The discrete multicomponent, multienzyme cellulosome complex of anaerobic cellulolytic bacteria provides enhanced synergistic activity among the different resident enzymes to efficiently hydrolyze intractable cellulosic and hemicellulosic substrates of the plant cell wall. A pivotal noncatalytic subunit called scaffoldin secures the various enzymatic subunits into the complex via the cohesin-dockerin interaction. The specificity characteristics and tenacious binding between the scaffoldin-based cohesin modules and the enzyme-borne dockerin domains dictate the supramolecular architecture of the cellulosome. The diversity in cellulosome architecture among the known cellulosome-producing bacteria is manifest in the arrangement of their genes in either multiple-scaffoldin or enzyme-linked clusters on the genome. The recently described three-dimensional crystal structure of the cohesin-dockerin heterodimer sheds light on the critical amino acids that contribute to this high-affinity protein-protein interaction. In addition, new information regarding the regulation of cellulosome-related genes, budding genetic tools, and emerging genomics of cellulosome-producing bacteria promises new insight into the assembly and consequences of the multienzyme complex.

A library of 75 different chimeric cellulosomes was constructed as an extension of our previously described approach for the production of model functional complexes (Fierobe, H.-P., Mechaly, A., Tardif, C., Bé laïch, A., Lamed, R., Shoham, Y., Bé laïch, J.-P., and Bayer, E. A. (2001) J. Biol. Chem. 276, 21257-21261), based on the high affinity species-specific cohesin-dockerin interaction. Each complex contained three protein components: (i) a chimeric scaffoldin possessing an optional cellulosebinding module and two cohesins of divergent specificity, and (ii) two cellulases, each bearing a dockerin complementary to one of the divergent cohesins. The activities of the resultant ternary complexes were assayed using different types of cellulose substrates. Organization of cellulolytic enzymes into cellulosome chimeras resulted in characteristically high activities on recalcitrant substrates, whereas the cellulosome chimeras showed little or no advantage over free enzyme systems on tractable substrates. On recalcitrant cellulose, the presence of a cellulose-binding domain on the scaffoldin and enzyme proximity on the resultant complex contributed almost equally to their elevated action on the substrate. For certain enzyme pairs, however, one effect appeared to predominate over the other. The results also indicate that substrate recalcitrance is not necessarily a function of its crystallinity but reflects the overall accessibility of reactive sites.A number of cellulolytic anaerobic microorganisms degrade plant cell wall cellulose by means of macromolecular complexes termed cellulosomes (1-8). In addition to a collection of cellulases, these large complexes can also include enzymes specialized for the degradation of other plant cell wall polymers, such as hemicellulases and pectinases (9, 10). Bacterial cellulosomes are typically composed of a scaffolding protein containing several cohesin domains, which bind to the dockerin domains of the catalytic subunits. The complete dissociation of all known bacterial cellulosomes into individual components requires harsh treatments, such as elevated temperatures and/or the presence of chaotropic agents, thus reflecting the strength of the cohesin-dockerin interaction. In the case of Clostridium cellulolyticum and Clostridium thermocellum, the interaction is Ca 2ϩ -dependent (11, 12) and of high affinity (Ն10 MϪ1 ; see Refs. 11 and 13).The scaffoldins produced by C. cellulolyticum and C. thermocellum contain multiple cohesin domains and a single family 3A cellulose-binding domain (CBD).1 The latter is located at the N terminus of the C. cellulolyticum scaffoldin, whereas the scaffoldin CBD from C. thermocellum adopts an internal position (14, 15). It has been shown for both species that the cohesins can interact with any of the dockerin domains of the same species, suggesting a random incorporation of the catalytic subunits along the scaffoldin (16 -18). The cohesin-dockerin interaction, however, is species-specific, at least between these two clostridia (19).In a previous st...

