CELLULOSOMAL CELLULASES

The cellulosome shown in Fig. 4 is an extracellular, multi-protein complex that is produced by a wide range of cellulolytic micro-organisms. It is believed to have the feature of “collecting” and “positioning” cellulose degrading enzymes onto a substrate (Bayer et al., 1994). The functional unit of the cellulosome is the “scaffoldin,” which is a non-catalytic protein containing repetitive domains (cohesins) for specific interaction with other protein domains, called dockerins.

Cellulosomal enzymes contain both a catalytic domain and a binding domain (dockerin). The cellulosome then self-assembles by type-specific recognition of

Table 2. Cellulase Families, Structure, Activity, and Distribution GH Structure Activity Catalytic

Mechanism

Nucleophile /Base

Proton Donor

Bacteria Fungi Plant

1 (/8 −glucosidase Retaining Glu Glu + + +

3 −glucosidase Retaining Asp Glu + + +

5 (/8 Endoglucanase, Retaining Glu Glu + + +

6 Endoglucanase,

cellobiohydrolase

Inverting Asp Asp + +

7 −jelly roll Endoglucanase, cellobiohydrolase

Retaining Glu Glu +

9 (/₆ Endoglucanase,

cellobiohydrolase

Inverting Asp Glu + + +

10 (/₈ Cellobiohydrolase Retaining Glu Glu + + +

12 −jelly roll Endoglucanase Retaining Glu Glu + +

16 −jelly roll Endo-1,3(4)--glucanase

Retaining Glu Glu + + +

44 Endoglucanase Inverting N/A N/A +

45 Endoglucanase Inverting Asp Asp + +

48 (/6 Endoglucanase,

cellobiohydrolase

Inverting N/A Glu + +

51 (/8 Endoglucanase Retaining Glu Glu + + +

61 N/A Endoglucanase N/A N/A N/A +

74 7-fold -propeller

Endoglucanase, cellobiohydrolase

Inverting Asp Asp + +

cohesin/dockerin pairs. The scaffoldins can also contain the carbohydrate-binding module (CBM) which serves as an attachment device for harnessing the cellulosome to the cell surface and/or for its targeting to substrate.

6.1. Non-Catalytic Subunit: Scaffoldin

The cellulosome is one of the best-studied protein complexes known to form self-assembled extracellular scaffolds (Bayeret al., 2004). The molecular mass of the cellulosome complex was determined to be several MDa. Two types of subunits have been identified from the bacterial cellulosome complex.Non-catalytic subunits, called “scaffoldins”, serve to position and organize theenzymatic subunitsand to attach the cellulosome to the cell surface and/or to the substrate –i.e. plant cell wall polysaccharides.

The scaffoldins contain multiple copies ofcohesins, which interact withdockerin domains of the enzymatic subunits to form the cellulosome assembly. The cohesins are about 140 amino acids in length and highly conserved in sequence and domain structure. The dockerin domains comprise about 70 amino acids and contain two 22-amino acid duplicated regions, each of which includes an “F-hand” modifi-cation of the EF-hand calcium-binding motif. To date, several hundred cohesin and dockerin sequences have been found, mostly from anaerobic bacteria. More than

Figure 4.Schematic structure (not scaled) of an example cellulosome complex fromClostridium thermo-cellum. The cellulosome complex are composed of two groups of proteins. One group is non-catalytic proteins (scaffoldin) including CipA, SdbA, Orf2p, OlpA, and OlpB, each of these scaffoldins contain various number of function domains,i.e.cohesin domain interacts with same type of dockerin domain (Type-I cohesin-dockerin pair are showing in black and Type-II pair in grey); carbohydrate-binding module (CBM) recognizes polysaccharide substrate; S-layer homologous (SLH) binds to cell surface; and linker between these domains. Another group is catalytic proteins (enzymes), each cellulosomal enzyme contains a Type-I dockerin domain recognizing Type-I cohesin of scaffoldin proteins. InClostridium thermocellum, more than twenty enzymes with various catalytic activities have been identified to be involved in cellulosome complex

a dozen different specificities are currently known which will enable the design and production of numerous types of nano-component systems.

Bacterial cellulosomes are organized by means of a special type of subunit, the scaffoldin, which is comprised of an array of cohesin modules. The cohesin interacts selectively and tenaciously with a complementary type of domain, the dockerin, which is borne by each of the cellulosomal enzyme subunits. The integrity of the complex is thus maintained by the cohesin-dockerin interaction. The first scaffoldin was sequenced fromClostridium cellulovorans(Shoseyovet al., 1992).

The relationship to the duplicated sequences of cellulosomal enzymes (Salamitou et al., 1992) was later realized when a second scaffoldin, derived fromC. thermo-cellum, was sequenced (Gerngross et al., 1993). Today, many scaffoldin genes has been sequenced and characterized from C. thermocellum, C. josui (Fujino et al., 1993),B. cellulosolvens(Xuet al., 2004a),A. cellulolyticus(Xuet al., 2004b), and R. flavefaciens(Dinget al., 2001).

