-rich sequence

The nucleotide sequence of the xynA gene of Ruminococcus flavefaciens 17 was determined and found to consist of a 2862bp open reading frame beginning with a TTG start codon. The predicted product, XYLA, consisted of distinct amino-terminal (A) and carboxy terminal (C) domains (248 amino acids, including a putative signal sequence, and 332 amino acids, respectively) linked by a repetitive sequence (B, 374 amino acids) extraordinarily rich in asparagine (45%) and glutamine (26%) residues. Domains A and C were shown to be capable of expressing xylanase activity independently of each other when suitably truncated derivatives of the xynA coding region were expressed as lacZ fusions. The activities associated with the two domains were shown to differ with respect to the average size of hydrolysis products formed from oat-spelt xylan, with domain C releasing relatively more xylose and domain A more xylo-oligosaccharides. The amino acid sequence of domain A of XYLA closely resembled that of the Bacillus pumilus xynA enzyme (45% identical residues). On the other hand domain C showed significant similarity (33% to 40% identical residues) to a different group of bacterial xylanases and exoglucanases exemplified by the Caldocellum saccharolyticum xynA and celB products. The xynA product is, therefore, a bifunctional enzyme having two dissimilar catalytic domains capable of acting on xylan.     

Evidence for a general role for high-affinity non-catalytic cellulose binding domains in microbial plant ceil wall hydroiases

Cellulases expressed by Cellulomonas fimi consist of a catalytic domain and a discrete non-catalytic cellulose-binding domain (CBD). To establish whether CBDs are common features of plant cell-wall hydroiases from C. fimi, the molecular architecture of xylanase D (XYLD) from this bacterium was investigated. The gene encoding XYLD, designated xynD, consisted of an open reading frame of 1936 bp encoding a protein of M_r 68000. The deduced primary sequence of XYLD was confirmed by the size (64kDa) and N-terminal sequence of the purified recombinant xylanase. Biochemical analysis of the purified enzyme revealed that XYLD is an endo-acting xylanase which displays no detectable activity against polysaccharides other than xylan. The predicted primary structure of XYLD comprised an /V-terminal signal peptide followed by a 190-residue domain that exhibited significant homology to Family-G xylanases. Truncated derivatives of xynD, encoding the W-terminal 193 amino acids of mature XYLD directed the synthesis of a functional xylanase, confirming that the 190-residue N-terminal sequence constitutes the catalytic domain. The remainder of the enzyme consisted of two approximately 90-residue domains, which exhibited extensive homology with each other, and limited sequence identity with CBDs from other polysaccharide hydrolases. Between the two putative CBDs is a 197-amino-acid sequence that exhibits substantial homology with Rhizobium NodB proteins. The four discrete domains in XYLD were separated by either threonine/prolineor novel glycine-rich linker regions. Although full-length XYLD adsorbed to cellulose, truncated derivatives of the enzyme lacking the C-terminal CBD hydrolysed xylan but did not bind to cellulose. Fusion of the C-terminal domain to glutathione-Stransferase generated hybrid proteins that bound to crystalline cellulose, but not to amorphous cellulose or xylan. The location of CBDs in a C. fimi xylanase indicates that domains of this type are not restricted to cellulases, but are widely distributed between hemicellutases also, and therefore play a pivotal role in the activity of the whole repertoire of plant cell-wall hydrolases. The role of the NodB homologue in XYLD is less certain.