Three structurally and functionally distinct β-glucuronidases from the human gut microbe Bacteroides uniformis

The glycoside hydrolases encoded by the human gut microbiome play an integral role in processing a variety of exogenous and endogenous glycoconjugates. Here we present three structurally and functionally distinct β-glucuronidase (GUS) glycoside hydrolases from a single human gut commensal microbe, Bacteroides uniformis. We show using nine crystal structures, biochemical, and biophysical data that whereas these three proteins share similar overall folds, they exhibit different structural features that create three structurally and functionally unique enzyme active sites. Notably, quaternary structure plays an important role in creating distinct active site features that are hard to predict via structural modeling methods. The enzymes display differential processing capabilities toward glucuronic acid–containing polysaccharides and SN-38-glucuronide, a metabolite of the cancer drug irinotecan. We also demonstrate that GUS-specific and nonselective inhibitors exhibit varying potencies toward each enzyme. Together, these data highlight the diversity of GUS enzymes within a single Bacteroides gut commensal and advance our understanding of how structural details impact the specific roles microbial enzymes play in processing drug-glucuronide and glycan substrates.

[1]  R. Linhardt,et al.  Homogeneous low-molecular-weight heparins with reversible anticoagulant activity. , 2014, Nature chemical biology.

[2]  S. Brunak,et al.  SignalP 4.0: discriminating signal peptides from transmembrane regions , 2011, Nature Methods.

[3]  M. Redinbo,et al.  Glucuronides in the gut: Sugar-driven symbioses between microbe and host , 2017, The Journal of Biological Chemistry.

[4]  M. Redinbo,et al.  Bacterial β-glucuronidase inhibition protects mice against enteropathy induced by indomethacin, ketoprofen or diclofenac: mode of action and pharmacokinetics , 2014, Xenobiotica; the fate of foreign compounds in biological systems.

[5]  Benjamin P. Westover,et al.  Glycan Foraging in Vivo by an Intestine-Adapted Bacterial Symbiont , 2005, Science.

[6]  Randy J Read,et al.  Electronic Reprint Biological Crystallography Phenix: Building New Software for Automated Crystallographic Structure Determination Biological Crystallography Phenix: Building New Software for Automated Crystallographic Structure Determination , 2022 .

[7]  Frank DiMaio,et al.  Automatic structure prediction of oligomeric assemblies using Robetta in CASP12 , 2018, Proteins.

[8]  S. E. West,et al.  Fermentation of mucin and plant polysaccharides by strains of Bacteroides from the human colon , 1977, Applied and environmental microbiology.

[9]  M. Redinbo,et al.  Pharmacologic Targeting of Bacterial β-Glucuronidase Alleviates Nonsteroidal Anti-Inflammatory Drug-Induced Enteropathy in Mice , 2012, Journal of Pharmacology and Experimental Therapeutics.

[10]  M. Penttilä,et al.  Characterization of a novel Agrobacterium tumefaciens Galactarolactone Cycloisomerase Enzyme for Direct Conversion of d-Galactarolactone to 3-Deoxy-2-keto-l-threo-hexarate* , 2012, The Journal of Biological Chemistry.

[11]  Kevin Cowtan,et al.  research papers Acta Crystallographica Section D Biological , 2005 .

[12]  T. Gibson,et al.  Malectin: a novel carbohydrate-binding protein of the endoplasmic reticulum and a candidate player in the early steps of protein N-glycosylation. , 2008, Molecular biology of the cell.

[13]  M. Redinbo,et al.  Understanding and modulating mammalian-microbial communication for improved human health. , 2014, Annual review of pharmacology and toxicology.

[14]  J. Gordon,et al.  Olfactory receptor responding to gut microbiota-derived signals plays a role in renin secretion and blood pressure regulation , 2013, Proceedings of the National Academy of Sciences.

[15]  N. Mcneil.  The contribution of the large intestine to energy supplies in man. , 1984, The American journal of clinical nutrition.

