A Low-Volume, Parallel Copper-Bicinchoninic Acid (BCA) Assay for Glycoside Hydrolases

Arnal, Grégory; Attia, Mohamed Adel; Asohan, Jathavan; Brumer, Harry

doi:10.1007/978-1-4939-6899-2_1

Cited by 19 publications

(11 citation statements)

References 15 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Enzymatic assays. Polysaccharide hydrolysis was quantified using a BCA reducing-sugar assay (74). Assays were conducted in a final volume of 100 l at the optimum pH and 37°C for 10 min.…”

Section: Discussionmentioning

confidence: 99%

Synergy between Cell Surface Glycosidases and Glycan-Binding Proteins Dictates the Utilization of Specific Beta(1,3)-Glucans by Human Gut Bacteroides

Déjean

Tamura

Cabrera

et al. 2020

mBio

Self Cite

117

View full text Add to dashboard Cite

The human gut microbiota (HGM) has far-reaching impacts on human health and nutrition, which are fueled primarily by the metabolism of otherwise indigestible complex carbohydrates commonly known as dietary fiber. However, the molecular basis of the ability of individual taxa of the HGM to address specific dietary glycan structures remains largely unclear. In particular, the utilization of β(1,3)-glucans, which are widespread in the human diet as yeast, seaweed, and plant cell walls, had not previously been resolved. Through a systems-based approach, here we show that the symbiont Bacteroides uniformis deploys a single, exemplar polysaccharide utilization locus (PUL) to access yeast β(1,3)-glucan, brown seaweed β(1,3)-glucan (laminarin), and cereal mixed-linkage β(1,3)/β(1,4)-glucan. Combined biochemical, enzymatic, and structural analysis of PUL-encoded glycoside hydrolases (GHs) and surface glycan-binding proteins (SGBPs) illuminates a concerted molecular system by which B. uniformis recognizes and saccharifies these distinct β-glucans. Strikingly, the functional characterization of homologous β(1,3)-glucan utilization loci (1,3GUL) in other Bacteroides further demonstrated that the ability of individual taxa to utilize β(1,3)-glucan variants and/or β(1,3)/β(1,4)-glucans arises combinatorially from the individual specificities of SGBPs and GHs at the cell surface, which feed corresponding signals to periplasmic hybrid two-component sensors (HTCSs) via TonB-dependent transporters (TBDTs). These data reveal the importance of cooperativity in the adaptive evolution of GH and SGBP cohorts to address individual polysaccharide structures. We anticipate that this fine-grained knowledge of PUL function will inform metabolic network analysis and proactive manipulation of the HGM. Indeed, a survey of 2,441 public human metagenomes revealed the international, yet individual-specific, distribution of each 1,3GUL. IMPORTANCE Bacteroidetes are a dominant phylum of the human gut microbiota (HGM) that target otherwise indigestible dietary fiber with an arsenal of polysaccharide utilization loci (PULs), each of which is dedicated to the utilization of a specific complex carbohydrate. Here, we provide novel insight into this paradigm through functional characterization of homologous PULs from three autochthonous Bacteroides species, which target the family of dietary β(1,3)-glucans. Through detailed biochemical and protein structural analysis, we observed an unexpected diversity in the substrate specificity of PUL glycosidases and glycan-binding proteins with regard to β(1,3)-glucan linkage and branching patterns. In combination, these individual enzyme and protein specificities support taxon-specific growth on individual β(1,3)-glucans. This detailed metabolic insight, together with a comprehensive survey of individual 1,3GULs across human populations, further expands the fundamental roadmap of the HGM, with potential application to the future development of microbial intervention therapies.

show abstract

“…Enzymatic assays. Polysaccharide hydrolysis was quantified using a BCA reducing-sugar assay (74). Assays were conducted in a final volume of 100 l at the optimum pH and 37°C for 10 min.…”

Section: Discussionmentioning

confidence: 99%

Synergy between Cell Surface Glycosidases and Glycan-Binding Proteins Dictates the Utilization of Specific Beta(1,3)-Glucans by Human Gut Bacteroides

Déjean

Tamura

Cabrera

et al. 2020

mBio

Self Cite

117

View full text Add to dashboard Cite

show abstract

“…The bicinchoninic acid‐copper (BCA) assay was performed as previously described (Arnal et al ., ). The pH activity optimum of VvEG16(ΔV152) was determined by incubating 1 mg ml −1 tXyG with 5 μg ml −1 VvEG16(ΔV152) for 15 min at room temperature (22–25°C) in 50 m m buffer containing 1 m m EDTA.…”

