Structure and function of novel cellulosic, hemicellulosic and pectic glycoside hydrolases

Cellulose is a major component of the plant cell wall and is the most abundant organic molecule in the biosphere. Efficient degradation of this polysaccharide is required if the plant cell wall is to be used as a viable source of renewable biofuels. Bacteria express an arsenal of different cellulases that catalyse the degradation of cellulose. The reason why many different cellulases are expressed rather than one highly active cellulase is unclear, but probably lies in the structural diversity displayed by cellulose, which is much greater than its invariant chemical composition suggests. Part of this work describes a novel cellulase from the plant cell wall degrading bacterium Clostridium thermocellum. This cellulase, CtCel119, is the first of this class of enzyme to display the α8 helical fold, is the founding member of a new GH family, performs catalysis through a possible “Grothuss style mechanism” and shares features typical of lytic transglycoslases. Structural data also seem to suggest that the enzyme may attack a novel structure in crystalline cellulose, which could contribute to understanding why bacteria such as C. thermocellum employ a diverse variety of cellulases. A study on glycoside hydrolase family (GH) 26, which consists mainly of endo-β-1,4 mannanases, was also conducted. The work presented in this thesis focused on two novel members of the family. One component of this section provided a thorough kinetic analysis of mutants of active site residues of a GH26 endo-β-1,4-1,3-glucanase. This identified crucial interactions at the -2 subsite, which contribute to the stabilisation of the 4H3 transition state. The other component of this section was the identification and characterisation of an exo-acting mannanase, CjMan26C, also termed a mannobiohydrolase. CjMan26C is the only mannanase characterised to date to release mannobiose. The mannobiohydrolase displayed an extremely high catalytic efficiency of 3 x 109 min-1 M-1 against mannotetraose. The crystal structure revealed a -1 sugar in a 1S5 pre transition state, providing further support for a B2,5 transition state in GH26. The exo activity was conferred by a four amino acid insertion in loop 3 at the -2 subsite. Mutation of D130, to Gly or Ala, in loop 3 was enough to partially convert the enzyme to an endo-mode of action, while removal of D130 plus two flanking resides caused a full conversion to an endo-mode of action. The gene expansion observed in family GH43 enzymes was also investigated. Eleven genes encoding GH43 enzymes were cloned, expressed and investigated for catalytic activity. Three arabinofuranosidases were characterised, two exo α-1,5-L-arabinofuranosidases and a novel sugar beet arabinan specific α-1,2-L-arabinofuranosidase that could attack both single and double substitutions named CjAbf43B. The crystal structure of CjAbf43B was solved in complex with ligand. The structure revealed a curved binding cleft, around a deep active site pocket, that was specific for the curved nature of sugar beet arabinan backbone. The curved binding cleft also had a groove into which α-1,3-L-arabinofuranosides could be accommodated, explaining how the enzyme has plasticity for single and double substitutions. A β-1,4 xylosidase was also characterised, while four enzymes were identified that displayed „trace‟ activity against xylans. Three of these appeared to display endo-activity, while the fourth enzyme displayed very weak arabinfuranosidase activity against xylans.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

What is ‘research-led teaching’ and how can we deliver it in University STEM degrees?

Universities advertise STEM degrees as being delivered via ‘research-led teaching’. We worked with students at the University of Liverpool to ascertain what students understood ‘research-led teaching’ to mean, and how it could be improved. We found that most students considered it important to be taught by active researchers and expected that their lecturers be experts in their fields. We found that students craved a more practical, research-based curriculum. Students believed that a research-heavy degree would better prepare them for future employment and provide skills that are more difficult to attain through lecture-based courses, but those who had commitments outside of the University were more hesitant. Our findings highlight the importance of considering underserved communities such as carers and single parents in course design. Overall, we found that the way in which teaching is delivered has a critical impact on students’ confidence and future career choices. We also report that students’ confidence and perceived learning opportunities have been significantly affected by the COVID-19 pandemic. We propose that this is something that needs to be factored in when recruiting for postgraduate degrees and graduate jobs to protect future opportunities for students whose degrees were completed during the pandemic

