Nucleotide sequence and deletion analysis of the xylanase gene (xynZ) of Clostridium thermocellum

Grépinet, Olivier; Chebrou, M C; Béguin, Pierre

doi:10.1128/jb.170.10.4582-4588.1988

Cited by 188 publications

(94 citation statements)

References 52 publications

(36 reference statements)

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“…Similar structures were found in an endoglucanase sequence from C. cellulolyticum, egccA (Faure et al, 1989), and a xylanase gene from C. thermocellum, cloxynZ (Hall et al, 1988). The function of these conserved, reiterated regions of homology is unknown (Faure et al, 1989) but it is not essential for catalytic activity (Grepinet et al, 1988;Hall et al, 1988). The truncated C. thermocellum enzyme (Chavaux et al, 1990) without its conserved C-terminal region was also very similar to the enzyme, EgD, expressed from the intact gene in terms of activity and calcium-binding effects.…”

Section: Discussionmentioning

confidence: 67%

Nucleotide sequence and characteristics of endoglucanase gene engB from Clostridium cellulovorans

Foong

Hamamoto²,

Shoseyov³

et al. 1991

Journal of General Microbiology

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An endoglucanase gene, engB, from ClostridYum cellulovorans, previously cloned into pUCl9, has been further characterized and its product investigated. The enzyme, EngB, encoded by the gene was secreted into the periplasmic space of Escherichiu 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 ribosomebinding 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 cellulolytic bacteria. According to our deletion analyses, this region is not needed for catalytic activity.

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Section: Discussionmentioning

confidence: 67%

Nucleotide sequence and characteristics of endoglucanase gene engB from Clostridium cellulovorans

Foong

Hamamoto²,

Shoseyov³

et al. 1991

Journal of General Microbiology

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“…The activity of many cellulolytic enzymes of C. thermocellum increases in the presence of calcium ions (Lamed & Bayer, 1988;Grepinet et al, 1988). The highest increase in the activity of recombinant truncated Lic16A proteins due to calcium stimulation was observed for constructs that contained CBMX.…”

Section: Discussionmentioning

confidence: 99%

Carbohydrate-binding properties of a separately folding protein module from β-1,3-glucanase Lic16A of Clostridium thermocellum

et al. 2009

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The multi-modular non-cellulosomal endo-1,3(4)-b-glucanase Lic16A from Clostridium thermocellum contains a so-called X module (denoted as CBMX) near the N terminus of the catalytic module (191-426 aa). Melting of X-module-containing recombinant proteins revealed an independent folding of the module. CBMX was isolated and studied as a separate fragment. It was shown to bind to various insoluble polysaccharides, including xylan, pustulan, chitin, chitosan, yeast cell wall glucan, Avicel and bacterial crystalline cellulose. CBMX thus contains a hitherto unknown carbohydrate-binding module (CBM54). It did not bind soluble polysaccharides on which Lic16A is highly active. Ca 2+ ions had effects on the binding, e.g. stimulated complex formation with chitosan, which was observed only in the presence of Ca 2+ . The highest affinity to CBMX was shown for xylan (binding constant K53.1¾10 4 M "1 ), yeast cell wall glucan (K51.4¾10 5 M "1 ) and chitin (K53.3.10 5 M "1 in the presence of Ca 2+ ). Lic16A deletion derivatives lacking CBMX had lower affinity to lichenan and laminarin and a slight decrease in optimum temperature and thermostability. However, the specific activity was not significantly affected.

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“…6) a bond between Cys129 and Cysl60 of C. albidus Xyn [25] (corresponding to the 208 -240 bond of Cex) is probable. Similarly, bonds between Cys782 and Cys788 of C. thermocellum XynZ [26] and between Cys247 and Cys253 of C. albidus Xyn (corresponding to the 302-308 bond of Cex) are likely. Corresponding Cys residues are not discernible in other family F enzymes and overall it appears that disulfide bonds are less well conserved than among the known family B enzymes.…”

Section: Discussionmentioning

confidence: 99%

“…6. Sequence alignment,for /3-1,4-glucanases offamily F. The sequences shown are from the catalytic domains or pulative catalytic domains of (a) Cex, C.,fimi [38]; (b) XynZ, Clostridium thermocellum [26]; (c) XynA, ~seudomonus,fluorescens subsp. cellulosa [39]; (d) CelB, exoglucanase domain, Cuidocellum sacchurolyticum [40]; (e) XynA, C. saccharolyticum [41]; (0 XynA, Bucillus sp.…”

Section: Discussionmentioning

confidence: 99%

Structural and functional relationships in two families of β‐1,4‐glycanases

Gilkes

Claeyssens

Aebersold

et al. 1991

European Journal of Biochemistry

111

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CenA and Cex are /3-1 ,Cglycanases produced by the cellulolytic bacterium Cellulomonasfimi. Both enzymes are composed of two domains and contain six Cys residues. Two disulfide bonds were assigned in both enzymes by peptide analysis of the isolated catalytic domains. A further disulfide bond was deduced in both cellulosebinding domains from the absence of free thiols under denaturing conditions. Corresponding Cys residues are conserved in eight of nine other known C.fimi-type cellulose-binding domains. CenA and Cex belong to families B and F, respectively, in the classification of p-1,4-glucanases and p-1 ,Cxylanases based on similarities in catalytic domain primary structure. Disulfide bonds in the CenA catalytic domain correspond to the two disulfide bonds in the catalytic domain of Trichoderma reesei cellobiohydrolase I1 (family B) which stabilize loops forming the active-site tunnel. Sequence alignment indicates the probable occurrence of disulfides at equivalent positions in the two other family B enzymes. Partial resequencing of the gene encoding Streptomyces KSM-9 fl-1,4-glucanase CasA (family B) revealed five errors in the original nucleotide sequence analysis. The corrected amino acid sequence contains an Asp residue corresponding to the proposed proton donor in hydrolysis catalysed by cellobiohydrolase 11. Cys residues which form disulfide bonds in the Cex catalytic domain are conserved in XynZ of Clostridium thermocellum and Xyn of Cryptococcus alhidus but not in the other eight known family F enzymes. Like other members of its family, Cex catalyses xylan hydrolysis. The catalytic efficiency (kCat/Km) for hydrolysis of the heterosidic bond ofp-nitrophenyI-fl-D-xylobioside is 14385 min-l . mM-l at 25 "C; the corresponding kc,,/ K, for p-nitrophenyl-fi-D-cellobioside hydrolysis is 296 min-l . mM-'.Cellulose and xylan are the major components of plant biomass. Many bacteria and fungi exploit the natural abundance of these P-l,Cglycans to obtain carbon and energy. Such microorganisms produce an array of degradative enCorrespondence t o N. R. Gilkes,

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Nucleotide sequence and deletion analysis of the xylanase gene (xynZ) of Clostridium thermocellum

Cited by 188 publications

References 52 publications

Nucleotide sequence and characteristics of endoglucanase gene engB from Clostridium cellulovorans

Nucleotide sequence and characteristics of endoglucanase gene engB from Clostridium cellulovorans

Carbohydrate-binding properties of a separately folding protein module from β-1,3-glucanase Lic16A of Clostridium thermocellum

Structural and functional relationships in two families of β‐1,4‐glycanases

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