scholarly journals Cloning and DNA Sequencing of the Genes EncodingClostridium josui Scaffolding Protein CipA and Cellulase CelD and Identification of Their Gene Products as Major Components of the Cellulosome

1998 ◽  
Vol 180 (16) ◽  
pp. 4303-4308 ◽  
Author(s):  
Motohide Kakiuchi ◽  
Ayako Isui ◽  
Katsuhisa Suzuki ◽  
Tsuchiyoshi Fujino ◽  
Emi Fujino ◽  
...  
Keyword(s):  
Signal Peptide ◽  
Catalytic Domain ◽  
Glycosyl Hydrolase ◽  
Scaffolding Protein ◽  
Scaffolding Proteins ◽  
Molecular Architecture ◽  
Amino Acid Sequence Analysis ◽  
Genes Encoding ◽  
Terminal Amino ◽  
Cellulose Binding

ABSTRACT The Clostridium josui cipA and celD genes, encoding a scaffolding-like protein (CipA) and a putative cellulase (CelD), respectively, have been cloned and sequenced. CipA, with an estimated molecular weight of 120,227, consists of an N-terminal signal peptide, a cellulose-binding domain of family III, and six successive cohesin domains. The molecular architecture of C. josuiCipA is similar to those of the scaffolding proteins reported so far, such as Clostridium thermocellum CipA, Clostridium cellulovorans CbpA, and Clostridium cellulolyticumCipC, but C. josui CipA is considerably smaller than the other scaffolding proteins. CelD consists of an N-terminal signal peptide, a family 48 catalytic domain of glycosyl hydrolase, and a dockerin domain. N-terminal amino acid sequence analysis of theC. josui cellulosomal proteins indicates that both CipA and CelD are major components of the cellulosome.

scholarly journals Molecular characterization of hypothetical scaffolding-like protein S1 in multienzyme complex produced by Paenibacillus curdlanolyticus B-6

AMB Express ◽  
2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Patthra Pason ◽  
Junjarus Sermsathanaswadi ◽  
Rattiya Waeonukul ◽  
Chakrit Tachaapaikoon ◽  
Sirilak Baramee ◽  
...  
Keyword(s):  
Glycosyl Hydrolase ◽  
Xylanase Activity ◽  
Scaffolding Protein ◽  
Scaffolding Proteins ◽  
Multienzyme Complex ◽  
Amino Acid Residues ◽  
Carbohydrate Polymers ◽  
Enzyme Subunits ◽  
Paenibacillus Curdlanolyticus ◽  
Cellulose Binding

Abstract Paenibacillus curdlanolyticus B-6 produces an extracellular multienzyme complex containing a hypothetical scaffolding-like protein and several xylanases and cellulases. The largest (280-kDa) component protein, called S1, has cellulose-binding ability and xylanase activity, thus was considered to function like the scaffolding proteins found in cellulosomes. S1 consists of 863 amino acid residues with predicted molecular mass 91,029 Da and includes two N-terminal surface layer homology (SLH) domains, but most of its sequence shows no homology with proteins of known function. Native S1 (nS1) was highly glycosylated. Purified nS1 and recombinant Xyn11A (rXyn11A) as a major xylanase subunit could assemble in a complex, but recombinant S1 (rS1) could not interact with rXyn11A, indicating that S1 glycosylation is necessary for assembly of the multienzyme complex. nS1 and rS1 showed weak, typical endo-xylanase activity, even though they have no homology with known glycosyl hydrolase family enzymes. S1 and its SLH domains bound tightly to the peptide-glycan layer of P. curdlanolyticus B-6, microcrystalline cellulose, and insoluble xylan, indicating that the SLHs of S1 bind to carbohydrate polymers and the cell surface. When nS1 and rXyn11A were co-incubated with birchwood xylan, the degradation ability was synergistically increased compared with that for each protein; however synergy was not observed for rS1 and rXynA. These results indicate that S1 may have a scaffolding protein-like function by interaction with enzyme subunits and polysaccharides through its glycosylated sites and SLH domains.

