Christian Gaudin

Crystal structure of the catalytic domain of a bacterial cellulase belonging to family 5.

BACKGROUND Cellulases are glycosyl hydrolases--enzymes that hydrolyze glycosidic bonds. They have been widely studied using biochemical and microbiological techniques and have attracted industrial interest because of their potential in biomass conversion and in the paper and textile industries. Glycosyl hydrolases have lately been assigned to specific families on the basis of similarities in their amino acid sequences. The cellulase endoglucanase A produced by Clostridium cellulolyticum (CelCCA) belongs to family 5. RESULTS We have determined the crystal structure of the catalytic domain of CelCCA at a resolution of 2.4 A and refined it to 1.6 A. The structure was solved by the multiple isomorphous replacement method. The overall structural fold, (alpha/beta)8, belongs to the TIM barrel motif superfamily. The catalytic centre is located at the C-terminal ends of the beta strands; the aromatic residues, forming the substrate-binding site, are arranged along a long cleft on the surface of the globular enzyme. CONCLUSIONS Strictly conserved residues within family 5 are described with respect to their catalytic function. The proton donor, Glu170, and the nucleophile, Glu307, are localized on beta strands IV and VII, respectively, and are separated by 5.5 A, as expected for enzymes which retain the configuration of the substrates anomeric carbon. Structure determination of the catalytic domain of CelCCA allows a comparison with related enzymes belonging to glycosyl hydrolase families 2, 10 and 17, which also display an (alpha/beta)8 fold.

The cellulolytic system of Clostridium cellulolyticum

Recent findings on the cellulolytic system of the mesophilic Clostridium cellulolyticum are reviewed. Six cellulases and the scaffolding protein, which are, at the present time, the known components of the cellulosome have been cloned. The catalytic and structural properties of the cloned enzymes CelA, CelC, CelD and CelF are described. It was shown that the grafting of the cellulases onto the scaffolding protein was performed using the dockerin-cohesin attachment device and was strictly dependent on the integrity of both components of the complex. The amino-acid sequences of dockerin and cohesin domains of C. cellulolyticum were compared to that of C. cellulovorans and C. thermocellum. This sequence analysis shows that domains belonging to the thermophilic or the mesophilic bacteria can be placed into two well defined groups. The genetic organization of the gene cluster of C. cellulolyticum is discussed.

Sequence analysis of a gene cluster encoding cellulases from Clostridium cellulolyticum

The sequence of a 5633-bp EcoRI-PvuII DNA fragment from Clostridium cellulolyticum was determined. This fragment contains two complete endo-beta-1,4-glucanase-encoding genes, designated celCCC and celCCG. These two genes are flanked by two other partial open reading frames (ORF1 and celCCE) that probably encode two cellulases or related enzymes. The celCCC and celCCG genes appear to be present in a polycistronic transcriptional unit. Northern blot hybridisations with intragenic probes derived from celCCC and celCCG gave similar patterns. Two transcripts of about 5 and 6 kb were identified. The celCCC and celCCG ORFs extend over 1380 bp and 2175 bp, respectively. They are separated by only 87 nt. A typical signal sequence is present at the N terminus of the deduced polypeptides. The mature CelCCC and CelCCG proteins have M(r)s 47,201 and 76,101, respectively. Comparisons between their amino acid (aa) sequences and other known cellulase sequences revealed that: first, they both contain the repeated 24-aa sequence characteristic of clostridial beta-glycanases, secondly, the N-terminal catalytic domains of CelCCC and CelCCG can be classified into the D and E2 families, respectively, and thirdly, the largest CelCCG contains an additional internal domain which is very similar to that of the Bacillus-type cellulose-binding domain (CBD). The ORF1-C-terminal-encoded sequence also contains the clostridial 24-aa repeat. The CelCCE N-terminus consists of a typical signal sequence followed by a 168-aa domain homologous to the N-terminal repeated domain of Cellulomonas fimi CenC. This domain is connected to an incomplete catalytic domain of family E1 by a Pro-rich junction linker.

CelE, a Multidomain Cellulase from Clostridium cellulolyticum: a Key Enzyme in the Cellulosome?

