scholarly journals Evidence for a general role for non-catalytic thermostabilizing domains in xylanases from thermophilic bacteria

1995 ◽  
Vol 307 (1) ◽  
pp. 151-158 ◽  
Author(s):  
C M G A Fontes ◽  
G P Hazlewood ◽  
E Morag ◽  
J Hall ◽  
B H Hirst ◽  
...  
Keyword(s):  
Cell Wall ◽  
Plant Cell ◽  
Plant Cell Wall ◽  
Catalytic Domain ◽  
Genomic Library ◽  
Thermophilic Bacteria ◽  
Cross Hybridization ◽  
General Role ◽  
Sequence Identity ◽  
Cell Wall Hydrolases

A genomic library of Clostridium thermocellum DNA constructed in lambda ZAPII was screened for xylanase-expressing clones. Cross-hybridization experiments revealed a new xylanase gene isolated from the gene library, which was designated xyn Y. The encoded enzyme, xylanase Y (XYLY), displayed features characteristic of an endo-beta1,4-xylanase: the enzyme rapidly hydrolysed oat spelt, wheat and rye arabinoxylans and was active against methyl-umbelliferyl-beta-D-cellobioside, but did not hydrolyse any cellulosic substrates. The pH and temperature optima of the enzyme were 6.8 and 75 degrees C respectively, and the recombinant XYLY, expressed by Escherichia coli had a maximum Mr of 116000. The nucleotide sequence of xyn Y contained an open reading frame of 3228 bp encoding a protein of predicted Mr 120 105. The encoded enzyme contained a typical N-terminal 26-residue signal peptide, followed by a 164 amino acid sequence, designated domain A, that was not essential for catalytic activity. Downstream of domain A was a 351-residue xylanase Family F catalytic domain, followed by a 180-residue sequence that exhibited 28% sequence identity with a thermostable domain of Thermoanaerobacterium saccharolyticum xylanase A. The C-terminal portion of XYLY comprised the 23-residue duplicated docking sequence found in all other C. thermocellum plant cell wall hydrolases that are constituents of the bacterium's multienzyme complex, termed the cellulosome, followed by a 286-residue domain which exhibited 32% sequence identity with the N-terminal region of C. thermocellum xylanase Z. The enzyme did not contain linker sequences found in other C. thermocellum plant cell wall hydrolases. Analysis of truncated forms of XYLY and hybrid proteins, comprising segments of XYLY fused to the E. coli maltose binding domain, confirmed that XYLY contained a central catalytic domain and an adjacent thermostable domain. The C-terminal domain did not bind to cellulose or xylan. Western blot analysis using antiserum raised against XYLY showed that the xylanase was located in the cellulosome and did not appear to be extensively glycosylated. The non-catalytic domains of XYLY are discussed in relation to the general stability of thermophilic xylanases.

scholarly journals A non-modular endo-β-1,4-mannanase from Pseudomonas fluorescens subspecies cellulosa

1995 ◽  
Vol 305 (3) ◽  
pp. 1005-1010 ◽  
Author(s):  
K L Braithwaite ◽  
G W Black ◽  
G P Hazlewood ◽  
B R S Ali ◽  
H J Gilbert
Keyword(s):  
Cell Wall ◽  
Pseudomonas Fluorescens ◽  
Plant Cell ◽  
Plant Cell Wall ◽  
Catalytic Domain ◽  
Genomic Library ◽  
Glycosyl Hydrolase ◽  
Ph Optimum ◽  
Reading Frame ◽  
Protein Motifs

