scholarly journals The type II and X cellulose-binding domains of Pseudomonas xylanase A potentiate catalytic activity against complex substrates by a common mechanism

1999 ◽  
Vol 342 (2) ◽  
pp. 473-480 ◽  
Author(s):  
Jaitinder GILL ◽  
Jane E. RIXON ◽  
David N. BOLAM ◽  
Simon MCQUEEN-MASON ◽  
Peter J. SIMPSON ◽  
...  
Keyword(s):  
Microcrystalline Cellulose ◽  
Plant Cell Wall ◽  
Catalytic Domain ◽  
Wall Material ◽  
Common Mechanism ◽  
Type Ii ◽  
Binding Domains ◽  
Covalently Linked ◽  
Cellulose Binding ◽  
Internal Domain

Xylanase A (Pf Xyn10A), in common with several other Pseudomonas fluorescens subsp. cellulosa polysaccharidases, consists of a Type II cellulose-binding domain (CBD), a catalytic domain (Pf Xyn10ACD) and an internal domain that exhibits homology to Type X CBDs. The Type X CBD of Pf Xyn10A, expressed as a discrete entity (CBDX) or fused to the catalytic domain (Pf Xyn10A′), bound to amorphous and bacterial microcrystalline cellulose with a Ka of 2.5×105 M-1. CBDX exhibited no affinity for soluble forms of cellulose or cello-oligosaccharides, suggesting that the domain interacts with multiple cellulose chains in the insoluble forms of the polysaccharide. Pf Xyn10A′ was 2-3 times more active against cellulose-hemicellulose complexes than Pf Xyn10ACD; however, Pf Xyn10A′ and Pf Xyn10ACD exhibited the same activity against soluble substrates. CBDX did not disrupt the structure of plant-cell-wall material or bacterial microcrystalline cellulose, and did not potentiate Pf Xyn10ACD when not covalently linked to the enzyme. There was no substantial difference in the affinity of full-length Pf Xyn10A and the enzyme's Type II CBD for cellulose. The activity of Pf Xyn10A against cellulose-hemicellulose complexes was similar to that of Pf Xyn10A′, and a derivative of Pf Xyn10A in which the Type II CBD is linked to the Pf Xyn10ACD via a serine-rich linker sequence [Bolam, Cireula, McQueen-Mason, Simpson, Williamson, Rixon, Boraston, Hazlewood and Gilbert (1998) Biochem J. 331, 775-781]. These data indicate that CBDX is functional in Pf Xyn10A and that no synergy, either in ligand binding or in the potentiation of catalysis, is evident between the Type II and X CBDs of the xylanase.

scholarly journals Pseudomonas cellulose-binding domains mediate their effects by increasing enzyme substrate proximity

1998 ◽  
Vol 331 (3) ◽  
pp. 775-781 ◽  
Author(s):  
David N. BOLAM ◽  
Antonio CIRUELA ◽  
Simon McQUEEN-MASON ◽  
Peter SIMPSON ◽  
Michael P. WILLIAMSON ◽  
...  
Keyword(s):  
Plant Cell ◽  
Mode Of Action ◽  
Plant Cell Wall ◽  
Catalytic Domain ◽  
Cellulase Activity ◽  
Type Ii ◽  
Binding Domains ◽  
Enzyme Substrate ◽  
Tryptophan Residues ◽  
Cellulose Binding