A genomic DNA fragment from Desulfovibrio fructosovorans, which strongly hybridized with the hydAB genes from Desulfovibrio vulgaris Hildenborough, was cloned and sequenced. This fragment was found to contain four genes, named hndA, hndB, hndC, and hndD. Analysis of the sequence homologies indicated that HndA shows 29, 21, and 26% identity with the 24-kDa subunit from Bos taurus complex I, the 25-kDa subunit from Paracoccus denitrificans NADH dehydrogenase type I, and the N-terminal domain of HoxF subunit of the NAD-reducing hydrogenase from Alcaligenes eutrophus, respectively. HndB does not show any significant homology with any known protein. HndC shows 37 and 33% identity with the C-terminal domain of HoxF and the 51-kDa subunit from B. taurus complex I, respectively, and has the requisite structural features to be able to bind one flavin mononucleotide, one NAD, and three [4Fe-4S] clusters. HndD has 40, 42, and 48% identity with hydrogenase I from Clostridium pasteurianum and HydC and HydA from D. vulgaris Hildenborough, respectively. The 4.5-kb length of the transcripts expressed in D. fructosovorans and in Escherichia coli (pSS13) indicated that all four genes were present on the same transcription unit. The sizes of the four polypeptides were measured by performing heterologous expression of hndABCD in E. coli, using the T7 promoter/polymerase system. The products of hndA, hndB, hndC, and hndD were 18.8, 13.8, 52, and 63.4 kDa, respectively. One hndC deletion mutant, called SM3, was constructed by performing marker exchange mutagenesis. Immunoblotting studies carried out on cell extracts from D. fructosovorans wild-type and SM3 strains, using antibodies directed against HndC, indicated that the 52-kDa protein was recognized in extracts from the wild-type strain only. In soluble extracts from D. fructosovorans wild type, a 10-fold induction of NADP reduction was observed when H 2 was present, but no H 2 -dependent NAD reduction ever occurred. This H 2 -dependent NADP reductase activity disappeared completely in extracts from SM3. These results indicate that the hnd operon actually encodes an NADP-reducing hydrogenase in D. fructosovorans.Hydrogenases (Hyds) are iron-sulfur enzymes which are responsible for the oxidation of molecular hydrogen, as well as for the reduction of protons during molecular hydrogen production. Hyds are key enzymes in the energy metabolism of the sulfate-reducing bacteria belonging to the genus Desulfovibrio, which use oxidized sulfur compounds as their terminal electron acceptors (31). Many Desulfovibrio species can use molecular hydrogen as their sole energy source (2); thus, the hydrogen oxidation which is thought to take place on the periplasmic side of the inner membrane would be involved in the electron transfer across the membrane and the creation of a proton motive force. Alternatively, Desulfovibrio species can grow on lactate or pyruvate, the oxidation of which in the cytoplasm may result in H 2 production (7,15,26 (class 4). This diversity makes the role of these variou...

The recombinant form of the cellulase CelF of Clostridium cellulolyticum, tagged by a C-terminal histine tail, was overproduced in Escherichia coli. The fusion protein was purified by affinity chromatography on a Ninitrilotriacetic acid column. The intact form of CelF (M r , 79,000) was rapidly degraded at the C terminus, giving a shorter stable form, called truncated CelF (M r , 71,000). Both the entire and the truncated purified forms degraded amorphous cellulose (k cat ‫؍‬ 42 and 30 min ؊1 , respectively) and microcrystalline cellulose (k cat ‫؍‬ 13 and 10 min ؊1 , respectively). The high ratio of soluble reducing ends to insoluble reducing ends released by truncated CelF from amorphous cellulose showed that CelF is a processive enzyme. Nevertheless, the diversity of the cellodextrins released by truncated CelF from phosphoric acid-swollen cellulose at the beginning of the reaction indicated that the enzyme might randomly hydrolyze ␤-1,4 bonds. This hypothesis was supported by viscosimetric measurements and by the finding that CelF and the endoglucanase CelA are able to degrade some of the same cellulose sites. CelF was therefore called a processive endocellulase. The results of immunoblotting analysis showed that CelF was associated with the cellulosome of C. cellulolyticum. It was identified as one of the three major components of cellulosomes. The ability of the entire form of CelF to interact with CipC, the cellulosome integrating protein, or mini-CipC 1 , a recombinant truncated form of CipC, was monitored by interaction Western blotting (immunoblotting) and by binding assays using a BIAcore biosensor-based analytical system.