[16]  Eric C. Martens,et al.  Complex Glycan Catabolism by the Human Gut Microbiota: The Bacteroidetes Sus-like Paradigm , 2009, The Journal of Biological Chemistry.

[17]  Yang Zhang,et al.  mTM‐align: an algorithm for fast and accurate multiple protein structure alignment , 2018, Bioinform..

[18]  Vincent Lombard,et al.  A hierarchical classification of polysaccharide lyases for glycogenomics. , 2010, The Biochemical journal.

[19]  L. Comstock,et al.  Longitudinal Analysis of the Prevalence, Maintenance, and IgA Response to Species of the Order Bacteroidales in the Human Gut , 2011, Infection and Immunity.

[20]  B. Henrissat,et al.  Complex pectin metabolism by gut bacteria reveals novel catalytic functions , 2017, Nature.

[21]  M. Redinbo,et al.  Multiple NSAID-induced hits injure the small intestine: underlying mechanisms and novel strategies. , 2013, Toxicological sciences : an official journal of the Society of Toxicology.

[22]  Masashi Yanagisawa,et al.  Effects of the gut microbiota on host adiposity are modulated by the short-chain fatty-acid binding G protein-coupled receptor, Gpr41 , 2008, Proceedings of the National Academy of Sciences.

[23]  M. Redinbo,et al.  An Atlas of β-Glucuronidases in the Human Intestinal Microbiome. , 2017, Structure.

[24]  G. A. Levvy,et al.  Inhibition of glycosidases by aldonolactones of corresponding configuration: Preparation of (1-->5)-lactones by catalytic oxidation of pyranoses and study of their inhibitory properties. , 1967, The Biochemical journal.

[25]  Frank DiMaio,et al.  Protein structure prediction using Rosetta in CASP12 , 2018, Proteins.

[26]  N. Sreerama,et al.  Estimation of protein secondary structure from circular dichroism spectra: comparison of CONTIN, SELCON, and CDSSTR methods with an expanded reference set. , 2000, Analytical biochemistry.

[27]  Brandi L. Cantarel,et al.  The Carbohydrate-Active EnZymes database (CAZy): an expert resource for Glycogenomics , 2008, Nucleic Acids Res..

[28]  F. Scott Mathews,et al.  Structure of human β-glucuronidase reveals candidate lysosomal targeting and active-site motifs , 1996, Nature Structural Biology.

[29]  Matthew R. Redinbo,et al.  Alleviating Cancer Drug Toxicity by Inhibiting a Bacterial Enzyme , 2010, Science.

[30]  Bernard Henrissat,et al.  The abundance and variety of carbohydrate-active enzymes in the human gut microbiota , 2013, Nature Reviews Microbiology.

[31]  M. Redinbo,et al.  Molecular Insights into Microbial β-Glucuronidase Inhibition to Abrogate CPT-11 Toxicity , 2013, Molecular Pharmacology.

[32]  David Baker,et al.  Protein homology model refinement by large-scale energy optimization , 2018, Proceedings of the National Academy of Sciences.

[33]  Pedro M. Coutinho,et al.  The carbohydrate-active enzymes database (CAZy) in 2013 , 2013, Nucleic Acids Res..

[34]  M. Redinbo,et al.  The human microbiome is a source of therapeutic drug targets. , 2013, Current opinion in chemical biology.

[35]  R. Xavier,et al.  Regulation of inflammatory responses by gut microbiota and chemoattractant receptor GPR43 , 2009, Nature.

[36]  H. Flint,et al.  Microbial degradation of complex carbohydrates in the gut , 2012, Gut microbes.

[37]  S. Frye,et al.  Structure and Inhibition of Microbiome β-Glucuronidases Essential to the Alleviation of Cancer Drug Toxicity. , 2015, Chemistry & biology.

[38]  E. Myers,et al.  Basic local alignment search tool. , 1990, Journal of molecular biology.