Abstract
The vast majority of glycosidases characterized to date follow one of the variations of the ‘Koshland’ mechanisms1 to hydrolyse glycosidic bonds through substitution reactions. Here we describe a large-scale screen of a human gut microbiome metagenomic library using an assay that selectively identifies non-Koshland glycosidase activities2. Using this, we identify a cluster of enzymes with extremely broad substrate specificities and thoroughly characterize these, mechanistically and structurally. These enzymes not only break glycosidic linkages of both α and β stereochemistry and multiple connectivities, but also cleave substrates that are not hydrolysed by standard glycosidases. These include thioglycosides, such as the glucosinolates from plants, and pseudoglycosidic bonds of pharmaceuticals such as acarbose. This is achieved through a distinct mechanism of hydrolysis that involves oxidation/reduction and elimination/hydration steps, each catalysed by enzyme modules that are in many cases interchangeable between organisms and substrate classes. Homologues of these enzymes occur in both Gram-positive and Gram-negative bacteria associated with the gut microbiome and other body parts, as well as other environments, such as soil and sea. Such alternative step-wise mechanisms appear to constitute largely unrecognized but abundant pathways for glycan degradation as part of the metabolism of carbohydrates in bacteria.
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Data availability
Data supporting the findings of this study are available within the Article and the Supplementary Information. All sequencing data are available at the end of the Supplementary Information and also from GenBank database with accession numbers PP693324 and PP693325. Structural models are deposited at the Protein Data Bank under accession codes 8TCD, 8TCR, 8TCS, 8TCT, 8TDA, 8TDE, 8TDF, 8TDH, 8TDI and 8V31. Source data are provided with this paper.
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Acknowledgements
This work was supported by operating grants from the Natural Sciences and Engineering Research Council of Canada (NSERC) and the Canadian Institutes of Health Research (CIHR) as well as infrastructure funds from the Canada Foundation for Innovation and BC Knowledge Development Fund. S.A.N. was supported by a Vanier scholarship from NSERC; A.C.L. by a GAP scholarship from the UBC Centre for Blood Research; and L.B. by an Early Postdoc Mobility grant from Swiss National Science Foundation. N.C.J.S. is a Tier I Canada Research Chair in Structure-guided Antibiotic Discovery. Part of the research described in this paper was performed using beamline CMCF-BM at the Canadian Light Source, a national research facility of the University of Saskatchewan, which is supported by the Canada Foundation for Innovation (CFI), the Natural Sciences and Engineering Research Council (NSERC), the National Research Council (NRC), the Canadian Institutes of Health Research (CIHR), the Government of Saskatchewan and the University of Saskatchewan. We thank the GM/CA beamline staff at beamline 23-ID-B at the APS for access and support. GM/CA@APS has been funded in whole or in part by Federal funds from the National Cancer Institute (ACB-12002) and the National Institute of General Medical Sciences (AGM-12006). We thank A. M. Deutschbauer for providing access to the B.T. mutant strains18; T. Warren for the initial inspiration for these studies; P. Rahfeld for guidance about the screening process; A. Noonan and S. Hallam for guidance with processing of the sequencing data; M. Ezhova for assistance with the NMR experiments; and F. Liu, S. Macdonald, P. Danby, B. Herring, C. Olagnon, J. Wardman, R. Bains and Y. Tian for suggestions and discussions.
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S.G.W., S.A.N. and L.B. conceived and developed the screen. Screening was performed by I.L.L. and S.A.N.; S.A.N., C.Y.Z. and E.B. performed the cloning, purification and biochemical characterization of the enzymes. S.A.N., I.L.L., C.Y.Z. and H.-M.C. synthesized all of the chemicals. A.C.L., L.S., L.J.W. and N.C.J.S. performed the crystallography analysis. S.A.N. and D.P. performed microbial growth and RT–qPCR assays under the guidance of H.B.; S.A.N., A.C.L. and S.G.W. wrote the manuscript with input from all of the authors.
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Extended data figures and tables
Extended Data Fig. 1 Reactions catalysed by AL/BT3 and AL/BT1.
1H-NMR characterization of the reactions of a) AL3 and b) AL1.
Extended Data Fig. 2 Reaction catalysed by AL/BT2 and proposed transition states for AL/BT 1 and 2.
a) 1H-NMR characterization of the reaction of BT2 and b) proposed transition states for AL/BT1 (top) and AL/BT2 (bottom), B refers to protein residues capable of acid/base catalysis and M refers to the metal ions that coordinate the 3-keto groups.
