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Metabolism of a hybrid algal galactan by members of the human gut microbiome

Nature Chemical Biology volume 18, pages 501–510 (2022)Cite this article

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Abstract

Native porphyran is a hybrid of porphryan and agarose. As a common element of edible seaweed, this algal galactan is a frequent component of the human diet. Bacterial members of the human gut microbiota have acquired polysaccharide utilization loci (PULs) that enable the metabolism of porphyran or agarose. However, the molecular mechanisms that underlie the deconstruction and use of native porphyran remains incompletely defined. Here, we have studied two human gut bacteria, porphyranolytic Bacteroides plebeius and agarolytic Bacteroides uniformis, that target native porphyran. This reveals an exo-based cycle of porphyran depolymerization that incorporates a keystone sulfatase. In both PULs this cycle also works together with a PUL-encoded agarose depolymerizing machinery to synergistically reduce native porphyran to monosaccharides. This provides a framework for understanding the deconstruction of a hybrid algal galactan, and insight into the competitive and/or syntrophic relationship of gut microbiota members that target rare nutrients.

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Fig. 1: Identification of initiating exo-porphyranase enzymes.
Fig. 2: Structural analysis of initiating exo-porphyranase enzymes.
Fig. 3: An exo-β-d-porphyraoligosaccharide hydrolase leads to the exo-based model of neoporphyraoligosaccharide hydrolysis.
Fig. 4: Depolymerization of native porphyran.
Fig. 5: Native porphyran as a growth substrate.
Fig. 6: Schematics of the depolymerization of native porphyran and oligosaccharides.

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Data availability

Structure coordinates and structure factors determined in this study have been deposited in the PDB with the accession codes of 7LHA, 7LJ2, 7LJJ, 7LK7, 7LNP, 5T98, 5T99 and 7LH6. Existing structure coordinates used in this study that are already deposited in the PDB are 2WVU, 1YNP, 5T9G, 7CWD, 4PSR, 5K9H, 4ZRX, 3GZA, 4OUE, 1ODU, 3UET and 2G2V. All other data are available by request.

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Acknowledgements

This research was supported by Natural Sciences and Engineering Research Council of Canada Discovery Grants (nos. FRN 04355 to A.B.B. and FRN 03929 to W.F.Z.), funding from Agriculture and Agri-Food Canada (project grant no. J-002262 to D.W.A.) and Beef and Cattle Research Council (grant no. FOS.04.17 / J-001973 to D.W.A.). G.R. was supported by funding from the European Union’s Horizons 2020 research and innovations program under the Marie Skłodowska-Curie grant agreement no. 840804. We thank the staff at the CLS where diffraction data were collected. The CLS is supported by the Natural Sciences and Engineering Research Council of Canada, the National Research Council of Canada, the Canadian Institutes of Health Research, the Province of Saskatchewan, Western Economic Diversification Canada and the University of Saskatchewan.

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Author notes

  1. Chelsea Vickers

    Present address: School of Biological Sciences, Victoria University, Wellington, New Zealand

  2. These authors contributed equally: Craig S. Robb, Joanne K. Hobbs.

Authors and Affiliations

  1. Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia, Canada

    Craig S. Robb, Joanne K. Hobbs, Benjamin Pluvinage, Chelsea Vickers, Andrew G. Hettle, Rory Hills & Alisdair B. Boraston

  2. Department of Biochemistry and Molecular Biology and Centre for Blood Research, The University of British Columbia, Vancouver, British Columbia, Canada

    Craig S. Robb

  3. Lethbridge Research and Development Centre, Agriculture and Agri-Food Canada, Lethbridge, Alberta, Canada

    Greta Reintjes, Leeann Klassen, Stephanie Monteith, Carolyn Amundsen, Xiaohui Xing & D. Wade Abbott

  4. Max Planck Institute for Marine Microbiology, Bremen, Germany

    Greta Reintjes & Greta Giljan

  5. Irving K Barber Faculty of Science, The University of British Columbia, Department of Chemistry, Kelowna, British Columbia, Canada

    Nitin & Wesley F. Zandberg

  6. Southern Alberta Genome Sciences Centre, Department of Chemistry and Biochemistry, University of Lethbridge, Lethbridge, Alberta, Canada

    Tony Montina

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Contributions

C.S.R and A.B.B. initiated the project. A.B.B. and D.W.A directed the project. C.S.R performed structural analyses of BuS1_11 and BpGH29 and activity analyses of the S1_11 and GH29 enzymes. J.K.H. performed all galactose release assays for the enzymes in this study and kinetic assays of BpLGDH. B.P. determined the structures of BuGH2A. G.R. and G.G. performed glycan uptake studies. L.K., S.M. and C.A. performed growth assays. C.V. determined the structure of BpLGDH. A.G.H. performed kinetic analysis of BpS1_11. R.H. performed binding studies of SusE-like proteins. N., X.X., T.M. and W.F.Z. performed carbohydrate analysis by NMR, GC–MS and LC–MS. A.B.B. wrote the paper with input from C.S.R. and D.W.A. All authors read and approved the final paper.

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Correspondence to Alisdair B. Boraston.

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Extended data

Extended Data Fig. 1 Structures of algal galactans and the polysaccharide utilization loci (PUL) that target them.