The human gut microbe <i>Bacteroides thetaiotaomicron</i> encodes the founding member of a novel glycosaminoglycan-degrading polysaccharide lyase family PL29

Glycosaminoglycans (GAGs) and GAG-degrading enzymes have wide-ranging applications in the medical and biotechnological industries. The former are also an important nutrient source for select species of the human gut microbiota (HGM), a key player in host-microbial interactions. How GAGs are metabolized by the HGM is therefore of interest and has been extensively investigated in the model human gut microbe Bacteroides thetaiotaomicron. The presence of yet uncharacterized GAG-inducible genes in its genome and that of related species however, is testament to our incomplete understanding of this process. Nevertheless, it presents a potential opportunity for the discovery of additional GAG-degrading enzymes. Here, we investigated a gene of unknown function (BT_3328) from the chondroitin sulfate (CS) utilization locus of B. thetaiotaomicron. NMR and UV spectroscopic assays revealed that it encodes a novel polysaccharide lyase (PL), hereafter referred to as BtCDH, reflecting its source (B. thetaiotaomicron or Bt) and ability to degrade the GAGs CS, dermatan sulfate (DS) and hyaluronic acid (HA). When incubated with HA, BtCDH generated a series of unsaturated HA sugars including Δ4,5UA-GlcNAc, Δ4,5UA-GlcNAc-GlcA-GlcNac, Δ4,5UA-[GlcNAc-GlcA]2-GlcNac and Δ4,5UA-[GlcNAc-GlcA]3-GlcNac) as end products and hence was classed as endo-acting. A combination of genetic and biochemical assays revealed that BtCDH localizes to the cell surface of B. thetaiotaomicron where it enables extracellular GAG degradation. BtCDH homologues were also detected in several other HGM species and we therefore propose that it represents the founding member of a new polysaccharide lyase family (PL29). The current discovery also contributes new insights into CS metabolism by the HGM

Structural and functional analyses of glycoside hydrolase 138 enzymes targeting chain A galacturonic acid in the complex pectin rhamnogalacturonan II

The metabolism of carbohydrate polymers drives microbial diversity in the human gut microbiome. The selection pressures in this environment have spurred the evolution of a complex reservoir of microbial genes encoding carbohydrate-active enzymes (CAZymes). Previously, we have shown that the human gut bacterium Bacteroides thetaiotaomicron (Bt) can depolymerize the most structurally complex glycan, the plant pectin rhamnogalacturonan II (RGII), commonly found in the human diet. Previous investigation of the RGII-degrading apparatus in Bt identified BT0997 as a new CAZyme family, classified as glycoside hydrolase 138 (GH138). The mechanism of substrate recognition by GH138, however, remains unclear. Here, using synthetic substrates and biochemical assays, we show that BT0997 targets the D-galacturonic acid-α-1,2-L-rhamnose linkage in chain A of RGII and that it absolutely requires the presence of a second D-galacturonic acid side chain (linked β-1,3 to L-rhamnose) for activity. NMR analysis revealed that BT0997 operates through a double displacement retaining mechanism. We also report the crystal structure of a BT0997 homolog, BPA0997 from Bacteroides paurosaccharolyticus, in complex with ligands at 1.6 Å resolution. The structure disclosed that the enzyme comprises four domains, including a catalytic TIM (α/β)8 barrel. Characterization of several BT0997 variants identified Glu-294 and Glu-361 as the catalytic acid/base and nucleophile, respectively, and we observed a chloride ion close to the active site. The three-dimensional structure and bioinformatic analysis revealed that two arginines, Arg-332 and Arg-521, are key specificity determinants of BT0997 in targeting D-galacturonic acid residues. In summary, our study reports the first structural and mechanistic analyses of GH138 enzymes

Mobility shift-based electrophoresis coupled with fluorescent detection enables real-time enzyme analysis of carbohydrate sulfatase activity

