An Escherichia colz clone was constructed to overproduce endoglucanase C (CelCCC) from Clostridium cellulolyticum. This construction made it easier to isolate the enzyme but, as observed in the case of endoglucanase A (CelCCA) from the same organism, the purification led to the isolation of two forms of the cellulase differing in their molecular masses, 48 kDa and 41 kDa. Nterminal sequence analysis of both purified enzymes showed that the shorter form was probably the result of partial proteolysis near the COOH-extremity. The difference in mass indicated that the shorter protein lacks the C-terminal reiterated domains (20-24-amino-acid twice-repeated sequences). These particular domains are characteristic of clostridial cellulases acting on cellulose by the mean of cellulosomal particles. Biochemical and enzymic studies were performed on each form of CelCCC, and revealed that their temperature and pH optima were identical, but their catalytic parameters were quite different. Furthermore, the differences of enzymic behavior observed between the two forms of CelCCC are almost identical to those already noted in the case of the two forms of CelCCA. The stereoselectivity of the reaction catalysed by CelCCC and CelCCA was determined using proton NMR spectroscopy ; CelCCC acts by configuration inversion, whereas CelCCA acts by configuration retention. The degradation patterns on cellodextrins (ranging from cellotriose to cellohexaose) and chromophoric cellodextrins (from p-nitrophenyl-cellobiose to p-nitrophenyl-cellopentaose) were also investigated in both forms of CelCCC and CelCCA. It emerged that the natural cellodextrins degradation patterns of CelCCC and CelCCA were very similar but the utilization of p-nitrophenyl-cellodextrins showed the existence of considerable differences between these two endoglucanases in terms of cleavage-site position and catalytic parameters. CelCCC and CelCCA were found not to act synergistically on the tested substrates.Clostridiurn cellulolyticum is a mesophilic Clostridium which is able to completely degrade crystalline cellulose. The four known cellulase sequences of this organism [l-31 exhibit reiterated domains. These particular sequences are thought to be characteristic of enzymes integrated into the cellulosome structure [4], and yet they are observed with cellulolytic systems of Clostridium thermocellum [4, 51 and Clostridium cellulovorans [6]. These organisms secrete highmolecular-mass particles named cellulosomes which are active against cellulose, and they possess an assembly factor, CbpA in C. cellulovorans [7] and CipA in C. thermocellum [8, 91. Four genes, celCCA, celCCC, coding for enzymes of the cellulolytic system of C. cellulolyticum have been completely sequenced and the four corresponding proteins are members of three different fami-Correspondence to H

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Sequence analysis of the endoglucanase EGCCA of Clostridium cellulolyticum indicates the existence of two domains: a catalytic domain extending from residue 1 to residue 376 and a reiterated domain running from residue 390 to 450. A small deletion in the C terminal end of the catalytic domain inactivated the protein. From the analysis of the sequences of 26 endoglucanases belonging to family A, we focused on seven amino acids which were totally conserved in all the catalytic domains compared. The roles of two of these, Arg-79 and His-122, were studied and defined on the basis of the mutants obtained by introducing various substitutions. Our findings suggest that Arg-79 is involved in the structural organization of the protein; the His-122 residue seems to be more essential for catalysis. The role of His-123, which is conserved only in subfamily A4, was also investigated.Clostridium cellulolyticum is a mesophilic anaerobic bacterium which is able to degrade crystalline cellulose (13). Studies of the various components of the cellulolytic complex of this bacterium are in progress, and previous studies have dealt with the cloning (8) and sequencing (9) of the celCCA gene, which encodes the endoglucanase EGCCA. Numerous genes of cellulases have now been sequenced, and the corresponding enzymes have been divided into nine different families on the basis of sequence comparisons and hydrophobic cluster analysis (14,17,18). According to this classification, EGCCA is a member of family A. At least 26 genes of cellulases belonging to family A have been sequenced to date, but none of their three-dimensional (3-D) structures have been determined. At present, the only 3-D structure described in the literature is that of cellobiohydrolase II from Trichoderma reesei (25), which belongs to family B. A second structure description should be published soon, as Clostridium thermocellum endoglucanase CELD (family E) has been crystallized (19) and its 3-D structure has been determined (20). Since the structure of the cellulases of family A is unknown, information about the structurefunction relationships must be deduced from the primary sequences. Using hydrophobic cluster analysis, Henrissat et al. (17) found that five segments were conserved, including several amino acids which were particularly well conserved. In the present study, we explored the roles of three of these amino acids: arginine 79 and histidine 122, which are conserved throughout the family, and histidine 123, which is conserved only in subfamily A4. MATERIALS AND METHODSStrains and media. Escherichia coli TG1 (30)