The chemical structures of a, agarose, b, porphyran, and c, native porphyran comprising blocks of porphyran and agarose. d, The PorPUL is shown on top with the AgaPUL beneath. Individual genes are labelled by general function according to the provided legend. Glycoside hydrolases with known agarose and porphyran activity have blue and green labels, respectively. Black labels indicate gene products whose activity are identified in this study. Gray labels indicate unknowns. See also Supplementary Table 1.

Extended Data Fig. 2 Activity analysis of BpLGDH.

a, screen of BpLGDH activity against various monosaccharides using NAD+ as a co-factor. n = 3 independent reactions were performed with each replicate shown along with the mean ± standard deviation. b and c show the kinetics using NAD+ and NADP+, respectively, as a co-factor when excess L-galactose is used as a substrate. d, the kinetics of L-galactose oxidation using excess NADP+ as a co-factor. The data is shown as the mean ± standard deviation of n = 3 independent reactions (see Supplementary Fig. 1 for independent data points). The solid line shows the best fit line to the Michaelis-Menton equation.

Extended Data Fig. 3 Structural analysis of BpLGDH.

a, structure of the BpLGDH dimer with one monomer shown in cartoon and the other as a solvent accessible surface. The putative catalytic histidine is shown in blue. b, overlap of BpLGDH (blue) with AKR11C1 from Bacillus halodurans (grey, PDB ID 1YNP). c, overlap of the BpLGDH (blue) active site with the active site of AKR11C1 (grey). Conserved residues, including the putative catalytic histidine (H119 in AKR11C1), are shown as sticks. In panels c and d the NADPH bound to AKR11C1 is shown in stick representation.

Extended Data Fig. 4 Kinetic and structural analysis of BuS1_11.

a, kinetic analysis of neoporphyrabiose hydrolysis by BpS1_11*. The data is shown as the mean ± standard deviation of n = 3 independent reactions (see Supplementary Fig. 2 for independent data points). The solid line shows the best fit line to the Michaelis-Menton equation. b, the overall fold of BpS1_11 shown as a cartoon. The bound calcium ion is shown as a green sphere and key residues in the active site are shown as blue sticks. c, electron density maps of NP2 (yellow sticks) bound to the BuS1_11 active site are shown with the 2Fo-Fc map at 1σ in blue (top) and the Fo-Fc omit map at 3σ in green (bottom). d, the solvent accessible surface of the active site is shown in transparent grey with the bound NP2 as yellow sticks. The subsites of the active site are labeled in red according to the nomenclature of Hettle et al.27. e, structural overlap of BuS1_11 (blue) with BT4656 (grey, 5G2V). NP2 is shown as yellow sticks and 2-N,6-O-disulfo-d-glucosamine bound to BT4656 shown as green sticks. Relevant inserted structural motifs in each protein that contribute to carbohydrate specificity are shown as solid cartoons.

Extended Data Fig. 5 Structural analysis of BpGH29.

a, cartoon representation of uncomplexed BpGH29. b, electron density maps of L-galactose (yellow sticks) bound to the BpGH29 active site are shown with the 2Fo-Fc map at 1σ in blue (top) and the Fo-Fc omit map at 3σ in green (bottom). c, the specific interactions between the active site and L-galactose. The nucleophile (N) and acid/base (A/B) are colored blue and organge respectively. d, cartoon representation of BpGH29 in complex with L-galactose. In panels a and c the sidechain proposed to act as the nucleophile (D264) is shown as blue sticks, the sidechain proposed to act as the acid/base in orange sticks, and mobile loops that help form the active site in orange and blue. e, electron density maps of pNP-α-L-galactopyranoside (yellow sticks) bound to the BpGH29D264N active site are shown with the 2Fo-Fc map at 1σ in blue (top) and the Fo-Fc omit map at 3σ in green (bottom). f, pNP-α-L-galactopyranoside bound to the BpGH29 D264N active site shown with the solvent accessible surface of the active site shown in grey.

Extended Data Fig. 6 Enzymatic activity and sequencing of a purified tetrasaccharide derived from porphyran.

The enzyme combinations are indicated on the left with the amounts of d-galactose and l-galactose release displayed as a bar chart. The sites of bond hydrolysis are indicated in the schematics on the right side of the figure. Thirteen nanomoles (13 nmoles) of tetrasaccharide were used in each reaction and each reaction was performed in independent triplicate (n = 3) reactions with each replicate shown along with the mean ± standard deviation.

Extended Data Fig. 7 Structural analysis of BuGH2A.

a, cartoon representation of BuGH2A showing the central (α/β)8-barrel sitting in a nest of four Ig-like domains. b, electron density maps of galactoisofagomine (green sticks) bound to the BuGH2A active site are shown with the 2Fo-Fc map at 1σ in blue (top) and the Fo-Fc omit map at 3σ in green (bottom).

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Supplementary Figs. 1–16, Tables 1–8 and References.

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Robb, C.S., Hobbs, J.K., Pluvinage, B. et al. Metabolism of a hybrid algal galactan by members of the human gut microbiome. Nat Chem Biol 18, 501–510 (2022). https://doi.org/10.1038/s41589-022-00983-y

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