Novel architecture of family-9 glycoside hydrolases identified in cellulosomal enzymes of<i>Acetivibrio cellulolyticus</i>and<i>Clostridium thermocellum</i>

Jindou, Sadanari; Xu, Qi; Kenig, Rina; Shulman, Michal; Shoham, Yuval; Bayer, Edward A.; Lamed, Raphael

doi:10.1111/j.1574-6968.2005.00040.x

Cited by 50 publications

(47 citation statements)

References 33 publications

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“…The architectural diversity of GH9 modules have been assigned to four different groups known as theme A, B, C, and D (29). In CbCel9B/Man5A, the GH9 catalytic module is linked to an accessory CBM3c at its C terminus, and this is the architecture of the members of theme B1.…”

Section: Resultsmentioning

confidence: 99%

“…Our results suggest that a CBM3c can indeed bind to insoluble cellulose although the binding appeared weak. A sequence alignment of CbCel9B/ Man5A with its homologs revealed that considerable differences exist in the amino acid residues proposed to interact with the ligand (29,32,33) between CbCel9B/Man5A CBM3c with its homologues. Notably, the Q553, R557, E559, and R563 residues in ThefuCel9A, proposed to interact with the ligand, are replaced by E545, K549, Q561, and K565, respectively, in the CBM3c of CbCel9B/Man5A (see Fig.…”

Section: Discussionmentioning

confidence: 99%

“…A three-dimensional structure of a CBM3c in complex with a ligand is still lacking, which hinders accurate designation of residues important for ligand binding. Due to the diversity of CBM3c modules (29), one may postulate that there are variants of CBM3c that are capable of holding a cellooligosaccharide tightly enough to capture this complex in a crystal.…”

Section: Discussionmentioning

confidence: 99%

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Biochemical and Mutational Analyses of a Multidomain Cellulase/Mannanase from Caldicellulosiruptor bescii

Mackie

Cann

2012

Appl Environ Microbiol

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Thermophilic cellulases and hemicellulases are of significant interest to the biofuel industry due to their perceived advantages over their mesophilic counterparts. We describe here biochemical and mutational analyses of Caldicellulosiruptor bescii Cel9B/ Man5A (CbCel9B/Man5A), a highly thermophilic enzyme. As one of the highly secreted proteins of C. bescii, the enzyme is likely to be critical to nutrient acquisition by the bacterium. CbCel9B/Man5A is a modular protein composed of three carbohydratebinding modules flanked at the N terminus and the C terminus by a glycoside hydrolase family 9 (GH9) module and a GH5 module, respectively. Based on truncational analysis of the polypeptide, the cellulase and mannanase activities within CbCel9B/ Man5A were assigned to the N-and C-terminal modules, respectively. CbCel9B/Man5A and its truncational mutants, in general, exhibited a pH optimum of ϳ5.5 and a temperature optimum of 85°C. However, at this temperature, thermostability was very low. After 24 h of incubation at 75°C, the wild-type protein maintained 43% activity, whereas a truncated mutant, TM1, maintained 75% activity. The catalytic efficiency with phosphoric acid swollen cellulose as a substrate for the wild-type protein was 7.2 s ؊1 ml/mg, and deleting the GH5 module led to a mutant (TM1) with a 2-fold increase in this kinetic parameter. Deletion of the GH9 module also increased the apparent k cat of the truncated mutant TM5 on several mannan-based substrates; however, a concomitant increase in the K m led to a decrease in the catalytic efficiencies on all substrates. These observations lead us to postulate that the two catalytic activities are coupled in the polypeptide.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Biochemical and Mutational Analyses of a Multidomain Cellulase/Mannanase from Caldicellulosiruptor bescii

Mackie

Cann

2012

Appl Environ Microbiol

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“…Moreover, many cellulosomal enzymes contain different types of CBMs, which may provide the ability to bind to different substrates. Some CBMs seem to have an anchor function, whereas others have been hypothesized to be "helper" CBMs capable of holding a single cellulose chain and feeding it to its catalytic module partner (49). The immunoglobulin-like modules on Family 9 enzymes are believed to stabilize the catalytic modules (50).…”