scholarly journals Cloning and Sequence Analysis of a New Cellulase Gene Encoding CelK, a Major Cellulosome Component of Clostridium thermocellum: Evidence for Gene Duplication and Recombination

1999 ◽  
Vol 181 (17) ◽  
pp. 5288-5295 ◽  
Author(s):  
Irina Kataeva ◽  
Xin-Liang Li ◽  
Huizhong Chen ◽  
Sang-Ki Choi ◽  
Lars G. Ljungdahl
Keyword(s):  
Amino Acid ◽  
Gene Duplication ◽  
Signal Peptide ◽  
Clostridium Thermocellum ◽  
Catalytic Domain ◽  
Glycosyl Hydrolase ◽  
Size Difference ◽  
Amino Acid Residues ◽  
Cellulase Gene ◽  
Original Activity

ABSTRACT The cellulolytic and hemicellulolytic complex of Clostridium thermocellum, termed cellulosome, consists of up to 26 polypeptides, of which at least 17 have been sequenced. They include 12 cellulases, 3 xylanases, 1 lichenase, and CipA, a scaffolding polypeptide. We report here a new cellulase gene, celK, coding for CelK, a 98-kDa major component of the cellulosome. The gene has an open reading frame (ORF) of 2,685 nucleotides coding for a polypeptide of 895 amino acid residues with a calculated mass of 100,552 Da. A signal peptide of 27 amino acid residues is cut off during secretion, resulting in a mature enzyme of 97,572 Da. The nucleotide sequence is highly similar to that of cbhA(V. V. Zverlov et al., J. Bacteriol. 180:3091–3099, 1998), having an ORF of 3,690 bp coding for the 1,230-amino-acid-residue CbhA of the same bacterium. Homologous regions of the two genes are 86.5 and 84.3% identical without deletion or insertion on the nucleotide and amino acid levels, respectively. Both have domain structures consisting of a signal peptide, a family IV cellulose binding domain (CBD), a family 9 glycosyl hydrolase domain, and a dockerin domain. A striking distinction between the two polypeptides is that there is a 330-amino-acid insertion in CbhA between the catalytic domain and the dockerin domain containing a fibronectin type 3-like domain and family III CBD. This insertion, missing in CelK, is responsible for the size difference between CelK and CbhA. Upstream and downstream flanking sequences of the two genes show no homology. The data indicate thatcelK and cbhA in the genome of C. thermocellum have evolved through gene duplication and recombination of domain coding sequences. celK without a dockerin domain was expressed in Escherichia coli and purified. The enzyme had pH and temperature optima at 6.0 and 65°C, respectively. It hydrolyzedp-nitrophenyl-β-d-cellobioside with aKm and a V max of 1.67 μM and 15.1 U/mg, respectively. Cellobiose was a strong inhibitor of CelK activity, with a Ki of 0.29 mM. The enzyme was thermostable, after 200 h of incubation at 60°C, 97% of the original activity remained. Properties of the enzyme indicated that it is a cellobiohydrolase.

scholarly journals Cell-Surface-Anchoring Role of N-Terminal Surface Layer Homology Domains of Clostridium cellulovorans EngE

2002 ◽  
Vol 184 (4) ◽  
pp. 884-888 ◽  
Author(s):  
Akihiko Kosugi ◽  
Koichiro Murashima ◽  
Yutaka Tamaru ◽  
Roy H. Doi
Keyword(s):  
Cell Wall ◽  
Surface Layer ◽  
Cell Surface ◽  
Catalytic Domain ◽  
Glycosyl Hydrolase ◽  
Sodium Dodecyl ◽  
Scaffolding Protein ◽  
Clostridium Cellulovorans ◽  
Surface Anchoring