Section: Methodsmentioning

confidence: 97%

Crystallographic insight into the evolutionary origins of xyloglucan endotransglycosylases and endohydrolases

et al. 2017

Self Cite

View full text Add to dashboard Cite

SUMMARY The xyloglucan endo-transglycosylase/hydrolase (XTH) gene family encodes enzymes of central importance to plant cell wall remodelling. The evolutionary history of plant XTH gene products is incompletely understood vis-à-vis the larger body of bacterial endo-glycanases in Glycoside Hydrolase Family 16 (GH16). To provide molecular insight into this issue, high-resolution X-ray crystal structures and detailed enzyme kinetics of an extant transitional plant endo-glucanase (EG) were determined. Functionally intermediate between plant XTH gene products and bacterial licheninases of GH16, Vitis vinifera EG16 (VvEG16) effectively catalyzes the hydrolysis of the backbones of two dominant plant cell wall matrix glycans, xyloglucan (XyG) and β(1,3)/β(1,4)-mixed-linkage glucan (MLG). Crystallographic complexes with extended oligosaccharide substrates reveal the structural basis for the accommodation of both unbranched, mixed-linked (MLG) and highly decorated, linear (XyG) polysaccharide chains in a broad, extended active-site cleft. Structural comparison with representative bacterial licheninases, a xyloglucan endo-tranglycosylase (XET), and a xyloglucan endo-hydrolase (XEH) outline the functional ramifications of key sequence deletions and insertions across the phylogenetic landscape of GH16. Although the biological role(s) of EG16 orthologs remains to be fully resolved, the present biochemical and tertiary structural characterization provides key insight into plant cell wall enzyme evolution, which will continue to inform genomic analyses and functional studies across species.

show abstract

“…For all enzyme assays on polysaccharides, the activity was determined using the BCA assay as described previously (84). Substrate specificity was determined in 50 mM sodium phosphate buffer, pH 7.0, using 0.5 mg/ml substrate and 1 g/ml enzyme overnight at 37°C.…”

Section: Carbohydrate Sourcesmentioning

confidence: 99%

Substrate specificity, regiospecificity, and processivity in glycoside hydrolase family 74

Arnal

Stogios

Asohan

et al. 2019

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Glycoside hydrolase family 74 (GH74) is a historically important family of endo-␤-glucanases. On the basis of early reports of detectable activity on cellulose and soluble cellulose derivatives, GH74 was originally considered to be a "cellulase" family, although more recent studies have generally indicated a high specificity toward the ubiquitous plant cell wall matrix glycan xyloglucan. Previous studies have indicated that GH74 xyloglucanases differ in backbone cleavage regiospecificities and can adopt three distinct hydrolytic modes of action: exo, endo-dissociative, and endo-processive. To improve functional predictions within GH74, here we coupled in-depth biochemical characterization of 17 recombinant proteins with structural biology-based investigations in the context of a comprehensive molecular phylogeny, including all previously characterized family members. Elucidation of four new GH74 tertiary structures, as well as one distantly related dual seven-bladed ␤propeller protein from a marine bacterium, highlighted key structure-function relationships along protein evolutionary trajectories. We could define five phylogenetic groups, which delineated the mode of action and the regiospecificity of GH74 members. At the extremes, a major group of enzymes diverged to hydrolyze the backbone of xyloglucan nonspecifically with a dissociative mode of action and relaxed backbone regiospecificity. In contrast, a sister group of GH74 enzymes has evolved a large hydrophobic platform comprising 10 subsites, which facilitates processivity. Overall, the findings of our study refine our understanding of catalysis in GH74, providing a framework for future experimentation as well as for bioinformatics predictions of sequences emerging from (meta)genomic studies. Terrestrial plants harbor ϳ80% of the biomass on Earth, some 450 gigatons of carbon, in the form of lignocellulose (cell walls comprised of cellulose, matrix glycans, lignin, and other polymers) (1). Although terrestrial biomass represents an attractive renewable resource for the production of fuels, chemicals, and materials for human consumption, the controlled degradation of lignocellulose, whether (thermo)chemical or enzymatic, is hindered by its heterogeneous composition and complex organization (2). Hence, significant efforts have been made to identify enzymes able to efficiently modify and deconstruct this complex material. Xyloglucans (XyGs) 3 comprise a prominent family of cell wall matrix glycans (hemicelluloses). XyGs are ubiquitous in land plants, in which they constitute up to 20% of the dry weight of cell walls (3, 4). Notably, XyGs are secreted by roots of diverse plant species and are therefore likely to actively influence rhizobiota (5). XyGs are also found as storage polysaccharides comprising ϳ50% of the mass of some seeds (e.g. tamarind and nasturtium) and therefore represent important agricultural byproducts with applications in the food, biomaterial, and medical sectors (6, 7). XyGs have a ␤-1,4-linked glucosyl backbone ("G" unit), some of which a...

show abstract

A Low-Volume, Parallel Copper-Bicinchoninic Acid (BCA) Assay for Glycoside Hydrolases

Cited by 19 publications

References 15 publications

Synergy between Cell Surface Glycosidases and Glycan-Binding Proteins Dictates the Utilization of Specific Beta(1,3)-Glucans by Human Gut Bacteroides

Synergy between Cell Surface Glycosidases and Glycan-Binding Proteins Dictates the Utilization of Specific Beta(1,3)-Glucans by Human Gut Bacteroides

Crystallographic insight into the evolutionary origins of xyloglucan endotransglycosylases and endohydrolases

Substrate specificity, regiospecificity, and processivity in glycoside hydrolase family 74

Contact Info

Product

Resources

About