The tertiary structure of a bacterial cellulase determined by small-angle X-ray-scattering analysis

Pilz, Ingrid; Schwarz, Erika; Kilburn, Douglas G.; Miller, Robert C.; Warren, R. A. J.; Gilkes, Neil R.

doi:10.1042/bj2710277

Cited by 42 publications

(33 citation statements)

References 17 publications

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“…A method for the isolation of p30 (the CenA catalytic domain) using C. fimi protease has been described [3]. Proteolysis of Cex by C.,finti protease and by immobilized papain were examined in order to design a convenient procedure for the preparation of p33 (the Cex catalytic domain).…”

Section: Pro Teo Lysis Q F Cexmentioning

confidence: 99%

“…Approximately 3 pg of the late-eluting chromatographic peak (p8) were also examined (A, lane 4). The CenA catalytic domain (p30) was prepared as previously described [3]; SDSjPAGE analysis of an aliquot containing 50 pg is also shown (A, lane 5). Gels contained 12.5% acrylamide and the positions of molecular mass standards are shown on the left.…”

Section: Pro Teo Lysis Q F Cexmentioning

confidence: 99%

“…Cex concentration in kinetic assays was determined from absorbance at 280 nm. An absorption coefficient (0.1%, 1 cm, 280 nm) of 1.61 for Cex was determined as previously described [3] using the far-ultraviolet method of Scopes [18]. Ainino acid sequences were aligned with the program MALIGN [I91 and confirmed by hydrophobic cluster analysis…”

Section: Other Proceduresmentioning

confidence: 99%

“…The CenA catalytic domain (p30) was prepared by digestion of CenA with C-Ji'mi protease followed by size-exclusion chromatography in Mes buffer, as previously described [3].…”

Section: Preparation Of the Isolated Catalytic Domains From Cex And mentioning

confidence: 99%

“…Although their mechanisms of action remain elusive, it is now known that many /3-1,4-glycanases contain two or more structural domains: a catalytic domain joined to a domain which mediates binding to cellulose is a common arrangement [I, 21. For example, the tertiary structure of CenA, an end0-/3-1,4-glucanase from the bacterium Cellulomonas fimi, resembles a tadpole : the globular head is the catalytic domain, while the cellulose-binding domain (CBD) and a 23-amino-acid linker which contains only proline and threonine residues (the Pro-Thr box) comprise the extended tail [3]. All available evidence indicates that Cex, a C. fimi exo-$1 ,bglycanase, has the same general structural and functional organisation [l].…”

mentioning

confidence: 99%

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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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Section: Pro Teo Lysis Q F Cexmentioning

confidence: 99%

Section: Pro Teo Lysis Q F Cexmentioning

confidence: 99%

Section: Other Proceduresmentioning

confidence: 99%

“…The CenA catalytic domain (p30) was prepared by digestion of CenA with C-Ji'mi protease followed by size-exclusion chromatography in Mes buffer, as previously described [3].…”

Section: Preparation Of the Isolated Catalytic Domains From Cex And mentioning

confidence: 99%

mentioning

confidence: 99%

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