ABSTRACT engE, coding for endoglucanase E, one of the three major subunits of the Clostridium cellulovorans cellulosome, has been cloned and sequenced (Y. Tamaru and R. H. Doi, J. Bacteriol. 181:3270-3276, 1999). The N-terminal-half region of EngE possesses three repeated surface layer homology (SLH) domains, which are homologous to those of some bacterial S-layer proteins. Also, the C-terminal-half region consists of a catalytic domain of glycosyl hydrolase family 5 and a duplicated sequence (dockerin) for binding EngE to scaffolding protein CbpA. Our hypothesis is that the SLH domains serve in the role of anchoring to the cell surface. This model was investigated by using recombinant EngEs (rEngE) with and without SLH domains that were synthesized in Escherichia coli and cell wall preparations from C. cellulovorans. When rEngE and SLH polypeptides of EngE were incubated with cell wall fragments prepared by sodium dodecyl sulfate treatment, these proteins bound strongly to the cell wall. However, rEngEs without SLH domains lost their ability to bind to cell walls. When rEngE was incubated with mini-CbpA, consisting of two cohesin domains, and cell wall fragments, the mini-CbpA was able to bind to the cell wall with rEngE. However, the binding of mini-CbpA was dramatically inhibited by addition of a chelating reagent, such as EDTA, which prevents cohesin-dockerin interactions. These results suggest not only that the SLH domains of EngE can bind to the cell surface but also that EngE plays an anchoring role for cellulosomes through the interaction of its dockerin domain with a CbpA cohesin.

scholarly journals The non-catalytic cellulose-binding domain of a novel cellulase from Pseudomonas fluorescens subsp. cellulosa is important for the efficient hydrolysis of Avicel

1995 ◽  
Vol 309 (3) ◽  
pp. 749-756 ◽  
Author(s):  
J Hall ◽  
G W Black ◽  
L M A Ferreira ◽  
S J Millward-Sadler ◽  
B R S Ali ◽  
...  
Keyword(s):  
Pseudomonas Fluorescens ◽  
Primary Structure ◽  
Catalytic Domain ◽  
Genomic Library ◽  
Glycosyl Hydrolase ◽  
Cellulase Activity ◽  
Full Length ◽  
Type I ◽  
Catalytic Domains ◽  
Cellulose Binding

A genomic library of Pseudomonas fluorescens subsp. cellulosa DNA, constructed in lambda ZAPII, was screened for carboxymethyl-cellulase activity. The pseudomonad insert from a recombinant phage which displayed elevated cellulase activity in comparison with other cellulase-positive clones present in the library, was excised into pBluescript SK- to generate the plasmid pC48. The nucleotide sequence of the cellulase gene, designated celE, revealed a single open reading frame of 1710 bp that encoded a polypeptide, defined as endoglucanase E (CelE), of M(r) 59663. The deduced primary structure of CelE revealed an N-terminal signal peptide followed by a 300-amino-acid sequence that exhibited significant identity with the catalytic domains of cellulases belonging to glycosyl hydrolase Family 5. Adjacent to the catalytic domain was a 40-residue region that exhibited strong sequence identity to non-catalytic domains located in two other endoglucanases and a xylanase from P. fluorescens. The C-terminal 100 residues of CelE were similar to Type-I cellulose-binding domains (CBDs). The three domains of the cellulase were joined by linker sequences rich in serine residues. Analysis of the biochemical properties of full-length and truncated derivatives of CelE confirmed that the enzyme comprised an N-terminal catalytic domain and a C-terminal CBD. Analysis of purified CelE revealed that the enzyme had an M(r) of 56000 and an experimentally determined N-terminal sequence identical to residues 40-54 of the deduced primary structure of full-length CelE. The enzyme exhibited an endo mode of action in hydrolysing a range of cellulosic substrates including Avicel and acid-swollen cellulose, but did not attack xylan or any other hemicelluloses. A truncated form of the enzyme, which lacked the C-terminal CBD, displayed the same activity as full-length CelE against soluble cellulose and acid-swollen cellulose, but exhibited substantially lower activity than the full-length cellulase against Avicel. The significance of these data in relation to the role of the CBD is discussed.