Pseudomonas fluorescens subsp. cellulosa when cultured in the presence of carob galactomannan degraded the polysaccharide. To isolate gene(s) from P. fluorescens subsp. cellulosa encoding endo-beta-1,4-mannanase (mannanase) activity, a genomic library of Pseudomonas DNA, constructed in lambda ZAPII, was screened for mannanase-expressing clones using the dye-labelled substrate, azo-carob galactomannan. The nucleotide sequence of the pseudomonad insert from a mannanase-positive clone revealed a single open reading frame of 1257 bp encoding a protein of M(r) 46,938. The deduced N-terminal sequence of the putative polypeptide conformed to a typical prokaryotic signal peptide. Truncated derivatives of the mannanase, lacking 54 and 16 residues from the N- and C-terminus respectively of the mature form of the enzyme, did not exhibit catalytic activity. Inspection of the primary structure of the mannanase did not reveal any obvious linker sequences or protein motifs characteristic of the non-catalytic domains located in other Pseudomonas plant cell wall hydrolases. These data indicate that the mannanase is non-modulator, comprising a single catalytic domain. Comparison of the mannanase sequence with those in the SWISSPROT database revealed greatest sequence homology with the mannanase from Bacillus sp. Thus the Pseudomonas enzyme belongs to glycosyl hydrolase Family 26, a family containing mannanases and endoglucanases. Analysis of the substrate specificity of the mannanase showed that the enzyme hydrolysed mannan and galactomannan, but displayed little activity towards other polysaccharides located in the plant cell wall. The enzyme had a pH optimum of approx. 7.0, was resistant to proteolysis and had an M(r) of 46,000 when expressed by Escherichia coli.

scholarly journals Crystallization and preliminary crystallographic studies of a novel noncatalytic carbohydrate-binding module from theRuminococcus flavefacienscellulosome

2015 ◽  
Vol 71 (1) ◽  
pp. 45-48 ◽  
Author(s):  
Immacolata Venditto ◽  
Arun Goyal ◽  
Andrew Thompson ◽  
Luis M. A. Ferreira ◽  
Carlos M. G. A. Fontes ◽  
...  
Keyword(s):  
Cell Wall ◽  
Plant Cell ◽  
Plant Cell Wall ◽  
Catalytic Domain ◽  
Glycoside Hydrolase Family ◽  
Carbohydrate Binding ◽  
Cellulolytic Bacterium ◽  
Modular Architecture ◽  
Carbohydrate Binding Modules ◽  
Fundamental Biological Process

Microbial degradation of the plant cell wall is a fundamental biological process with considerable industrial importance. Hydrolysis of recalcitrant polysaccharides is orchestrated by a large repertoire of carbohydrate-active enzymes that display a modular architecture in which a catalytic domain is connectedvialinker sequences to one or more noncatalytic carbohydrate-binding modules (CBMs). CBMs direct the appended catalytic modules to their target substrates, thus potentiating catalysis. The genome of the most abundant ruminal cellulolytic bacterium,Ruminococcus flavefaciensstrain FD-1, provides an opportunity to discover novel cellulosomal proteins involved in plant cell-wall deconstruction. It encodes a modular protein comprising a glycoside hydrolase family 9 catalytic module (GH9) linked to two unclassified tandemly repeated CBMs (termed CBM-Rf6A and CBM-Rf6B) and a C-terminal dockerin. The novel CBM-Rf6A from this protein has been crystallized and data were processed for the native and a selenomethionine derivative to 1.75 and 1.5 Å resolution, respectively. The crystals belonged to orthorhombic and cubic space groups, respectively. The structure was solved by a single-wavelength anomalous dispersion experiment using theCCP4 program suite andSHELXC/D/E.

scholarly journals Updating Insights into the Catalytic Domain Properties of Plant Cellulose synthase (CesA) and Cellulose synthase-like (Csl) Proteins

Molecules ◽  
2021 ◽  
Vol 26 (14) ◽  
pp. 4335
Author(s):  
Gerasimos Daras ◽  
Dimitris Templalexis ◽  
Fengoula Avgeri ◽  
Dikran Tsitsekian ◽  
Konstantina Karamanou ◽  
...  
Keyword(s):  
Cell Wall ◽  
Plant Cell ◽  
Plant Cell Wall ◽  
Catalytic Domain ◽  
Economic Value ◽  
Cellulose Synthase ◽  
Protein Isoforms ◽  
Research Progress ◽  
Cellulose Synthesis ◽  
Biotic Stresses

