29 28 D I A M O N D L I G H T S O U R C E A N N U A L R E V I E W 2 0 2 0 / 2 1 D I A M O N D L I G H T S O U R C E A N N U A L R E V I E W 2 0 2 0 / 2 1 Macromolecular Crystallography Group BeamlinesVMXi, I03 and I04 Sweet enzymes fromgut bacteria: fromdiscovery to diagnostics Related publication: Wu H., Rebello O., Crost E. H., Owen C. D.,Walpole S., Bennati-Granier C., Ndeh D., Monaco S., HicksT., Colvile A., Urbanowicz P. A.,WalshM. A., Angulo J., Spencer D. I. R. & Juge N. Fucosidases from the human gut symbiont Ruminococcus gnavus . Cell. Mol. Life Sci. 78 , 675–693 (2020). DOI: 10.1007/s00018-020-03514-x Publication keywords: Antennary fucose; Glycoside hydrolase; Gut microbiota; Lewis epitopes; Mucin glycosylation; Mucus T hemicroorganisms that live in thehumangut haveaprofound impact onour health.The microbes living in intestinalmucus, a complex network of proteins and attached sugars, act as‘gatekeepers’bymaintaining gut barrier function. However, themechanisms by which they interact with the host remain largely unknown. Researchers investigated Ruminococcus gnavus , a common resident of the human gut that has amajor role in health and disease. They investigated its potential to process the sugar fucose using enzymes called fucosidases. They also explored the pool of fucosidases produced by different R. gnavus strains. UsingDiamondLightSource’sVersatileMacromolecularCrystallography insitu (VMXi)beamlineallowedtheteamtotracktheircrystallisation experiments in real time. They were also able to test crystal diffraction quickly and easily, rapidly optimising crystallisation conditions to produce consistent, high-quality protein crystals for X-ray diffraction experiments on MX beamlines I03 and I04. The high-resolution diffraction datasets of the R. gnavus fucosidase they collected allowed themto solve its structure and locate the part that performs the sugar cleaving chemical reaction. Fucosylation (adding a fucose sugar to a molecule) is a common modification involved in many human physiological processes including ABO blood grouping, antibody effector functions, cancer progression, and lymphocyte development and adhesion. As such, fucosidases are relevant to many aspects of human health and disease. This work may have potential applications in diagnosing several diseases, including diabetes and certain cancers. The microbial community inhabiting the human gut (gut microbiota) exerts a profound effect on human health 1 . In the adult colon, gut bacteria have not only access to non-digestible polysaccharides from the diet, but also to complex oligosaccharides from host mucins 2,3 . Mucins are large glycoproteins with a high carbohydrate content of up to 80%. Mucin O-glycan chains are based on core structures which are further elongated with galactose (Gal), N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc) modified by fucosylation, sialylation and sulfation 3 . The main source of glycan diversity is provided by the peripheral terminal epitopes, sialic acid and fucose. These sugars show considerable variation along the GI tract and provide a food source to gut bacteria species inhabiting different regions of the gut 3 . To access this source of nutrients, gut bacteria encode glycoside hydrolases (GHs) with α-L-fucosidases (fucosidases) catalysing the hydrolysis of terminal α-L-fucosidic linkages. Based on sequence identity, α-fucosidases are classified into GH29 and GH95 families of the CAZy database (www.cazy.org) . To date, functionally characterised GH95 enzymes show strict substrate specificity to the terminal Fuc α1-2Gal linkage and hydrolyse the linkage via an inverting mechanism whereas GH29 enzymes show relatively relaxed substrate specificities for α-1–3, α-1–4 and α-1–6 linked fucose with hydrolysis proceeding via a retaining mechanism (www.cazy.org) . Ruminococcus gnavus is a prevalent member of the gut microbial community belonging to the Firmicutes division 4 . R. gnavus is an early coloniser of the human gut but persists in healthy adults where it is strongly