Limits...
Glycan complexity dictates microbial resource allocation in the large intestine.

Rogowski A, Briggs JA, Mortimer JC, Tryfona T, Terrapon N, Lowe EC, Baslé A, Morland C, Day AM, Zheng H, Rogers TE, Thompson P, Hawkins AR, Yadav MP, Henrissat B, Martens EC, Dupree P, Gilbert HJ, Bolam DN - Nat Commun (2015)

Bottom Line: We show here, using xylan as a model, that sharing the breakdown products of complex carbohydrates by key members of the microbiota, such as Bacteroides ovatus, is dependent on the complexity of the target glycan.Characterization of the extensive xylan degrading apparatus expressed by B. ovatus reveals that the breakdown of the polysaccharide by the human gut microbiota is significantly more complex than previous models suggested, which were based on the deconstruction of xylans containing limited monosaccharide side chains.Our report presents a highly complex and dynamic xylan degrading apparatus that is fine-tuned to recognize the different forms of the polysaccharide presented to the human gut microbiota.

View Article: PubMed Central - PubMed

Affiliation: Institute for Cell and Molecular Biosciences, The Medical School, Newcastle University, Newcastle upon Tyne NE2 4HH, UK.

ABSTRACT
The structure of the human gut microbiota is controlled primarily through the degradation of complex dietary carbohydrates, but the extent to which carbohydrate breakdown products are shared between members of the microbiota is unclear. We show here, using xylan as a model, that sharing the breakdown products of complex carbohydrates by key members of the microbiota, such as Bacteroides ovatus, is dependent on the complexity of the target glycan. Characterization of the extensive xylan degrading apparatus expressed by B. ovatus reveals that the breakdown of the polysaccharide by the human gut microbiota is significantly more complex than previous models suggested, which were based on the deconstruction of xylans containing limited monosaccharide side chains. Our report presents a highly complex and dynamic xylan degrading apparatus that is fine-tuned to recognize the different forms of the polysaccharide presented to the human gut microbiota.

No MeSH data available.


Related in: MedlinePlus

Activity and structure of BACOVA_03438 GH95 α-L-galactosidase and comparison to the Bifidobacterium bifidum GH95 α-L-fucosidase.(a,b) TLC and HPAEC profiles, respectively, of the reaction product (L-galactose) generated by incubating CX at 1 mg ml−1 with 1 μM BACOVA_03438 for 16 h in 20 mM sodium phosphate buffer, pH 7.0, at 37 °C. In (a) the lanes are as follows: (−) CX no enzyme; (+) CX incubated with BACOVA_03438. The asterisk marks the position of the single reaction product released from CX by the GH95. (c) The product of the GH95 is resistant to oxidation by D-galactose dehydrogenase (GDH). (d) Schematic of BACOVA_03438, colour-ramped from blue (N-terminus) to red (C-terminus), with L-Gal bound in the active site (carbohydrate carbons shown as green sticks). (e) Solvent exposed surface representation of B. ovatus GH95. The active site pocket that houses β-L-Gal product is boxed. (f) Side chains of the active site residues of BACOVA_03438 (magenta carbons) and the GH95 α-L-fucosidase (blue carbons; PDB: 2EAE) from Bifidobacterium bifidum, bound to L-Gal (green carbons) and L-Fuc (orange carbons), respectively. Predicted hydrogen bonds between the amino-acid side chains and carbohydrates are shown as yellow dotted lines, except for the H-bond between the Oγ of BACOVA_03438 Thr370 and O6 of L-Gal, which is shown as a red dotted line. (g) Overlay of the active site residues displayed in panel (f) with the side-chains of the Thr/His polymorphism that may play a role in specificity for L-Gal over L-Fuc labelled. A stereo image of a portion of the electron density map is shown in Supplementary Fig. 9.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4491172&req=5

f5: Activity and structure of BACOVA_03438 GH95 α-L-galactosidase and comparison to the Bifidobacterium bifidum GH95 α-L-fucosidase.(a,b) TLC and HPAEC profiles, respectively, of the reaction product (L-galactose) generated by incubating CX at 1 mg ml−1 with 1 μM BACOVA_03438 for 16 h in 20 mM sodium phosphate buffer, pH 7.0, at 37 °C. In (a) the lanes are as follows: (−) CX no enzyme; (+) CX incubated with BACOVA_03438. The asterisk marks the position of the single reaction product released from CX by the GH95. (c) The product of the GH95 is resistant to oxidation by D-galactose dehydrogenase (GDH). (d) Schematic of BACOVA_03438, colour-ramped from blue (N-terminus) to red (C-terminus), with L-Gal bound in the active site (carbohydrate carbons shown as green sticks). (e) Solvent exposed surface representation of B. ovatus GH95. The active site pocket that houses β-L-Gal product is boxed. (f) Side chains of the active site residues of BACOVA_03438 (magenta carbons) and the GH95 α-L-fucosidase (blue carbons; PDB: 2EAE) from Bifidobacterium bifidum, bound to L-Gal (green carbons) and L-Fuc (orange carbons), respectively. Predicted hydrogen bonds between the amino-acid side chains and carbohydrates are shown as yellow dotted lines, except for the H-bond between the Oγ of BACOVA_03438 Thr370 and O6 of L-Gal, which is shown as a red dotted line. (g) Overlay of the active site residues displayed in panel (f) with the side-chains of the Thr/His polymorphism that may play a role in specificity for L-Gal over L-Fuc labelled. A stereo image of a portion of the electron density map is shown in Supplementary Fig. 9.