The wall is the last frontier of a plant cell involved in modulating growth, development and defense against biotic stresses. Cellulose and additional polysaccharides of plant cell walls are the most abundant biopolymers on earth, having increased in economic value and thereby attracted significant interest in biotechnology. Cellulose biosynthesis constitutes a highly complicated process relying on the formation of cellulose synthase complexes. Cellulose synthase (CesA) and Cellulose synthase-like (Csl) genes encode enzymes that synthesize cellulose and most hemicellulosic polysaccharides. Arabidopsis and rice are invaluable genetic models and reliable representatives of land plants to comprehend cell wall synthesis. During the past two decades, enormous research progress has been made to understand the mechanisms of cellulose synthesis and construction of the plant cell wall. A plethora of cesa and csl mutants have been characterized, providing functional insights into individual protein isoforms. Recent structural studies have uncovered the mode of CesA assembly and the dynamics of cellulose production. Genetics and structural biology have generated new knowledge and have accelerated the pace of discovery in this field, ultimately opening perspectives towards cellulose synthesis manipulation. This review provides an overview of the major breakthroughs gathering previous and recent genetic and structural advancements, focusing on the function of CesA and Csl catalytic domain in plants.

scholarly journals Characterization of Two Noncellulosomal Subunits, ArfA and BgaA, from Clostridium cellulovorans That Cooperate with the Cellulosome in Plant Cell Wall Degradation

2002 ◽  
Vol 184 (24) ◽  
pp. 6859-6865 ◽  
Author(s):  
Akihiko Kosugi ◽  
Koichiro Murashima ◽  
Roy H. Doi
Keyword(s):  
Cell Wall ◽  
Plant Cell ◽  
Plant Cell Wall ◽  
Genomic Library ◽  
Amino Acid Sequences ◽  
Glycosyl Hydrolases ◽  
Cell Wall Degradation ◽  
Clostridium Cellulovorans ◽  
Molecular Weights ◽  
Plant Cell Wall Degradation

ABSTRACT Plant cell wall degradation by Clostridium cellulovorans requires the cooperative activity of its cellulases and hemicellulases. To characterize the α-l-arabinosidases that are involved in hemicellulose degradation, we screened the C. cellulovorans genomic library for clones with α-l-arabinofuranosidase or α-l-arabinopyranosidase activity, and two clones utilizing different substrates were isolated. The genes from the two clones, arfA and bgaA, encoded proteins of 493 and 659 amino acids with molecular weights of 55,731 and 76,414, respectively, and were located on neighboring loci. The amino acid sequences for ArfA and BgaA were related to α-l-arabinofuranosidase and β-galactosidase, respectively, which are classified as family 51 and family 42 glycosyl hydrolases, respectively. Recombinant ArfA (rArfA) had high activity for p-nitrophenyl α-l-arabinofuranoside, arabinoxylan, and arabinan but not for p-nitrophenyl α-l-arabinopyranoside. On the other hand, recombinant BgaA (rBgaA) hydrolyzed not only p-nitrophenyl α-l-arabinopyranoside but also p-nitrophenyl β-d-galactopyranoside. However, when the affinities of rBgaA for p-nitrophenyl α-l-arabinopyranoside and p-nitrophenyl β-d-galactopyranoside were compared, the Km values were 1.51 and 6.06 mM, respectively, suggesting that BgaA possessed higher affinity for α-l-arabinopyranose residues than for β-d-galactopyranoside residues and possessed a novel enzymatic property for a family 42 β-galactosidase. Activity staining analyses revealed that ArfA and BgaA were located exclusively in the noncellulosomal fraction. When rArfA and rBgaA were incubated with β-1,4-xylanase A (XynA), a cellulosomal enzyme from C. cellulovorans, on plant cell wall polymers, the plant cell wall-degrading activity was synergistically increased compared with that observed with XynA alone. These results indicate that, to obtain effective plant cell wall degradation, there is synergy between noncellulosomal and cellulosomal subunits.

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