associated with human health and diseases such as inflammatory bowel disease. R. gnavus' ability to grow on mucins is strain-dependent, reflecting the distribution of GH families between R. gnavus strains 5 . The objectives of this work were to determine the substrate and linkage specificities of fucosidases across R. gnavus strains. Sequence similarity network identified strain-specific fucosidases in R. gnavus ATCC 29149 and E1 strains that were further validated structurally and enzymatically and against a range of defined oligosaccharides and glycoconjugates. X-ray crystallography combined with saturation transfer difference (STD) nuclear magnetic resonance (NMR) and molecular dynamics simulation unravelled the structural basis for R. gnavus unique fucosidase specificity. A sequence similarity network (SSN) analysis was first conducted to identify putative functional relationships between GH29 or GH95 fucosidases from R. gnavus and related protein sequences. The SSN analysis covered 6,736 amino acid sequences from the GH29 family extracted from Interpro database 66.0 and CAZy (www.cazy.org/GH29_characterised.html) and 825 GH95 sequences from Interpro IPR027414. Representative fucosidases were chosen for further characterisation, E1_10125, E1_10180, and ATCC_03833 from the GH29 family and ATCC_00842 and E1_10587 from the GH95 family (Fig. 1a,b). The genes encoding the selected GH29 and GH95 fucosidases from R. gnavus strains ATCC 29149 and E1 were heterologously expressed in Escherichia coli . Enzyme activity was first screened against the synthetic substrate pNP-α-L-fucopyranoside (pNP-Fuc). Fucosidase ATCC_03833 showed the highest catalytic efficiency with a k cat of 83.6 s -1 and a K M of 28.77 µM. Next, the substrate specificity of the recombinant fucosidases was tested on a range of fucosylated oligosaccharides including 2’FL (Fucα1,2Galβ1,4Glc), 3FL (Galβ1-4[Fucα1-3]Glc), Lewis A (LeA, Galβl-3[Fucα1-4]GlcNAc) and Lewis X (LeX, Galβ1-4[Fucα1-3]GlcNAc). These oligosaccharides represent different glycan structures present in the human host. Fucosidases E1_10125 and E1_10180 showed substrate specificity towards α1,3/4 fucosylated linkages while fucosidases ATCC_00842 and ATCC_03833 showed preference for α1,2 linkages. Activity against more complex oligosaccharides and glycoproteins was also assayed, including sialyl Lewis X (sLeX), sialyl Lewis A (sLeA), sialylated as well as non-sialylated human plasma N -glycans, horseradish peroxidase N -glycans containing core a-1,3-fucose (HRP), blood group A type II, blood group B type II and fucosylated IgG glycoform (FA2G2) and the product of the reactions analysed by LC- MS. These screens revealed that E1_10125 was active againstfucosylatedsubstrates presenting a terminal sialic acid modification.This unique specificity prompted further enzymatic and structural analyses. Using a combination of glycan microarrays, mass spectrometry, and isothermal titration calorimetry (ITC), we showed that R. gnavus E1_10125 fucosidase has the capacity to recognise sialic acid-terminated fucosylated glycans (sialyl Lewis X/A epitopes) and hydrolyse α1-3/4 fucosyl linkages in these substrates without the need to remove sialic acid (Fig. 2). The catalytically inactive mutant E1_10125 D221A bound to LeX with a Kd of 51.43 ± 1.93 μM and to sLeX (3’-Sialyl Lewis X) with a Kd of 6.12 ± 1.08 as determined by ITC. To gain structural insights into the unique ligand specificity of E1_10125, the enzyme was crystallised in the presence of 2'FL. The trisaccharide was cleaved by the fucosidase during the crystallisation experiment presenting a single fucose sugar bound in the crystal structure (Fig. 3a). This allowed us to locate the active site and confirmed the reaction mechanism. The fucose binding site was conserved with other homologous GH29 fucosidases, including from Streptococcus pneumoniae and Bifidobacterium longum however the structure highlighted a region nearby the catalytic residues, which is absent in fucosidases with different reaction specificities. As