Mutational Insights into the Roles of Amino Acid Residues in Ligand Binding for Two Closely Related Family 16 Carbohydrate Binding Modules

Su, Xiaoyun; Agarwal, Vinayak; Dodd, Dylan; Bae, Byungchan; Mackie, Roderick I.; Nair, Satish K.; Cann, Isaac

doi:10.1074/jbc.m110.168302

Cited by 12 publications

(4 citation statements)

References 40 publications

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“…AGE showed that the mutants K455A, E485A, and Q487A of CBM80 RfGH5 retained WT affinity for all of the ligands tested, which suggests that the predicted polar interactions between the protein and β-glycans have very little influence on affinity. These data are unusual among type B CBMs where direct polar interactions generally make an important contribution to ligand recognition (14,15). The data, however, are consistent with CfCBM2b-1, in which affinity is dominated by two tryptophans (16).…”

supporting

confidence: 62%

Complexity of the Ruminococcus flavefaciens cellulosome reflects an expansion in glycan recognition

Venditto

Luís

Rydahl

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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The breakdown of plant cell wall (PCW) glycans is an important biological and industrial process. Noncatalytic carbohydrate binding modules (CBMs) fulfill a critical targeting function in PCW depolymerization. Defining the portfolio of CBMs, the CBMome, of a PCW degrading system is central to understanding the mechanisms by which microbes depolymerize their target substrates. Ruminococcus flavefaciens, a major PCW degrading bacterium, assembles its catalytic apparatus into a large multienzyme complex, the cellulosome. Significantly, bioinformatic analyses of the R. flavefaciens cellulosome failed to identify a CBM predicted to bind to crystalline cellulose, a key feature of the CBMome of other PCW degrading systems. Here, high throughput screening of 177 protein modules of unknown function was used to determine the complete CBMome of R. flavefaciens. The data identified six previously unidentified CBM families that targeted β-glucans, β-mannans, and the pectic polysaccharide homogalacturonan. The crystal structures of four CBMs, in conjunction with site-directed mutagenesis, provide insight into the mechanism of ligand recognition. In the CBMs that recognize β-glucans and β-mannans, differences in the conformation of conserved aromatic residues had a significant impact on the topology of the ligand binding cleft and thus ligand specificity. A cluster of basic residues in CBM77 confers calcium-independent recognition of homogalacturonan, indicating that the carboxylates of galacturonic acid are key specificity determinants. This report shows that the extended repertoire of proteins in the cellulosome of R. flavefaciens contributes to an extended CBMome that supports efficient PCW degradation in the absence of CBMs that specifically target crystalline cellulose.

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supporting

confidence: 62%

Complexity of the Ruminococcus flavefaciens cellulosome reflects an expansion in glycan recognition

Venditto

Luís

Rydahl

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…S3 in the supplemental material). This observation may be akin to the fine-tuning demonstrated in a Caldanaerobius polysaccharolyticus family 16 CBM by mutating one polar residue (Q121 to E121) involved in hydrogen bonding with the ligand (47). The E545, K549, Q561, and K565 residues can also be found in four CBM3 modules from the related organisms C. kronotskyensis, C. saccharolyticus, C. obsidiansis, and Caldicellulosiruptor sp.…”

Section: Discussionmentioning

confidence: 92%

“…Single colonies were inoculated in 7 ml of LB medium supplemented with 100 g of ampicillin/ml and cultured for 10 h. The plasmids were extracted from the recombinant E. coli cells, and the inserts were sequenced (W. M. Keck Center for Comparative and Functional Genomics, University of Illinois) to confirm the presence of the desired mutation. Circular dichroism scans of mutated proteins were carried out using a J-815 CD spectropolarimeter (Jasco, Japan) equipped with a constant-temperature cell-holder as described in our previous report (47). Briefly, proteins (0.2 mg/ml) in a phosphate buffer (10 mM sodium phosphate [pH 7.0]) in a 1-mm quartz cuvette were kept at 25°C for 5 min before scanning.…”

Section: Methodsmentioning

confidence: 99%

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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“…Here we discuss the dynamics of Caldanaerobius polysaccharolyticus Man5B, an enzyme that cleaves both β-1,4 glucosidic and β-1,4 mannosidic linkages [ 5 ]. Man5B and Man5A, two glycoside hydrolase family 5 (GH5) enzymes from the same bacterium, were shown to act synergistically and at high temperature on enzymatic conversion of plant cell wall polysaccharides to fermentable sugars [ 5 , 6 ], a property that is highly desirable in the emerging biofuel industry [ 5 , 7 , 8 ].…”

Section: Introductionmentioning

confidence: 99%

Molecular dynamics study of enhanced Man5B enzymatic activity

Bernardi

Cann

Schulten

2014

Biotechnol Biofuels

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BackgroundBiofuels are a well-known alternative to the largely used fossil-derived fuels, however the competition with food production is an ethical dilemma. Fortunately a solution is offered by second-generation biofuels which can be produced from agricultural waste or, more specifically, from plant cell wall polysaccharides. The conversion process involves typically enzymatic hydrolysis of lignocellulosic biomass and then separation of its constituent sugars that are further fermented to produce ethanol. Over the years several technologies have been developed that allow this conversion process to occur and the objective is now to make this process cost-competitive in today’s markets.ResultsWe observe that reduction of enzymatic efficiency in the presence of gluco-oligosaccharides is associated with a loss of the enzyme’s flexibility, the latter being required to bind new substrate, while the presence of manno-oligosaccharides does not pose this problem. Molecular dynamics simulations identify key contacts between substrates and the enzyme catalytic pocket that might be modified through site-directed mutagenesis to prevent loss of enzymatic efficiency.ConclusionsBased on previous experimental studies and the new molecular dynamics data, we suggest that cellohexaose in the active site pocket slows down or even inhibits Man5B enzymatic activity. The assumption of such a mechanism is reasonable since when the gluco-oligosaccharide substrate is attached to the catalytic pocket it takes much longer to leave the pocket and thus prevents other substrates from reaching the active site. The insight is of crucial importance since the inhibition of enzymes by the enzymatic product or by an unsuitable substrate is a major technological problem in reducing the competitiveness of second-generation biofuel production.

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Mutational Insights into the Roles of Amino Acid Residues in Ligand Binding for Two Closely Related Family 16 Carbohydrate Binding Modules

Cited by 12 publications

References 40 publications

Complexity of the Ruminococcus flavefaciens cellulosome reflects an expansion in glycan recognition

Complexity of the Ruminococcus flavefaciens cellulosome reflects an expansion in glycan recognition

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

Molecular dynamics study of enhanced Man5B enzymatic activity

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