To investigate the mode of action of cellulose-binding domains (CBDs), the Type II CBD from Pseudomonas fluorescenssubsp. cellulosaxylanase A (XYLACBD) and cellulase E (CELECBD) were expressed as individual entities or fused to the catalytic domain of a Clostridium thermocellumendoglucanase (EGE). The two CBDs exhibited similar Ka values for bacterial microcrystalline cellulose (CELECBD, 1.62×106 M-1; XYLACBD, 1.83×106 M-1) and acid-swollen cellulose (CELECBD, 1.66×106 M-1; XYLACBD, 1.73×106 M-1). NMR spectra of XYLACBD titrated with cello-oligosaccharides showed that the environment of three tryptophan residues was affected when the CBD bound cellohexaose, cellopentaose or cellotetraose. The Ka values of the XYLACBD for C6, C5 and C4 cello-oligosaccharides were estimated to be 3.3×102, 1.4×102 and 4.0×101 M-1 respectively, suggesting that the CBD can accommodate at least six glucose molecules and has a much higher affinity for insoluble cellulose than soluble oligosaccharides. Fusion of either the CELECBD or XYLACBD to the catalytic domain of EGE potentiated the activity of the enzyme against insoluble forms of cellulose but not against carboxymethylcellulose. The increase in cellulase activity was not observed when the CBDs were incubated with the catalytic domain of either EGE or XYLA, with insoluble cellulose and a cellulose/hemicellulose complex respectively as the substrates. PseudomonasCBDs did not induce the extension of isolated plant cell walls nor weaken cellulose paper strips in the same way as a class of plant cell wall proteins called expansins. The XYLACBD and CELECBD did not release small particles from the surface of cotton. The significance of these results in relation to the mode of action of Type II CBDs is discussed.

scholarly journals Evidence that linker sequences and cellulose-binding domains enhance the activity of hemicellulases against complex substrates

1996 ◽  
Vol 319 (2) ◽  
pp. 515-520 ◽  
Author(s):  
Gary W BLACK ◽  
Jane E RIXON ◽  
Jonathan H. CLARKE ◽  
Geoffrey P. HAZLEWOOD ◽  
Michael K. THEODOROU ◽  
...  
Keyword(s):  
Cell Wall ◽  
Plant Cell ◽  
Plant Cell Wall ◽  
Full Length ◽  
Wall Material ◽  
Catalytic Domains ◽  
Binding Domains ◽  
Cellulose Binding ◽  
Derivatives Of

Xylanase A (XYLA) and arabinofuranosidase C (XYLC) from Pseudomonas fluorescens subsp. cellulosa are modular enzymes consisting of discrete cellulose-binding domains (CBDs) and catalytic domains joined by serine-rich linker sequences. To evaluate the role of the CBDs and interdomain regions, the capacity of full-length and truncated derivatives of the two enzymes, lacking either the linker sequences or CBDs, to hydrolyse a range of substrates, and bind to cellulose, was determined. Removal of the CBDs did not affect either the activity of XYLA or XYLC against soluble arabinoxylan. Similarly, deletion of the linker sequences did not alter the affinity of the enzymes for cellulose or their activity against soluble substrates, even when bound to cellulose via the CBDs. Truncated derivatives of XYLA lacking either the linker sequences or the CBD were less active against xylan contained in cellulose-hemicellulose complexes, compared with the full-length xylanase. Similarly, removal of the CBD from XYLC diminished the activity of the enzyme (XYLC´´´) against plant-cell-wall material containing highly substituted arabinoxylan. The role of CBDs and linker sequences in the catalytic activity of hemicellulases against the plant cell wall is discussed.

scholarly journals The type II and X cellulose-binding domains of Pseudomonas xylanase A potentiate catalytic activity against complex substrates by a common mechanism

1999 ◽  
Vol 342 (2) ◽  
pp. 473 ◽  
Author(s):  
Jaitinder GILL ◽  
Jane E. RIXON ◽  
David N. BOLAM ◽  
Simon MCQUEEN-MASON ◽  
Peter J. SIMPSON ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Common Mechanism ◽  
Type Ii ◽  
Binding Domains ◽  
Cellulose Binding

scholarly journals Homologous xylanases from Clostridium thermocellum: evidence for bi-functional activity, synergism between xylanase catalytic modules and the presence of xylan-binding domains in enzyme complexes

1999 ◽  
Vol 342 (1) ◽  
pp. 105-110 ◽  
Author(s):  
Ana C. FERNANDES ◽  
Carlos M. G. A. FONTES ◽  
Harry J. GILBERT ◽  
Geoffrey P. HAZLEWOOD ◽  
Tito H. FERNANDES ◽  
...  
Keyword(s):  
Clostridium Thermocellum ◽  
Plant Cell Wall ◽  
Catalytic Domain ◽  
Glycosyl Hydrolase ◽  
Biochemical Properties ◽  
Binding Domain ◽  
Binding Domains ◽  
A Cell ◽  
Cellulose Binding ◽  
Hydrolysis Of