Mentions: As discussed above the two xylan PULs encode debranching enzymes located in GH families typically associated with xylan degradation (GH43, GH67 and GH115) that cleave α-L-arabinofuranosyl and α-D-glucuronyl linkages. In addition, PUL-XylL encodes three periplasmic proteins that belong to GH31, GH97 and GH95, families that have not previously been implicated in the deconstruction of the polysaccharide. Assays showed that the GH31 enzyme, BACOVA_03422, released xylose from CX but displayed no activity against a range of other xylans (Supplementary Fig. 6). As GH31 enzymes target α-D-glycosides39, BACOVA_03422 is likely an α-xylosidase, consistent with the presence of α-D-Xyl in CX13. BACOVA_03422 is ∼3,000-fold more active against CX than a xyloglucan oligosaccharide (Supplementary Table 6), demonstrating that the enzyme is a xylan-specific α-xylosidase. In contrast, a previously characterized GH31 α-xylosidase, CjXyl31A40, exhibited minimal activity against CX, strongly preferring xyloglucan oligosaccharide (Supplementary Table 6). These data show that the xylan specificity displayed by the BACOVA_03422 enzyme is not a general property of GH31 enzymes. The GH97 enzyme, BACOVA_03423, was active against 4-nitrophenyl-α-D-galactopyranoside, consistent with the presence of α-D-Gal in some highly complex xylans13. The GH95 enzyme BACOVA_03438, although located in an ‘α-L-fucosidase specific family'41, released exclusively L-Gal from CX, displaying only trace activity against oligosaccharides containing terminal α-L-Fuc residues (Fig. 5a–c and Supplementary Table 7). Conversely, a previously characterized GH95 α-1,2-L-fucosidase (AfcA)42 exhibited minimal α-L-galactosidase activity against CX (Supplementary Table 7), showing that GH95 enzymes can be either L-Gal or L-Fuc specific. Although these data provide an initial example of a glycosidase specific for L-galactose, it should be noted that the GH117 family contains enzymes that target the 3,6-anhydro form of the sugar43.


Glycan complexity dictates microbial resource allocation in the large intestine.

Rogowski A, Briggs JA, Mortimer JC, Tryfona T, Terrapon N, Lowe EC, Baslé A, Morland C, Day AM, Zheng H, Rogers TE, Thompson P, Hawkins AR, Yadav MP, Henrissat B, Martens EC, Dupree P, Gilbert HJ, Bolam DN - Nat Commun (2015)

Activity and structure of BACOVA_03438 GH95 α-L-galactosidase and comparison to the Bifidobacterium bifidum GH95 α-L-fucosidase.(a,b) TLC and HPAEC profiles, respectively, of the reaction product (L-galactose) generated by incubating CX at 1 mg ml−1 with 1 μM BACOVA_03438 for 16 h in 20 mM sodium phosphate buffer, pH 7.0, at 37 °C. In (a) the lanes are as follows: (−) CX no enzyme; (+) CX incubated with BACOVA_03438. The asterisk marks the position of the single reaction product released from CX by the GH95. (c) The product of the GH95 is resistant to oxidation by D-galactose dehydrogenase (GDH). (d) Schematic of BACOVA_03438, colour-ramped from blue (N-terminus) to red (C-terminus), with L-Gal bound in the active site (carbohydrate carbons shown as green sticks). (e) Solvent exposed surface representation of B. ovatus GH95. The active site pocket that houses β-L-Gal product is boxed. (f) Side chains of the active site residues of BACOVA_03438 (magenta carbons) and the GH95 α-L-fucosidase (blue carbons; PDB: 2EAE) from Bifidobacterium bifidum, bound to L-Gal (green carbons) and L-Fuc (orange carbons), respectively. Predicted hydrogen bonds between the amino-acid side chains and carbohydrates are shown as yellow dotted lines, except for the H-bond between the Oγ of BACOVA_03438 Thr370 and O6 of L-Gal, which is shown as a red dotted line. (g) Overlay of the active site residues displayed in panel (f) with the side-chains of the Thr/His polymorphism that may play a role in specificity for L-Gal over L-Fuc labelled. A stereo image of a portion of the electron density map is shown in Supplementary Fig. 9.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4491172&req=5