Section: Architecturementioning

confidence: 99%

Modeling the Self-assembly of the Cellulosome Enzyme Complex

Bomble

Beckham

Matthews

et al. 2011

Journal of Biological Chemistry

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Most bacteria use free enzymes to degrade plant cell walls in nature. However, some bacteria have adopted a different strategy wherein enzymes can either be free or tethered on a protein scaffold forming a complex called a cellulosome. The study of the structure and mechanism of these large macromolecular complexes is an active and ongoing research topic, with the goal of finding ways to improve biomass conversion using cellulosomes. Several mechanisms involved in cellulosome formation remain unknown, including how cellulosomal enzymes assemble on the scaffoldin and what governs the population of cellulosomes created during self-assembly. Here, we present a coarse-grained model to study the self-assembly of cellulosomes. The model captures most of the physical characteristics of three cellulosomal enzymes (Cel5B, CelS, and CbhA) and the scaffoldin (CipA) from Clostridium thermocellum. The protein structures are represented by beads connected by restraints to mimic the flexibility and shapes of these proteins. From a large simulation set, the assembly of cellulosomal enzyme complexes is shown to be dominated by their shape and modularity. The multimodular enzyme, CbhA, binds statistically more frequently to the scaffoldin than CelS or Cel5B. The enhanced binding is attributed to the flexible nature and multimodularity of this enzyme, providing a longer residence time around the scaffoldin. The characterization of the factors influencing the cellulosome assembly process may enable new strategies to create designers cellulosomes.

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“…Amino acid sequences were aligned using DNAMAN. The secondary structures marked with horizontal arrows refer to those relevant to family 3 CBMs and the previous identified cellulose-binding residues (18,30) are boxed. Conserved amino acids are shown on a gray background, and the unique conserved amino acids of CBM3d are shown on a black background.…”

Section: Figmentioning

confidence: 99%

CBM3d, a Novel Subfamily of Family 3 Carbohydrate-Binding Modules Identified in Cel48A Exoglucanase of Cellulosilyticum ruminicola

2011

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Previously, we found that exoglucanase Cel48A from Cellulosilyticum ruminicola H1 bound intensively to Avicel; however, no known carbohydrate-binding module (CBM) was observed in the protein. Bioinformatics suggested that a C-terminal fragment of 127 amino acids, named the Cellulosilyticum-specific paralogous module (CPM), could function in binding. CPM-appended proteins are all putative (hemi)cellulases from Cellulosilyticum spp. In the present work, we demonstrated that Cel48A without the CPM retained only exoglucanase activity and lost the Avicel-binding ability, while the isolated CPM exhibited a high affinity for Avicel. In addition, the CPM bound to chitin, but not to soluble polysaccharides, making it a type A CBM, which binds only insoluble polysaccharides. Phylogenetic analysis clustered the CPM and its homologs as a separate branch that was distantly related to CBM subfamilies 3a (28% identity), 3b (24% identity), and 3c (21% identity). Sequence alignment revealed distinct secondary structures of the new CBM 3 group, in particular, a conserved Pro66-Trp67 insert preceding strand ␤4, a deletion preceding strand ␤6, and incomplete strands ␤8 and ␤9. An alanine scan for six aromatic and three nonaromatic amino acid residues (D66, P66, and R111) by site-directed mutagenesis determined that Phe62, Pro66, Trp67, Tyr68, Arg111, and Trp117 were the functional residues for binding. Among them, Phe62, Pro66, and Trp67 were the newly determined key sites in the CPM for binding. Three-dimensional homolog modeling revealed two types of substrate-binding sites, planar and groove, in the CPM. Thus, a new subfamily, CBM family 3d, is proposed.

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Novel architecture of family-9 glycoside hydrolases identified in cellulosomal enzymes ofAcetivibrio cellulolyticusandClostridium thermocellum

Cited by 50 publications

References 33 publications

Biochemical and Mutational Analyses of a Multidomain Cellulase/Mannanase from Caldicellulosiruptor bescii

Biochemical and Mutational Analyses of a Multidomain Cellulase/Mannanase from Caldicellulosiruptor bescii

Modeling the Self-assembly of the Cellulosome Enzyme Complex

CBM3d, a Novel Subfamily of Family 3 Carbohydrate-Binding Modules Identified in Cel48A Exoglucanase of Cellulosilyticum ruminicola

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