Extended Data Fig. 3 Additional structural figures for AL/BT 1-3.
a) Active site of BT1 bound with substrate analogue 3k-1,5-anhydroglucitol with coordinating residues labelled. b) Active site of AL1 bound with glucose with coordinating residues labelled. c) Metal binding site of apo AL1 with coordinating residues labelled. d) Front view of overall fold of BT2 with jelly roll in orange, fingers (purple) labelled F1-F5, and active site denoted with a dashed circle. e) Metal binding site of apo BT2 with coordinating residues labelled. f) BT2 active site with conserved possible active site residues labelled. g) Active site of AL3 bound with trehalose with key residues labelled. h) Binding sites of trehalose observed in AL3 crystal soaked with trehalose. Active site is labelled as 1 and circled, secondary sites are labelled 2–6.
Extended Data Fig. 4 In vivo hydrolysis of substrates by Bacteroides species.
a) Homologues of the proteins from Bacteroides thetaiotaomicron in some other Bacteroides strains. The percent similarity of each of the encoded proteins to the corresponding BT homologue is shown in white. b) The percent of hydrolysed selective non-Koshland substrates 1 and 3 by different Bacteroides strains. All the strains were grown anaerobically and incubated with the substrates overnight. Bars represent the mean and error bars represent standard deviations (n = 3 technical replicates, derived from the same original culture). c) Transcriptional response of BT genes BT2156-60 to trehalose and sucrose as sole carbon sources. Representative genes from the fructan utilization locus (BT1759 and BT1763, encoding GH32 and SusC homologues, respectively) serve as positive controls for sucrose regulation65. Data were acquired at mid-log phase (9 h for trehalose, 5 h for sucrose) and are normalized to transcript levels prior to inoculating cells into the respective carbohydrate medium. Bars represent the mean and error bars represent the standard error of the mean (n = 4 replicates, two technical replicates each derived from two separate original cultures). d) Resulting fluorescence from hydrolysis of substrates 1 and 3 by B. thetaiotaomicron cells grown in different media. Growth on α,α-trehalose results in significantly higher activities while growth on glucose represses them, (n = 3 technical replicates, derived from the same original pre-culture, data for all replicates are shown.) e) Growth of B. thetaiotaomicron cells on cellobiose in absence and presence of trehalose, performed in triplicate for all the conditions shown here. f) hydrolysis of substrates 1 and 3 by B. thetaiotaomicron cells grown on different glycosides as the sole carbon source. Bars represent the mean and error bars represent standard deviations (n = 3 technical replicates, derived from the same original culture).
Extended Data Fig. 5 Homologues of the proteins from P1C11 in bacteria from different environments.
Homologues of the proteins from P1C11 (Alistipes sp.) identified in bacteria from various environments (the source from which each bacterium is isolated is noted in parentheses below the name of bacterium). The percent similarity of each of the encoded proteins to the corresponding Alistipes homologue is shown in white. Dark blue denotes oxidoreductases that are not from the same family as AL3/AL4 and denoted in yellow are commonly co-occurring genes annotated as a DoxX-domain-containing protein, which is reported to form membrane-associated oxidoreductase complexes. Broken lines mean that a few genes are in between those shown and light grey shows other genes. The length of the arrows and the space between them does not correspond to the actual length of the DNA fragments in this figure. This is a representative list not an exhaustive one.
Extended Data Fig. 6 Homologues of the proteins from P2B11 in bacteria from different environments.
Homologues of the proteins from P2B11 identified in bacteria from various environments (the source from which each bacterium is isolated is noted in parentheses below the name of bacterium). The percent similarity of each of the encoded proteins to the corresponding P2B11 homologue is shown in white. Broken lines mean that a few genes are in between those shown and light grey shows other genes. The length of the arrows and the space between them does not correspond to the actual length of the DNA fragments in this figure. This is a representative list not an exhaustive one.
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Nasseri, S.A., Lazarski, A.C., Lemmer, I.L. et al. An alternative broad-specificity pathway for glycan breakdown in bacteria. Nature 631, 199–206 (2024). https://doi.org/10.1038/s41586-024-07574-y
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DOI: https://doi.org/10.1038/s41586-024-07574-y
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