it was confirmed by STD NMR that the sialic acid moiety of sLeX makes contacts with the enzyme, it was proposed that the sialic acid would be accommodated by this region, conferring the demonstrated specificity. Further, the high- resolution crystal structure, with data to 1.45 Å , provided a starting point for molecular dynamics (MD) simulations and in silico docking experiments that demonstrated that sLeX could be accommodated by the E1_10125 fucosidase binding site (Fig. 3b). This specificity of R. gnavus fucosidases characterised in this work may contribute to the adaptation of R. gnavus strains to the infant and adult gut and has potential applications in diagnostic glycomic assays for diabetes and certain cancers. In particular, the antennary fucosidase specificity reported here for sialic acid-terminated fucosylated glycans could be used as a discriminatory tool to identify N-glycan biomarkers of diseases and as a valuable tool for the purpose of glycoprofiling bio-pharmaceutical glycoproteins. References: 1. Thursby E. et al. Introduction to the human gut microbiota. Biochem. J. 474 , 1823–1836 (2017). DOI: 10.1042/BCJ20160510 2. Ndeh D. et al. Biochemistry of complex glycan depolymerisation by the human gut microbiota. FEMS Microbiol. Rev. 42 , 146–164 (2018). DOI: 10.1093/femsre/fuy002 3. Tailford L. E. et al. Mucin glycan foraging in the human gut microbiome. Front. Genet. 5 , 81 (2015). DOI: 10.3389/fgene.2015.00081 4. Qin J. et al. A human gut microbial gene catalogue established by metagenomic sequencing. Nature 464 , 59–65 (2010). DOI: 10.1038/nature08821 5. Crost E. H. Utilisation of Mucin Glycans by the Human Gut Symbiont Ruminococcus gnavus Is Strain-Dependent. PLOS ONE (2013). DOI: 10.1371/journal.pone.0076341 Funding acknowledgement: The authors gratefully acknowledge the support of the Biotechnology and Biological Sciences Research Council (BBSRC); this research was mostly funded by the Innovate UK Biocatalyst grant Glycoenzymes for Bioindustries (BB/ M029042/) with contribution from the Royal Society and the BBSRC Institute Strategic Programmes BB/J004529/1‘The Gut Health and Food Safety’and BB/R012490/1‘Gut Microbes and Health’. Corresponding authors: Prof. Nathalie Juge, Quadram Institute Biosciences,
[email protected]; Dr HaiyangWu, Quadram Instititute Biosciences,
[email protected] ; Dr David Owen, Diamond Light Source,
[email protected] Figure 1: The distribution of R. gnavus GH29 and GH95 fucosidases based on SSN analysis. (a) Partial representation of SSN analysis of GH29 family containing fucosidases from R. gnavus E1 and ATCC 29149 strains. (b) Representation of the SSN central cluster of GH95 family containing all GH95 from R. gnavus E1 and ATCC 29149 strains. Blue nodes sequences extracted from the CAZy database encoding functionally characterised enzymes. Red nodes sequences from R. gnavus E1 strain. Cyan nodes sequences from R. gnavus ATCC 29149 strain. Green nodes sequences common to both R. gnavus E1 and ATCC 29149 strains. Figure 3: Crystal structure of the R. gnavus E1_10125. (a) The location of the active site is highlighted by the presence of the fucose molecule, with its atoms shown as red spheres. The region proposed to accommodate the sialic acid moiety is shown in orange. (b) The substrate binding site with sLeX (fucose (red), GlcNAc (blue), galactose (yellow) and sialic acid (pink)) shown as positioned by MD simulations. Nearby binding site residues are shown in green. The spheres represent STD NMR transfer intensities. Red indicates greatest intensity (52%-100%), yellow medium (25%-50%) and blue low (0-24%). Stronger normalised STD intensities correlate with closer ligand contacts with the surface of the protein in the bound state. Figure 2: R. gnavus E1_10125 fucosidase can act on sialyl Lewis X/A epitopes. Schematic representation of the enzymatic reaction. Monosaccharide symbols follow the Symbol Nomenclature for Glycans system. Key: fucose (red triangle), GlcNAc (blue square), sialic acid (purple diamond), galactose (yellow circle).