f5: Activity and structure of BACOVA_03438 GH95 α-L-galactosidase and comparison to the Bifidobacterium bifidum GH95 α-L-fucosidase.(a,b) TLC and HPAEC profiles, respectively, of the reaction product (L-galactose) generated by incubating CX at 1 mg ml−1 with 1 μM BACOVA_03438 for 16 h in 20 mM sodium phosphate buffer, pH 7.0, at 37 °C. In (a) the lanes are as follows: (−) CX no enzyme; (+) CX incubated with BACOVA_03438. The asterisk marks the position of the single reaction product released from CX by the GH95. (c) The product of the GH95 is resistant to oxidation by D-galactose dehydrogenase (GDH). (d) Schematic of BACOVA_03438, colour-ramped from blue (N-terminus) to red (C-terminus), with L-Gal bound in the active site (carbohydrate carbons shown as green sticks). (e) Solvent exposed surface representation of B. ovatus GH95. The active site pocket that houses β-L-Gal product is boxed. (f) Side chains of the active site residues of BACOVA_03438 (magenta carbons) and the GH95 α-L-fucosidase (blue carbons; PDB: 2EAE) from Bifidobacterium bifidum, bound to L-Gal (green carbons) and L-Fuc (orange carbons), respectively. Predicted hydrogen bonds between the amino-acid side chains and carbohydrates are shown as yellow dotted lines, except for the H-bond between the Oγ of BACOVA_03438 Thr370 and O6 of L-Gal, which is shown as a red dotted line. (g) Overlay of the active site residues displayed in panel (f) with the side-chains of the Thr/His polymorphism that may play a role in specificity for L-Gal over L-Fuc labelled. A stereo image of a portion of the electron density map is shown in Supplementary Fig. 9.
Mentions: As discussed above the two xylan PULs encode debranching enzymes located in GH families typically associated with xylan degradation (GH43, GH67 and GH115) that cleave α-L-arabinofuranosyl and α-D-glucuronyl linkages. In addition, PUL-XylL encodes three periplasmic proteins that belong to GH31, GH97 and GH95, families that have not previously been implicated in the deconstruction of the polysaccharide. Assays showed that the GH31 enzyme, BACOVA_03422, released xylose from CX but displayed no activity against a range of other xylans (Supplementary Fig. 6). As GH31 enzymes target α-D-glycosides39, BACOVA_03422 is likely an α-xylosidase, consistent with the presence of α-D-Xyl in CX13. BACOVA_03422 is ∼3,000-fold more active against CX than a xyloglucan oligosaccharide (Supplementary Table 6), demonstrating that the enzyme is a xylan-specific α-xylosidase. In contrast, a previously characterized GH31 α-xylosidase, CjXyl31A40, exhibited minimal activity against CX, strongly preferring xyloglucan oligosaccharide (Supplementary Table 6). These data show that the xylan specificity displayed by the BACOVA_03422 enzyme is not a general property of GH31 enzymes. The GH97 enzyme, BACOVA_03423, was active against 4-nitrophenyl-α-D-galactopyranoside, consistent with the presence of α-D-Gal in some highly complex xylans13. The GH95 enzyme BACOVA_03438, although located in an ‘α-L-fucosidase specific family'41, released exclusively L-Gal from CX, displaying only trace activity against oligosaccharides containing terminal α-L-Fuc residues (Fig. 5a–c and Supplementary Table 7). Conversely, a previously characterized GH95 α-1,2-L-fucosidase (AfcA)42 exhibited minimal α-L-galactosidase activity against CX (Supplementary Table 7), showing that GH95 enzymes can be either L-Gal or L-Fuc specific. Although these data provide an initial example of a glycosidase specific for L-galactose, it should be noted that the GH117 family contains enzymes that target the 3,6-anhydro form of the sugar43.

Bottom Line: We show here, using xylan as a model, that sharing the breakdown products of complex carbohydrates by key members of the microbiota, such as Bacteroides ovatus, is dependent on the complexity of the target glycan.Characterization of the extensive xylan degrading apparatus expressed by B. ovatus reveals that the breakdown of the polysaccharide by the human gut microbiota is significantly more complex than previous models suggested, which were based on the deconstruction of xylans containing limited monosaccharide side chains.Our report presents a highly complex and dynamic xylan degrading apparatus that is fine-tuned to recognize the different forms of the polysaccharide presented to the human gut microbiota.

View Article: PubMed Central - PubMed

Affiliation: Institute for Cell and Molecular Biosciences, The Medical School, Newcastle University, Newcastle upon Tyne NE2 4HH, UK.

ABSTRACT
The structure of the human gut microbiota is controlled primarily through the degradation of complex dietary carbohydrates, but the extent to which carbohydrate breakdown products are shared between members of the microbiota is unclear. We show here, using xylan as a model, that sharing the breakdown products of complex carbohydrates by key members of the microbiota, such as Bacteroides ovatus, is dependent on the complexity of the target glycan. Characterization of the extensive xylan degrading apparatus expressed by B. ovatus reveals that the breakdown of the polysaccharide by the human gut microbiota is significantly more complex than previous models suggested, which were based on the deconstruction of xylans containing limited monosaccharide side chains. Our report presents a highly complex and dynamic xylan degrading apparatus that is fine-tuned to recognize the different forms of the polysaccharide presented to the human gut microbiota.

No MeSH data available.


Related in: MedlinePlus