Sulfated carbohydrate metabolism is a fundamental process, which occurs in all domains of life. Carbohydrate sulfatases are enzymes that remove sulfate groups from carbohydrates and are essential to the depolymerisation of complex polysaccharides. Despite their biological importance, carbohydrate sulfatases are poorly studied and challenges remain in accurately assessing the activity, specificity and kinetic parameters. Most notably, separation of desulfated products from sulfated substrates is currently a time-consuming process. In this paper, we describe the development of rapid capillary electrophoresis coupled to substrate fluorescence detection as a high-throughput and facile means of analysing carbohydrate sulfatase activity. The approach has utility for the determination of both kinetic and inhibition parameters and is based on existing microfluidic technology coupled to a new synthetic fluorescent 6S-GlcNAc carbohydrate substrate. Furthermore, we compare this technique in terms of both time and resources, to high performance anion exchange chromatography and NMR-based methods, which are the two current ‘gold standards’ for enzymatic carbohydrate sulfation analysis. Our study clearly demonstrates the advantages of mobility shift assays for the quantification of near real-time carbohydrate desulfation by purified sulfatases, and could support the search for small molecule inhibitors of these disease-associated enzymes. One sentence summary Sulfatases remove sulfate groups from biomolecules; in this study we report a rapid and robust capillary electrophoresis assay for the quantification of carbohydrate desulfation

How members of the human gut microbiota overcome the sulfation problem posed by glycosaminoglycans

The human microbiota, which plays an important role in health and disease, uses complex carbohydrates as a major source of nutrients. Utilization hierarchy indicates that the host glycosaminoglycans heparin (Hep) and heparan sulfate (HS) are high-priority carbohydrates for Bacteroides thetaiotaomicron, a prominent member of the human microbiota. The sulfation patterns of these glycosaminoglycans are highly variable, which presents a significant enzymatic challenge to the polysaccharide lyases and sulfatases that mediate degradation. It is possible that the bacterium recruits lyases with highly plastic specificities and expresses a repertoire of enzymes that target substructures of the glycosaminoglycans with variable sulfation or that the glycans are desulfated before cleavage by the lyases. To distinguish between these mechanisms, the components of the B. thetaiotaomicron Hep/HS degrading apparatus were analyzed. The data showed that the bacterium expressed a single-surface endo-acting lyase that cleaved HS, reflecting its higher molecular weight compared with Hep. Both Hep and HS oligosaccharides imported into the periplasm were degraded by a repertoire of lyases, with each enzyme displaying specificity for substructures within these glycosaminoglycans that display a different degree of sulfation. Furthermore, the crystal structures of a key surface glycan binding protein, which is able to bind both Hep and HS, and periplasmic sulfatases reveal the major specificity determinants for these proteins. The locus described here is highly conserved within the human gut Bacteroides, indicating that the model developed is of generic relevance to this important microbial community

Human gut Bacteroidetes can utilize yeast mannan through a selfish mechanism

Yeasts, which have been a component of the human diet for at least 7000 years, possess an elaborate cell wall α-mannan. The influence of yeast mannan on the ecology of the human microbiota is unknown. Here we show that yeast α-mannan is a viable food source for Bacteroides thetaiotaomicron (Bt), a dominant member of the microbiota. Detailed biochemical analysis and targeted gene disruption studies support a model whereby limited cleavage of α-mannan on the surface generates large oligosaccharides that are subsequently depolymerized to mannose by the action of periplasmic enzymes. Co-culturing studies showed that metabolism of yeast mannan by Bt presents a ‘selfish’ model for the catabolism of this recalcitrant polysaccharide. This report shows how a cohort of highly successful members of the microbiota has evolved to consume sterically-restricted yeast glycans, an adaptation that may reflect the incorporation of eukaryotic microorganisms into the human diet

Dietary pectic glycans are degraded by coordinated enzyme pathways in human colonic Bacteroides.

