Cell Surface Enzyme Attachment Is Mediated by Family 37 Carbohydrate-Binding Modules, Unique to Ruminococcus albus

Abstract

The rumen bacterium Ruminococcus albus binds to and degrades crystalline cellulosic substrates via a unique cellulose degradation system. A unique family of carbohydrate-binding modules (CBM37), located at the C terminus of different glycoside hydrolases, appears to be responsible both for anchoring these enzymes to the bacterial cell surface and for substrate binding.Ruminococcus albus is widely recognized as one of the specialist cellulose-degrading bacteria resident in the rumena and gastrointestinal tracts of herbivores. Most isolates have been shown to utilize cellulose, xylan, and cellobiose as carbon sources and found to produce a wide range of enzymatic activities, including ␤-glucosidase, ␤-xylosidase, ␣-galactosidase, ␣-arabinosidase, cellulase, polygalacturonase, and ␤-1,4-xylanase activities (2, 3, 5). Interestingly, many of these glycoside hydrolases bear a recently described family 37 carbohydratebinding module (CBM37), which appears to be exclusive to R. albus. These ϳ100-residue modules were first identified at the C-terminal ends of an exoglucanase (Cel48A) and a processive endocellulase (Cel9B) from R. albus strain 8 (1) and appear to be nondiscriminatory in their carbohydrate-binding properties, recognizing a variety of polysaccharides, including cellulose, xylan, chitin, and lichenan (21). This breadth of adhesive properties makes the CBM37 family unique among the CBM families known to date. A preliminary examination of the draft genome sequence for R. albus strain 8 suggests that CBM37 modules, which are grouped into three major subtypes, are present in numerous R. albus polysaccharide-degrading enzymes and other nonenzymatic proteins from this bacterium (http://blast.jcvi.org/rumenomics/index.cgi).Previous studies have shown that effective cellulose hydrolysis by R. albus strains is conditional on the provision of micromolar concentrations of phenylacetic and phenylpropionic acids (9,17,18). These compounds appear to influence capsule formation by the bacterium, and cellulase activity is retained as high-molecular-mass complexes on the bacterial cell surface. In the absence of phenylacetic and phenylpropionic acids, the adhesion of the bacterium to cellulose (and its hydrolysis) is negatively affected. Additionally, cellulase activity is secreted into the culture medium and, by size exclusion chromatography, is shown to be present in a form suggesting that there is no aggregation of activity into larger, multiprotein complexes (17). Although it was long believed that these characteristics were attributable to a cellulosomal mode of enzyme organization, the identification of CBM37 modules (rather than dockerins) in these two key enzymes suggests that the CBM37 modules might play some role(s) in protein retention to the bacterial cell surface. In the present study, we present evidence to validate this hypothesis, and we propose that an additional function for the CBM37 family is the attachment of the parent protein to the bacterium's cell surface.Three different CBM37 modules from R. albus, C...

The Biochemistry and Structural Biology of Plant Cell Wall Deconstruction

“…Thus, the CBM35 from the Amycolatopsis orientalis exo-b-D-glucosaminidase (Chi-CBM35) tethers the enzyme to the cell wall of the bacterium (Montanier et al, 2009b). Similarly, a family of CBMs unique to Ruminococcus albus, which bind to a wide spectrum of b-linked plant structural polysaccharides (CBM37), anchor their cognate enzymes to the surface of the bacterium (Ezer et al, 2008). The biological rationale for this CBM function appears to be to keep the enzymes in close proximity to the bacterium.…”

“…The biological rationale for this CBM function appears to be to keep the enzymes in close proximity to the bacterium. However, Ezer et al (2008) also proposed a model in which the CBM37 acts as a shuttle that transfers the appended enzymes from the bacterial surface to the plant cell wall. In any event, these recent reports of the cell adhesion role of CBMs, which was previously unconsidered, may prove to factor prominently in the function of these protein modules in the future.…”

Discrete and Structurally Unique Proteins (Tāpirins) Mediate Attachment of Extremely Thermophilic Caldicellulosiruptor Species to Cellulose

“…Additionally, a large (180 kDa) glycosylated protein from F. succinogenes binds cellulose (12) through a Fibrobacter-unique "fibro-slime domain" (11). Another ruminal bacterium, Ruminococcus albus, uses a unique CBM family (CBM37) to facilitate anchoring of enzymes to the cell surface (13,14). Other glycosylated proteins from R. albus also play a role in cellulose binding, including PilA1 homologs (15) from strains 8 (16) and 20 (17).…”

Complete Genome of the Cellulolytic Ruminal Bacterium Ruminococcus albus 7

“…The genome encodes a variety of cellulases and hemicellulases, including 3 glycosyl hydrolase (GH) family 2s (GH2s), 4 GH3s, 13 GH5s, 1 GH8, 8 GH9s, 5 GH10s, 5 GH11s, and 7 GH43s, as reported in the Carbohydrate-Active Enzyme (CAZy) database (3). A number of these cellulase and hemicellulase genes also encode carbohydrate-binding module 37 (CBM37), which is known to aid in substrate binding and is found exclusively in R. albus (6). Finally, genes encoding structural components of cellulosomes, such as dockerins, were found throughout the genome.…”