The cellulosome system characterized by multiple scaffoldins includes a primary scaffoldin, anchoring scaffoldins, and an “adaptor” scaffoldin. The primary scaffoldin incorporates the enzymatic subunits and usually bears a single CBM domain. The anchoring scaffoldin bears an SLH module for attaching the cellulosome to the cell

wall. The adaptor scaffoldin from A. cellulolyticus contains four cohesins and a dockerin, which effectively multiplies the number of enzymes that can be incor-porated into the complex. In contrast, the adaptor scaffoldin fromR. flavefaciens contains a single divergent cohesin and alters the specificity of the primary scaffoldin which expands the repertoire of cellulosomal subunits that can be incorporated into the complex. Scaffoldins have significant diversity in cellulosome architecture, as reflected by the number of cohesins in a given scaffodin and their disposition therein, the presence (or absence) and location of a CBM, and the presence (or absence) of a dockerin and/or SLH module. For example, theR. flavefaciensscaffoldin lack an identifiable CBM and SLH, although the cellulosome binds cellulose and is cell associated, an enzyme-bearing CBM might mediate this important function (Rincon et al., 2001); the scaffoldin (scaD) from A. cellulolyticus plays a dual role, both as a primary scaffoldin –capable of direct incorporation of a single dockerin-borne enzyme and as a secondary scaffoldin – one that anchors the major primary scaffoldin,ScaA, and its complement of enzymes to the cell surface (Xu et al., 2004b). In the case of mesophilicClostridia, their sacffoldins lack dockerins and conventional SLH domains. However, a similar type of module contained at theN -terminus of theC. cellulovoransenzyme family-9 enzyme, EngE, has been implicated in mediating cell surface attachment of its cellulosome (Kosugiet al., 2002).

6.2. The Cohesin-dockerin Interaction

The first biochemical analyses of the cellulosome complex fromC. thermocellum indicated an exceptionally strong interaction that rivaled the affinities of the most tenacious biochemical bonds (Lamed and Bayer, 1988; Lamedet al., 1983). Subse-quent analyses substantiated these claims, and the cohesin-dockerin interaction rates among the most potent protein-protein interactions known in nature (Fierobe et al., 2001; Mechaly et al., 2001). The interaction between the two components can be viewed as a kind of plug-and-socket arrangement, whereby the dockerin domain plugs into the cohesin module (Bayeret al., 2004).

6.3. Carbohydrate-Binding Modules

Glycosyl hydrolases attach to polysaccharides relatively inefficiently, as their target glycosidic bonds are often inaccessible to the active site of the appropriate enzymes.

In order to overcome these problems, many of the glycosyl hydrolases, primarily the noncellulosomal cellulases and related “free” enzymes that hydrolyze insoluble substrates, are modular and comprise catalytic modules appended to one or more non-catalytic CBMs (carbohydrate-binding modules). CBMs primarily promote the association of the enzyme with the substrate (Borastonet al., 2004; Bayeret al., 2004).

CBMs are divided into families based on amino acid sequence similarity. There are currently 43 defined families and these displayed substantial variation in ligand specificity (see http://afmb.cnrs-mrs.fr/CAZY/CBM.html). Thus there are characterized CBMs that recognize crystalline cellulose, non-crystalline cellulose,

chitin, -1,3-glucans and -(1,3)-(1,4) mixed linkage glucans, xylan, mannan, galactan, and starch. Some CBMs display “lectin-like” specificity and bind to a variety of cell-surface glycans (Borastonet al., 2004; Sorimachiet al., 1996, 1997;

Williamsonet al., 1997; Sigurskjoldet al., 1994). Based on structural and functional similarities, CBMs are been grouped into three types:

6.3.1. Type A Surface

Binding CBMs This class of CBMs binds to insoluble, highly crystalline cellulose and/or chitin. The aromatic amino acid residues play key role in the binding sites.

The planar architecture of the binding sites is thought to be complementary to the flat surfaces presented by cellulose or chitin crystals (Bayer et al., 1999).

The substrate binding site comprises the “hydrophobic” face of cellulose (Bayer et al., 1999). Upon binding to the substrate, the cellulosome is thought to undergo a supramolecular rearrangement so that the components redistribute to interact with the different target substrate. For this purpose, the various cellulosomal enzymes include different types of CBMs from different families that exhibit appropriate specificities that complement the action of the parent enzyme (Bayeret al., 2004).

6.3.2. Type B Polysaccharide-Chain-Binding CBMs

This class of CBMs binds to individual glycan chains. As with type A CBMs, aromatic residues play a pivotal role in ligand binding, and the orientation of these amino acids are key determinants of specificity. The binding sites often described as grooves or clefts, and comprise several sub-sites able to accommodate the individual sugar units of the polymeric ligand (Simpsonet al., 2000). In sharp contrast with the Type A CBMs, direct hydrogen bonds also play a key role in the defining the affinity and ligand specificity of Type B glycan chain binders (Notenboom et al., 2001; Xieet al., 2001).

6.3.3. Type C Small-Sugar-Binding CBMs

This class of CBMs has the lectin-like property of binding optimally to mono-, di-, or tri-saccharides and thus lacks the extended binding-site grooves of type B CBMs. The distinction between Type B CBMs and Type C CBMs can be subtle (Borastonet al., 2003).

6.3.4. Type D CBMs

This class of CBMs is always found in close spatial proximity with the catalytic domains of their respective proteins. Examples include the cellulase family 9 enzymes fromT. fusca(Sakonet al., 1997).