Clostridium thermocellum produces a consortium of plant-cell-wall hydrolases that form a cell-bound multi-enzyme complex called the cellulosome. In the present study two similar xylanase genes, xynU and xynV, were cloned from C. thermocellum strain YS and sequenced. The deduced primary structures of both xylanases, xylanase U (XylU) and xylanase V (XylV), were homologous with the previously characterized xylanases from C. thermocellum strain F1. Truncated derivatives of XylV were produced and their biochemical properties were characterized. The xylanases were shown to be remarkably thermostable and resistant to proteolytic inactivation. The catalytic domains hydrolysed xylan by a typical endo-mode of action. The type VI cellulose-binding domain (CBD) homologue of XylV bound xylan and, to a smaller extent, Avicel and acid-swollen cellulose. Deletion of the CBD from XylV abolished the capacity of the enzymes to bind polysaccharides. The polysaccharide-binding domain was shown to have a key role in the hydrolysis of insoluble substrates by XylV. The C-terminal domain of XylV, which is absent from XylU, removed acetyl groups from acetylated xylan and acted in synergy with the glycosyl hydrolase catalytic domain of the enzyme to elicit the hydrolysis of acetylated xylan.

scholarly journals Characterization of hybrid proteins consisting of the catalytic domains of Clostridium and Ruminococcus endoglucanases, fused to Pseudomonas non-catalytic cellulose-binding domains

1991 ◽  
Vol 279 (3) ◽  
pp. 787-792 ◽  
Author(s):  
D M Poole ◽  
A J Durrant ◽  
G P Hazlewood ◽  
H J Gilbert
Keyword(s):  
Catalytic Domain ◽  
Binding Capacity ◽  
Binding Domains ◽  
Hybrid Proteins ◽  
C Terminus ◽  
N Terminus ◽  
Fusion Enzymes ◽  
Cellulose Binding

The N-terminal 160 or 267 residues of xylanase A from Pseudomonas fluorescens subsp. cellulosa, containing a non-catalytic cellulose-binding domain (CBD), were fused to the N-terminus of the catalytic domain of endoglucanase E (EGE') from Clostridium thermocellum. A further hybrid enzyme was constructed consisting of the 347 N-terminal residues of xylanase C (XYLC) from P. fluorescens subsp. cellulosa, which also constitutes a CBD, fused to the N-terminus of endoglucanase A (EGA) from Ruminococcus albus. The three hybrid enzymes bound to insoluble cellulose, and could be eluted such that cellulose-binding capacity and catalytic activity were retained. The catalytic properties of the fusion enzymes were similar to EGE' and EGA respectively. Residues 37-347 and 34-347 of XYLC were fused to the C-terminus of EGE' and the 10 amino acids encoded by the multiple cloning sequence of pMTL22p respectively. The two hybrid proteins did not bind cellulose, although residues 39-139 of XYLC were shown previously to constitute a functional CBD. The putative role of the P. fluorescens subsp. cellulosa CBD in cellulase action is discussed.

scholarly journals Functional Analysis of the Carbohydrate-Binding Domains of Erwinia chrysanthemi Cel5 (Endoglucanase Z) and an Escherichia coli Putative Chitinase

1999 ◽  
Vol 181 (15) ◽  
pp. 4611-4616 ◽  
Author(s):  
Helen D. Simpson ◽  
Frederic Barras
Keyword(s):  
Escherichia Coli ◽  
Catalytic Domain ◽  
Three Dimensional ◽  
Site Directed Mutagenesis ◽  
Erwinia Chrysanthemi ◽  
Dimensional Structure ◽  
Carbohydrate Binding ◽  
Binding Domains ◽  
Cellulose Binding