scholarly journals Growth of Hyperthermophilic Archaeon Pyrococcus furiosus on Chitin Involves Two Family 18 Chitinases

2003 ◽  
Vol 69 (6) ◽  
pp. 3119-3128 ◽  
Author(s):  
Jun Gao ◽  
Michael W. Bauer ◽  
Keith R. Shockley ◽  
Marybeth A. Pysz ◽  
Robert M. Kelly
Keyword(s):  
Signal Peptide ◽  
Catalytic Domain ◽  
Pyrococcus Furiosus ◽  
Sequence Similarity ◽  
Glycosyl Hydrolase ◽  
Substrate Inhibition ◽  
Binding Domain ◽  
Synergistic Activity ◽  
Carbohydrate Source ◽  
Hyperthermophilic Archaeon

ABSTRACT Pyrococcus furiosus was found to grow on chitin, adding this polysacharide to the inventory of carbohydrates utilized by this hyperthermophilic archaeon. Accordingly, two open reading frames (chiA [Pf1234] and chiB [Pf1233]) were identified in the genome of P. furiosus, which encodes chitinases with sequence similarity to proteins from the glycosyl hydrolase family 18 in less-thermophilic organisms. Both enzymes contain multiple domains that consist of at least one binding domain and one catalytic domain. ChiA (ca. 39 kDa) contains a putative signal peptide, as well as a binding domain (ChiABD), that is related to binding domains associated with several previously studied bacterial chitinases. chiB, separated by 37 nucleotides from chiA and in the same orientation, encodes a polypeptide with two different proline-threonine-rich linker regions (6 and 3 kDa) flanking a chitin-binding domain (ChiBBD [11 kDa]), followed by a catalytic domain (ChiBcat [35 kDa]). No apparent signal peptide is encoded within chiB. The two chitinases share little sequence homology to each other, except in the catalytic region, where both have the catalytic glutamic acid residue that is conserved in all family 18 bacterial chitinases. The genes encoding ChiA, without its signal peptide, and ChiB were cloned and expressed in Escherichia coli. ChiA exhibited no detectable activity toward chitooligomers smaller than chitotetraose, indicating that the enzyme is an endochitinase. Kinetic studies showed that ChiB followed Michaelis-Menten kinetics toward chitotriose, although substrate inhibition was observed for larger chitooligomers. Hydrolysis patterns on chitooligosaccharides indicated that ChiB is a chitobiosidase, processively cleaving off chitobiose from the nonreducing end of chitin or other chitooligomers. Synergistic activity was noted for the two chitinases on colloidal chitin, indicating that these two enzymes work together to recruit chitin-based substrates for P. furiosus growth. This was supported by the observed growth on chitin as the sole carbohydrate source in sulfur-free media.

scholarly journals Functional Characterization of Two Cellulase Genes in the Gram-Positive Pathogenic Bacterium Clavibacter michiganensis for Wilting in Tomato

2019 ◽  
Vol 32 (4) ◽  
pp. 491-501 ◽  
Author(s):  
In Sun Hwang ◽  
Eom-Ji Oh ◽  
Han Beoyl Lee ◽  
Chang-Sik Oh
Keyword(s):  
Signal Peptide ◽  
Plant Pathogens ◽  
Catalytic Domain ◽  
Functional Characterization ◽  
Cellulase Activity ◽  
Cellulase Gene ◽  
Clavibacter Michiganensis ◽  
Gram Positive ◽  
Almost All ◽  
Cellulose Binding