The major nutrients available to human colonic Bacteroides species are glycans, exemplified by pectins, a network of covalently linked plant cell wall polysaccharides containing galacturonic acid (GalA). Metabolism of complex carbohydrates by the Bacteroides genus is orchestrated by polysaccharide utilization loci (PULs). In Bacteroides thetaiotaomicron, a human colonic bacterium, the PULs activated by different pectin domains have been identified; however, the mechanism by which these loci contribute to the degradation of these GalA-containing polysaccharides is poorly understood. Here we show that each PUL orchestrates the metabolism of specific pectin molecules, recruiting enzymes from two previously unknown glycoside hydrolase families. The apparatus that depolymerizes the backbone of rhamnogalacturonan-I is particularly complex. This system contains several glycoside hydrolases that trim the remnants of other pectin domains attached to rhamnogalacturonan-I, and nine enzymes that contribute to the degradation of the backbone that makes up a rhamnose-GalA repeating unit. The catalytic properties of the pectin-degrading enzymes are optimized to protect the glycan cues that activate the specific PULs ensuring a continuous supply of inducing molecules throughout growth. The contribution of Bacteroides spp. to metabolism of the pectic network is illustrated by cross-feeding between organisms.This work was supported in part by an Advanced Grant from the European Research Council (Grant No. 322820) awarded to H.J.G. and B.H. supporting A.S.L., D.N., A.C. and N.T., a Wellcome Trust Senior Investigator Award to H.J.G. (grant No. WT097907MA) that supported J.B. and E.C.L. a European Union Seventh Framework Initial Training Network Programme entitled the “WallTraC project” (Grant Agreement number 263916) awarded to M-C.R. and H.J.G, which supported X.Z. and J.S. The Biotechnology and Biological Research Council project ‘Ricefuel’ (grant numbers BB/K020358/1) awarded to H.J.G. supported A.L

Identification of d -arabinan-degrading enzymes in mycobacteria

Bacterial cell growth and division require the coordinated action of enzymes that synthesize and degrade cell wall polymers. Here, we identify enzymes that cleave the D-arabinan core of arabinogalactan, an unusual component of the cell wall of Mycobacterium tuberculosis and other mycobacteria. We screened 14 human gut-derived Bacteroidetes for arabinogalactan-degrading activities and identified four families of glycoside hydrolases with activity against the D-arabinan or D-galactan components of arabinogalactan. Using one of these isolates with exo-D-galactofuranosidase activity, we generated enriched D-arabinan and used it to identify a strain of Dysgonomonas gadei as a D-arabinan degrader. This enabled the discovery of endo- and exo-acting enzymes that cleave D-arabinan, including members of the DUF2961 family (GH172) and a family of glycoside hydrolases (DUF4185/GH183) that display endo-D-arabinofuranase activity and are conserved in mycobacteria and other microbes. Mycobacterial genomes encode two conserved endo-D-arabinanases with different preferences for the D-arabinan-containing cell wall components arabinogalactan and lipoarabinomannan, suggesting they are important for cell wall modification and/or degradation. The discovery of these enzymes will support future studies into the structure and function of the mycobacterial cell wall

Sulfated glycan recognition by carbohydrate sulfatases of the human gut microbiota

International audienceSulfated glycans are ubiquitous nutrient sources for microbial communities that have co-evolved with eukaryotic hosts. Bacteria metabolise sulfated glycans by deploying carbohydrate sulfatases that remove sulfate esters. Despite the biological importance of sulfatases, the mechanisms underlying their ability to recognise their glycan substrate remain poorly understood. Here, we utilise structural biology to determine how sulfatases from the human gut microbiota recognise sulfated glycans. We reveal 7 new carbohydrate sulfatase structures span four S1 sulfatase subfamilies. Structures of S1_16 and S1_46 represent the first structures of these subfamilies. Structures of S1_11 and S1_15 demonstrate how non-conserved regions of the protein drive specificity towards related but distinct glycan targets. Collectively, these data reveal that Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: https://www.springernature.com/gp/open-research/policies/accepted-manuscript-term