ABSTRACT The Cel5 cellulase (formerly known as endoglucanase Z) fromErwinia chrysanthemi is a multidomain enzyme consisting of a catalytic domain, a linker region, and a cellulose binding domain (CBD). A three-dimensional structure of the CBDCel5 has previously been obtained by nuclear magnetic resonance. In order to define the role of individual residues in cellulose binding, site-directed mutagenesis was performed. The role of three aromatic residues (Trp18, Trp43, and Tyr44) in cellulose binding was demonstrated. The exposed potential hydrogen bond donors, residues Gln22 and Glu27, appeared not to play a role in cellulose binding, whereas residue Asp17 was found to be important for the stability of Cel5. A deletion mutant lacking the residues Asp17 to Pro23 bound only weakly to cellulose. The sequence of CBDCel5 exhibits homology to a series of five repeating domains of a putative large protein, referred to as Yheb, from Escherichia coli. One of the repeating domains (Yheb1), consisting of 67 amino acids, was cloned from the E. coli chromosome and purified by metal chelating chromatography. While CBDCel5 bound to both cellulose and chitin, Yheb1 bound well to chitin, but only very poorly to cellulose. The Yheb protein contains a region that exhibits sequence homology with the catalytic domain of a chitinase, which is consistent with the hypothesis that the Yheb protein is a chitinase.

scholarly journals Cel9M, a New Family 9 Cellulase of the Clostridium cellulolyticum Cellulosome

2002 ◽  
Vol 184 (5) ◽  
pp. 1378-1384 ◽  
Author(s):  
Anne Belaich ◽  
Goetz Parsiegla ◽  
Laurent Gal ◽  
Claude Villard ◽  
Richard Haser ◽  
...  
Keyword(s):  
Microcrystalline Cellulose ◽  
Carboxymethyl Cellulose ◽  
Catalytic Domain ◽  
Soluble Sugar ◽  
Significant Activity ◽  
Cellulose Binding Domain ◽  
Clostridium Cellulolyticum ◽  
New Family ◽  
Cellulose Binding ◽  
Hydrolysis Of

ABSTRACT A new cellulosomal protein from Clostridium cellulolyticum Cel9M was characterized. The protein contains a catalytic domain belonging to family 9 and a dockerin domain. Cel9M is active on carboxymethyl cellulose, and the hydrolysis of this substrate is accompanied by a decrease in viscosity. Cel9M has a slight, albeit significant, activity on both Avicel and bacterial microcrystalline cellulose, and the main soluble sugar released is cellotetraose. Saccharification of bacterial microcrystalline cellulose by Cel9M in association with two other family 9 enzymes from C. cellulolyticum, namely, Cel9E and Cel9G, was measured, and it was found that Cel9M acts synergistically with Cel9E. Complexation of Cel9M with the mini-CipC1 containing the cellulose binding domain, the X2 domain, and the first cohesin domain of the scaffoldin CipC of the bacterium did not significantly increase the hydrolysis of Avicel and bacterial microcrystalline cellulose.

scholarly journals Adaptor Scaffoldins: An Original Strategy for Extended Designer Cellulosomes, Inspired from Nature

mBio ◽  
2016 ◽  
Vol 7 (2) ◽  
Author(s):  
Johanna Stern ◽  
Sarah Moraïs ◽  
Raphael Lamed ◽  
Edward A. Bayer
Keyword(s):  
Cell Wall ◽  
Plant Cell ◽  
Plant Cell Wall ◽  
Soluble Sugars ◽  
Single Type ◽  
Wall Material ◽  
Type I ◽  
Type Ii ◽  
Recombinant Enzymes ◽  
Designer Cellulosome