Diverse plant pathogens secrete cellulases to degrade plant cell walls. Previously, the plasmid-borne cellulase gene celA was shown to be important for the virulence of the gram-positive bacterium Clavibacter michiganensis in tomato. However, details of the contribution of cellulases to the development of wilting in tomato have not been well-determined. To better understand the contribution of cellulases to the virulence of C. michiganensis in tomato, a mutant lacking cellulase activity was generated and complemented with truncated forms of certain cellulase genes, and virulence of those strain was examined. A celA mutant of the C. michiganensis type strain LMG7333 lost its cellulase activity and almost all its ability to cause wilting in tomato. The cellulase catalytic domain and cellulose-binding domain of CelA together were sufficient for both cellulase activity and the development of wilting in tomato. However, the expansin domain did not affect virulence or cellulase activity. The celA ortholog of Clavibacter sepedonicus restored the full virulence of the celA mutant of C. michiganensis. Another cellulase gene, celB, located in the chromosome, carries a single-base deletion in most C. michiganensis strains but does not carry a functional signal peptide in its N terminus. Nevertheless, an experimentally modified CelB protein with a CelA signal peptide was secreted and able to cause wilting in tomato. These results indicate that cellulases are major virulence factors of C. michiganensis that causes wilting in tomato. Furthermore, there are natural variations among cellulase genes directly affecting their function.

scholarly journals Expression, purification, crystallization and preliminary X-ray analysis of CttA, a putative cellulose-binding protein fromRuminococcus flavefaciens

2015 ◽  
Vol 71 (6) ◽  
pp. 784-789 ◽  
Author(s):  
Immacolata Venditto ◽  
Pedro Bule ◽  
Andrew Thompson ◽  
Juan Sanchez-Weatherby ◽  
James Sandy ◽  
...  
Keyword(s):  
Plant Cell Wall ◽  
Binding Protein ◽  
Scaffolding Protein ◽  
Carbohydrate Binding ◽  
Bacterial Cells ◽  
X Ray ◽  
Anaerobic Microorganisms ◽  
Carbohydrate Binding Modules ◽  
Genes Encoding ◽  
Cellulose Binding

A number of anaerobic microorganisms produce multi-modular, multi-enzyme complexes termed cellulosomes. These extracellular macromolecular nanomachines are designed for the efficient degradation of plant cell-wall carbohydrates to smaller sugars that are subsequently used as a source of carbon and energy. Cellulolytic strains from the rumens of mammals, such asRuminococcus flavefaciens, have been shown to have one of the most complex cellulosomal systems known. Cellulosome assembly requires the binding of dockerin modules located in cellulosomal enzymes to cohesin modules located in a macromolecular scaffolding protein. Over 220 genes encoding dockerin-containing proteins have been identified in theR. flavefaciensgenome. The dockerin-containing enzymes can be incorporated into the primary scaffoldin (ScaA), which in turn can bind to adaptor scaffoldins (ScaB or ScaC) and subsequently to anchoring scaffoldin (ScaE), thereby attaching the whole complex to the cell surface. However, unlike other cellulosomes such as that fromClostridium thermocellum, theRuminococcusspecies lack a specific carbohydrate-binding module (CBM) on ScaA which recruits the entire complex onto the surface of the substrate. Instead, a cellulose-binding protein, CttA, comprising two putative tandem novel carbohydrate-binding modules and a C-terminal X-dockerin module, which can bind to the cohesin of ScaE, may mediate the attachment of bacterial cells to cellulose. Here, the expression, purification and crystallization of the carbohydrate-binding modular part of the CttA fromR. flavefaciensare described. X-ray data have been collected to resolutions of 3.23 and to 1.61 Å in space groupsP3121 orP3221 andP21, respectively. The structure was phased using bound iodide from the crystallization buffer by SAD experiments.

scholarly journals Novel cellulose-binding domains, NodB homologues and conserved modular architecture in xylanases from the aerobic soil bacteria Pseudomonas fluorescens subsp. cellulosa and Cellvibrio mixtus