CelE, one of the three major proteins of the cellulosome of Clostridium cellulolyticum, was characterized. The amino acid sequence of the protein deduced from celE DNA sequence led us to the supposition that CelE is a three-domain protein. Recombinant CelE and a truncated form deleted of the putative cellulose binding domain (CBD) were obtained. Deletion of the CBD induces a total loss of activity. Exhibiting rather low levels of activity on soluble, amorphous, and crystalline celluloses, CelE is more active on p-nitrophenyl-cellobiose than the other cellulases from this organism characterized to date. The main product of its action on Avicel is cellobiose (more than 90% of the soluble sugars released), and its attack on carboxymethyl cellulose is accompanied by a relatively small decrease in viscosity. All of these features suggest that CelE is a cellobiohydrolase which has retained a certain capacity for random attack mode. We measured saccharification of Avicel and bacterial microcrystalline cellulose by associations of CelE with four other cellulases from C. cellulolyticum and found that CelE acts synergistically with all tested enzymes. The positive influence of CelE activity on the activities of other cellulosomal enzymes may explain its relative abundance in the cellulosome.

Sequence analysis of the Clostridium cellulolyticum endoglucanase-A-encoding gene, celCCA

The nucleotide sequence of a Clostridium cellulolyticum endo-beta-1,4- glucanase (EGCCA)-encoding gene (celCCA) and its flanking regions, was determined. An open reading frame (ORF) of 1425 bp was found, encoding a protein of 475 amino acids (aa). This ORF began with an ATG start codon and ended with a TAA ochre stop codon. The N-terminal region of the EGCCA protein resembled a typical signal sequence of a Gram-positive bacterial extracellular protein. A putative signal peptidase cleavage site was determined. EGCCA, without a signal peptide, was found to be composed of more than 35% hydrophobic aa and to have an Mr of 50715. Comparison of the encoded sequence with other known cellulase sequences showed the existence of various kinds of aa sequence homologies. First, a strong homology was found between the C-terminal region of EGCCA, containing a reiterated stretch of 24 aa, and the conserved reiterated region previously found to exist in four Clostridium thermocellum endoglucanases and one xylanase from the same organism. This region was suspected of playing a role in organizing the cellulosome complex. Second, an extensive homology was found between EGCCA and the N-terminal region of the large endoglucanase, EGE, from C. thermocellum, which suggests that they may have a common ancestral gene. Third, a region, which extended for 21 aa residues beginning at aa + 127, was found to be homologous with regions of cellulases belonging to Bacilli, Clostridia and Erwinia chrysanthemi.

Molecular study and overexpression of the Clostridium cellulolyticum celF cellulase gene in Escherichia coli

The CelF-encoding sequence was isolated from Clostridium cellulolyticum genomic DNA using the inverse PCR technique. The gene lies between cipC (the gene encoding the cellulosome scaffolding protein) and celC (coding for the endoglucanase C) in the large cel cluster of this mesophilic cellulolytic Clostridium species. Comparisons between the deduced amino acid sequence of the mature CelF (693 amino acids, molecular mass 77626) and those of other beta-glycanases showed that this enzyme belongs to the recently proposed family L of cellulases (family 48 of glycosyl hydrolases). The protein was overproduced in Escherichia coli using the T7 expression system. It formed both cytoplasmic and periplasmic inclusion bodies when induction was performed at 37 degrees C. Surprisingly, the protein synthesized from the cytoplasmic production vector was degraded in the Ion protease-deficient strain BL21(DE3). The induction conditions were optimized with regard to the concentration of inductor, cell density, and temperature and time of induction in order to overproduce an active periplasmic protein (CelFp) which was both soluble and stable. It was collected using the osmotic shock method. The enzymic degradation of various cellulosic substrates by CelFp was studied. CelFp degraded swollen Avicel more efficiently than substituted soluble CM-cellulose or crystalline Avicel and was not active on xylan. Its activity is therefore quite different from that of endoglucanases, which are most active on CM-cellulose.

