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BoGH13ASus from Bacteroides ovatus represents a novel α-amylase used for  Bacteroides starch breakdown in the human gut

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Abstract

Members of the Bacteroidetes phylum in the human colon deploy an extensive number of proteins to capture and degrade polysaccharides. Operons devoted to glycan breakdown and uptake are termed polysaccharide utilization loci or PUL. The starch utilization system (Sus) is one such PUL and was initially described in Bacteroides thetaiotaomicron (Bt). BtSus is highly conserved across many species, except for its extracellular α-amylase, SusG. In this work, we show that the Bacteroides ovatus (Bo) extracellular α-amylase, BoGH13ASus, is distinguished from SusG in its evolutionary origin and its domain architecture and by being the most prevalent form in Bacteroidetes Sus. BoGH13ASus is the founding member of both a novel subfamily in the glycoside hydrolase family 13, GH13_47, and a novel carbohydrate-binding module, CBM98. The BoGH13ASus CBM98–CBM48–GH13_47 architecture differs from the CBM58 embedded within the GH13_36 of SusG. These domains adopt a distinct spatial orientation and invoke a different association with the outer membrane. The BoCBM98 binding site is required for Bo growth on polysaccharides and optimal enzymatic degradation thereof. Finally, the BoGH13ASus structure features bound Ca2+ and Mn2+ ions, the latter of which is novel for an α-amylase. Little is known about the impact of Mn2+ on gut bacterial function, much less on polysaccharide consumption, but Mn2+ addition to Bt expressing BoGH13ASus specifically enhances growth on starch. Further understanding of bacterial starch degradation signatures will enable more tailored prebiotic and pharmaceutical approaches that increase starch flux to the gut.

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Availability of data and material

BoGH13ASus native, maltoheptaose and acarbose-bound structures were deposited with the RCSB Protein Data Bank, with PDB IDs of: 8DGE, 8DL1 and 8DL2, respectively. All plasmids, proteins, bacterial strains and other reagents generated in this study will be made freely available to any researcher wishing to use them for non-commercial purposes.

Abbreviations

AP:

Potato amylopectin

Bo:

Bacteroides ovatus

Bt:

Bacteroides thetaiotaomicron

CBM:

Carbohydrate-binding module

G2:

Maltose

G3:

Maltotriose

G4:

Maltotetraose

G5:

Maltopentaose

G6:

Maltohexaose

G7:

Maltoheptaose

GH:

Glycoside hydrolase

Glc:

Glucose

PS:

Potato starch

PUL:

Polysaccharide utilization loci

Sus:

Starch utilization system

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Acknowledgements

We thank members of the Koropatkin, Martens and Ruotolo laboratories for helpful feedback on this work.

Funding

This work was supported by the following National Institutes of Health (NIH) grants from the National Institute of General Medical Sciences (NIGMS): R01-GM118475 to N.M.K. and R01-GM095832 to B.T.R. H.A.B was supported by an NIH Ruth L. Kirschstein Postdoctoral National Research Service Award F32-AT011278 from the National Center for Complementary and Integrative Health. The QE UMHR work was supported by the University of Michigan Biosciences Initiative. Use of the Pilatus 3 1 M detector was provided by grant 1S10OD018090-01 from NIGMS at the NIH. Use of the LS-CAT Sector 21 was supported by the Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor (grant no.: 085P1000817). N.T. and M.B. were supported by the Agence National de la Recherche [grant number ANR-20-CE20-0022].

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Authors and Affiliations

  1. Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI, 48109, USA

    Haley A. Brown, Anna L. DeVeaux, Amanda L. Photenhauer & Nicole M. Koropatkin

  2. Department of Chemistry, University of Michigan, Ann Arbor, MI, 48109, USA

    Brock R. Juliano, Russell E. Bornschein & Brandon T. Ruotolo

  3. Architecture et Fonction des Macromolécules Biologiques, UMR 7257, CNRS AMU; USC1408 INRAE, 13288, Marseille, France

    Matthieu Boulinguiez & Nicolas Terrapon

  4. Synchrotron Research Center, Life Science Collaborative Access Team, Northwestern University, Lemont, IL, USA

    Zdzislaw Wawrzak

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Contributions

HAB cloned, expressed and purified protein constructs as well as performed crystallography experiments, X-ray data collection and structural model refinement, cloned and generated all variant Bt and Bo strains and performed bacterial growth experiments, TLC, ITC, Native PAGE, Western blot, enzyme kinetics, protein sequence analysis, and wrote and edited the original manuscript. ALD cloned, expressed and purified protein constructs, performed TLC and crystallography experiments and did structural model refinement, as well as collected ITC data. BRJ collected and analyzed native MS and ICP-MS data. ALP performed ITC experiments. MB performed bioinformatics analyses of CBM98 and GH13_47. REB assisted with ICP-MS data collection. ZW collected and processed X-ray data. BTR helped direct research and analyzed MS data. NT performed bioinformatics analyses of CBM98 and GH13_47, created the CBM98 family and GH13_47 subfamily within CAZy and analyzed their distribution in the private CAZy database. HAB, BLJ, MB and NT were responsible for data visualization. NMK helped design and direct research and wrote the original manuscript. All authors provided input on the manuscript.

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Correspondence to Haley A. Brown or Nicole M. Koropatkin.

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Brown, H.A., DeVeaux, A.L., Juliano, B.R. et al. BoGH13ASus from Bacteroides ovatus represents a novel α-amylase used for  Bacteroides starch breakdown in the human gut. Cell. Mol. Life Sci. 80, 232 (2023). https://doi.org/10.1007/s00018-023-04812-w

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