1995 ◽  
Vol 312 (1) ◽  
pp. 39-48 ◽  
Author(s):  
S J Millward-Sadler ◽  
K Davidson ◽  
G P Hazlewood ◽  
G W Black ◽  
H J Gilbert ◽  
...  
Keyword(s):  
Pseudomonas Fluorescens ◽  
Soil Bacteria ◽  
Catalytic Domain ◽  
Glycosyl Hydrolase ◽  
Glycosyl Hydrolase Family ◽  
Sequence Identity ◽  
Binding Domains ◽  
N Terminus ◽  
Cellulose Binding ◽  
Hydrolase Family

To test the hypothesis that selective pressure has led to the retention of cellulose-binding domains (CBDs) by hemicellulase enzymes from aerobic bacteria, four new xylanase (xyn) genes from two cellulolytic soil bacteria, Pseudomonas fluorescens subsp. cellulosa and Cellvibrio mixtus, have been isolated and sequenced. Pseudomonas genes xynE and xynF encoded modular xylanases (XYLE and XYLF) with predicted M(r) values of 68,600 and 65000 respectively. XYLE contained a glycosyl hydrolase family 11 catalytic domain at its N-terminus, followed by three other domains; the second of these exhibited sequence identity with NodB from rhizobia. The C-terminal domain (40 residues) exhibited significant sequence identity with a non-catalytic domain of previously unknown function, conserved in all the cellulases and one of the hemicellulases previously characterized from the pseudomonad, and was shown to function as a CBD when fused to the reporter protein glutathione-S-transferase. XYLF contained a C-terminal glycosyl hydrolase family 10 catalytic domain and a novel CBD at its N-terminus. C. mixtus genes xynA and xynB exhibited substantial sequence identity with xynE and xynF respectively, and encoded modular xylanases with the same molecular architecture and, by inference, the same functional properties. In the absence of extensive cross-hybridization between other multiple cel (cellulase) and xyn genes from P. fluorescens subsp. cellulosa and genomic DNA from C. mixtus, similarity between the two pairs of xylanases may indicate a recent transfer of genes between the two bacteria.

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 Scaffolding proteins guide the evolution of algal light harvesting antennas

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Harry W. Rathbone ◽  
Katharine A. Michie ◽  
Michael J. Landsberg ◽  
Beverley R. Green ◽  
Paul M. G. Curmi
Keyword(s):  
Hydrophobic Surface ◽  
Scaffolding Protein ◽  
Secondary Endosymbiosis ◽  
Scaffolding Proteins ◽  
Α Subunit ◽  
Binding Partner ◽  
Photosynthetic Organisms ◽  
New Family ◽  
Red Algal ◽  
Multiple Copies

AbstractPhotosynthetic organisms have developed diverse antennas composed of chromophorylated proteins to increase photon capture. Cryptophyte algae acquired their photosynthetic organelles (plastids) from a red alga by secondary endosymbiosis. Cryptophytes lost the primary red algal antenna, the red algal phycobilisome, replacing it with a unique antenna composed of αβ protomers, where the β subunit originates from the red algal phycobilisome. The origin of the cryptophyte antenna, particularly the unique α subunit, is unknown. Here we show that the cryptophyte antenna evolved from a complex between a red algal scaffolding protein and phycoerythrin β. Published cryo-EM maps for two red algal phycobilisomes contain clusters of unmodelled density homologous to the cryptophyte-αβ protomer. We modelled these densities, identifying a new family of scaffolding proteins related to red algal phycobilisome linker proteins that possess multiple copies of a cryptophyte-α-like domain. These domains bind to, and stabilise, a conserved hydrophobic surface on phycoerythrin β, which is the same binding site for its primary partner in the red algal phycobilisome, phycoerythrin α. We propose that after endosymbiosis these scaffolding proteins outcompeted the primary binding partner of phycoerythrin β, resulting in the demise of the red algal phycobilisome and emergence of the cryptophyte antenna.

Sign in / Sign up

Export Citation Format

Share Document