Ribosomal DNA sequence analysis shows that the basidiomycete C30 belongs to the genus Trametes

The basidiomycete C30 was considered as an isolate of a population of Marasmius quercophilus collected on evergreen oak litter from the Mediterranean forest. Recent phenotypic studies have clearly shown that it differs from newly characterized M. quercophilus isolates. Subsequent analysis of laccase genes revealed that C30 sequences are similar to laccase encoding sequences from organisms belonging to the polyporoid clade. Comparison of sequences of the C30 ITS regions, including 5.8S rDNA, with those found in databanks confirmed that C30 is not a Marasmius. Finally, 25S rDNA analysis revealed that C30 is closely related to the Coriolaceae and, in particular, to Trametes trogii.

First Chemical Synthesis of Three Natural Depsides Involved in Flavonol Catabolism and Related to Quercetinase Catalysis

Abstract We report here the first chemical synthesis of three depsides related to quercetinase‐catalyzed degradation of kaempferol, quercetin, and myricetin. The three depsides were constructed through the coupling of suitably protected phloroglucinol carboxylic acid and hydroxy‐perbenzylated, derivatives of gallic, protocatechuic, and 4‐hydroxy benzoic acids. The three synthesized target compounds proved to be identical to their natural counterparts, arising from quercetinase action on corresponding flavonols.

Purification and partial characterization of two extracellular endoglucanases from Cellulomonas fermentans

Avicelase assay of gel slices after non-denaturing polyacrylamide gel electrophoresis of concentrated supernatants from Cellulomonas fermentans revealed four active bands. One of them corresponded to the principal active band on CM-cellulose. Among the three others, at least one did not correspond to any active band on CM-cellulose and might reflect the presence of an exoglucanase (EC 3.2.1.91). The active band on CM-cellulose was composed of two endoglucanases (EC 3.2.1.4), called CFA and CFB, which we purified by the means of DEAE-Trisacryl chromatography and high performance liquid chromatography (anion exchange chromatography and gel chromatography). These two monomeric enzymes differ in their molecular weights (40,000 and 57,000 for CFA and CFB, respectively) and in their catalytic constants in the reaction with CM-cellulose (Km were 1.5 g/l and 59 g/l for CFA and CFB, respectively), but have similar modes of action on this substrate and similar substrate specificities.

Physiological properties of Cellulomonas fermentans, a mesophilic cellulolytic bacterium

SummaryThe fermentation of cellobiose, glucose and cellulose MN 300 by Cellulomonas fermentans was studied. The molar growth yields (i.e. grams of cells per mole of hexose equivalent) were similar on cellobiose and cellulose at low sugar consumption levels (47.8 and 46.5 respectively), but was lower on glucose (38.0). The occurrence of cellobiose phosphorylase activity, detected in cellobiose- and cellulose-grown cells, might explain this result. The specific growth rates measured in cultures on cellobiose, glucose and cellulose were 0.055 h-1, 0.040 h-1 and 0.013 h-1 respectively. Growth inhibition was observed, and a drop in YH occurred after relatively low but different quantities of hexose were consumed (2.2 mM, 5 mM and 8 mM hexose equivalent with cellulose, glucose and cellobiose respectively), which coincided with a change in the fermentative metabolism from a typical mixed acid metabolism (1 ethanol, 1 acetate and 2 formate synthesized by consumed hexose) to a more ethanolic fermentation. When growth ceased in cellulose cultures, consumption of cellulose continued, as did production of ethanol.Molar growth yields of C. fermentans were similar in anaerobic and aerobic cellobiose cultures (47.8 g/mol and 42.2 g/mol respectively). Specific growth rates were also quite similar under both culture conditions (0.055±0.013 h-1 and 0.070±0.007 h-1 respectively). Aerobic metabolism was studied using 14C glucose. During the exponential growth phase, acetate, succinate and nonidentified compound(s) accumulated in the supernatant, but no 14CO2 was produced. During the stationary phase, acetate was oxidized and 14CO2 produced, but without any further biomass synthesis. It seems that a blocking of metabolite oxidation may have occurred in C. fermentans except in the case of acetate, but acetate oxidation was apparently not coupled with production of energy utilizable in biosynthesis.