ABSTRACTDesigner cellulosomes consist of chimeric cohesin-bearing scaffoldins for the controlled incorporation of recombinant dockerin-containing enzymes. The largest designer cellulosome reported to date is a chimeric scaffoldin that contains 6 cohesins. This scaffoldin represented a technical limit of sorts, since adding another cohesin proved problematic, owing to resultant low expression levels, instability (cleavage) of the scaffoldin polypeptide, and limited numbers of available cohesin-dockerin specificities—the hallmark of designer cellulosomes. Nevertheless, increasing the number of enzymes integrated into designer cellulosomes is critical, in order to further enhance degradation of plant cell wall material. Adaptor scaffoldins comprise an intermediate type of scaffoldin that can both incorporate various enzymes and attach to an additional scaffoldin. Using this strategy, we constructed an efficient form of adaptor scaffoldin that possesses three type I cohesins for enzyme integration, a single type II dockerin for interaction with an additional scaffoldin, and a carbohydrate-binding module for targeting to the cellulosic substrate. In parallel, we designed a hexavalent scaffoldin capable of connecting to the adaptor scaffoldin by the incorporation of an appropriate type II cohesin. The resultant extended designer cellulosome comprised 8 recombinant enzymes—4 xylanases and 4 cellulases—thereby representing a potent enzymatic cocktail for solubilization of natural lignocellulosic substrates. The contribution of the adaptor scaffoldin clearly demonstrated that proximity between the two scaffoldins and their composite set of enzymes is crucial for optimized degradation. After 72 h of incubation, the performance of the extended designer cellulosome was determined to be approximately 70% compared to that of native cellulosomes.IMPORTANCEPlant cell wall residues represent a major source of renewable biomass for the production of biofuels such as ethanol via breakdown to soluble sugars. The natural microbial degradation process, however, is inefficient for achieving cost-effective processes in the conversion of plant-derived biomass to biofuels, either from dedicated crops or human-generated cellulosic wastes. The accumulation of the latter is considered a major environmental pollutant. The development of designer cellulosome nanodevices for enhanced plant cell wall degradation thus has major impacts in the fields of environmental pollution, bioenergy production, and biotechnology in general. The findings reported in this article comprise a true breakthrough in our capacity to produce extended designer cellulosomes via synthetic biology means, thus enabling the assembly of higher-order complexes that can supersede the number of enzymes included in a single multienzyme complex.

scholarly journals Characterization of a Novel, Antifungal, Chitin-Binding Protein from Streptomyces tendae Tü901 That Interferes with Growth Polarity

1999 ◽  
Vol 181 (24) ◽  
pp. 7421-7429 ◽  
Author(s):  
Christiane Bormann ◽  
Daniel Baier ◽  
Ingmar Hörr ◽  
Claudia Raps ◽  
Jürgen Berger ◽  
...  
Keyword(s):  
Antifungal Activity ◽  
Plant Cell Wall ◽  
Binding Protein ◽  
Antifungal Protein ◽  
Multicopy Plasmid ◽  
Binding Domains ◽  
Streptomyces Tendae ◽  
Chitin Binding Protein ◽  
Cellulose Binding

ABSTRACT The afp1 gene, which encodes the antifungal protein AFP1, was cloned from nikkomycin-producing Streptomyces tendae Tü901, using a nikkomycin-negative mutant as a host and screening transformants for antifungal activity againstPaecilomyces variotii in agar diffusion assays. The 384-bpafp1 gene has a low G+C content (63%) and a transcription termination structure with a poly(T) region, unusual attributes forStreptomyces genes. AFP1 was purified from culture filtrate of S. tendae carrying the afp1 gene on the multicopy plasmid pIJ699. The purified protein had a molecular mass of 9,862 Da and lacked a 42-residue N-terminal peptide deduced from the nucleotide sequence. AFP1 was stable at extreme pH values and high temperatures and toward commercial proteinases. AFP1 had limited similarity to cellulose-binding domains of microbial plant cell wall hydrolases and bound to crab shell chitin, chitosan, and cell walls ofP. variotii but showed no enzyme activity. The biological activity of AFP1, which represents the first chitin-binding protein from bacteria exhibiting antifungal activity, was directed against specific ascomycetes, and synergistic interaction with the chitin synthetase inhibitor nikkomycin inhibited growth ofAspergillus species. Microscopy studies revealed that fluorescein-labeled AFP1 strongly bound to the surface of germinated conidia and to tips of growing hyphae, causing severe alterations in cell morphogenesis that gave rise to large spherical conidia and/or swollen hyphae and to atypical branching.

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