10 research outputs found
Molecular mechanisms of processive glycoside hydrolases underline catalytic pragmatism
Version of Record published: 2 June 2023Processive and distributive catalysis defines the conversion continuum, thus underpinning the transformation of oligo- and polymeric substrates by enzymes. Distributive catalysis follows an association–transformation–dissociation pattern during the formation of enzyme–reactant complexes, whereas during processive catalysis, enzymes partner with substrates and complete multiple catalytic events before dissociation from an enzyme–substrate complex. Here, we focus on processive catalysis in glycoside hydrolases (GHs), which ensures efficient conversions of substrates with high precision, and has the advantage over distributive catalysis in efficiency. The work presented here examines a recent discovery of substrate-product-assisted processive catalysis in the GH3 family enzymes with enclosed pocket-shaped active sites. We detail how GH3 β-D-glucan glucohydrolases exploit a transiently formed lateral pocket for product displacement and reactants sliding (or translocation motion) through the catalytic site without dissociation, including movements during nanoscale binding/unbinding and sliding. The phylogenetic tree of putative 550 Archaean, bacterial, fungal, Viridiplantae, and Metazoan GH3 entries resolved seven lineages that corresponded to major substrate specificity groups. This analysis indicates that two tryptophan residues in plant β-D-glucan glucohydrolases that delineate the catalytic pocket, and infer broad specificity, high catalytic efficiency, and substrate-product-assisted processivity, have evolved through a complex evolutionary process, including horizontal transfer and neo-functionalisation. We conclude that the definition of thermodynamic and mechano-structural properties of processive enzymes is fundamentally important for theoretical and practical applications in bioengineering applicable in various biotechnologies.Maria Hrmova and Julian G. Schwerd
Co-evolution of enzymes involved in plant cell wall metabolism in the grasses
There has been a dramatic evolutionary shift in the polysaccharide composition of cell walls in the grasses, with increases in arabinoxylans and (1,3;1,4)-β-glucans and decreases in pectic polysaccharides, mannans, and xyloglucans, compared with other angiosperms. Several enzymes are involved in the biosynthesis of arabinoxylans, but the overall process is not yet defined and whether their increased abundance in grasses results from active or reactive evolutionary forces is not clear. Phylogenetic analyses reveal that multiple independent evolution of genes encoding (1,3;1,4)-β-glucan synthases has probably occurred within the large cellulose synthase/cellulose synthase-like (CesA/Csl) gene family of angiosperms. The (1,3;1,4)-β-glucan synthases appear to be capable of inserting both (1,3)- and (1,4)-β-linkages in the elongating polysaccharide chain, although the precise mechanism through which this is achieved remains unclear. Nevertheless, these enzymes probably evolved from synthases that originally synthesized only (1,4)-β-linkages. Initially, (1,3;1,4)-β-glucans could be turned over through preexisting cellulases, but as the need for specific hydrolysis was required, the grasses evolved specific (1,3;1,4)-β-glucan endohydrolases. The corresponding genes evolved from genes for the more widely distributed (1,3)-β-glucan endohydrolases. Why the subgroups of CesA/Csl genes that mediate the synthesis of (1,3;1,4)-β-glucans have been retained by the highly successful grasses but by few other angiosperms or lower plants represents an intriguing biological question. In this review, we address this important aspect of cell wall polysaccharide evolution in the grasses, with a particular focus on the enzymes involved in noncellulosic polysaccharide biosynthesis, hydrolysis, and modification.Vincent Bulone, Julian G. Schwerdt and Geoffrey B. Finche
Barley Nodulin 26-like Intrinsic Protein permeates water, metalloids, saccharides, and ion pairs due to structural plasticity and diversification
OnlinePublAquaporins can facilitate the passive movement of water, small polar molecules and some ions. Here, we examined solute selectivity for the barley Nodulin 26-like Intrinsic Protein (HvNIP2;1) embedded in liposomes and examined through stopped-flow light scattering spectrophotometry and Xenopus laevis oocyte swelling assays. We found that HvNIP2;1 permeates water, boric and germanic acids, sucrose, and lactose but not d-glucose or d-fructose. Other saccharides, such as neutral (d-mannose, d-galactose, d-xylose, d-mannoheptaose) and charged (N-acetyl d-glucosamine, d-glucosamine, d-glucuronic acid) aldoses, disaccharides (cellobiose, gentiobiose, trehalose), trisaccharide raffinose, and urea, glycerol, and acyclic polyols were permeated to a much lower extent. We observed apparent permeation of hydrated KCl and MgSO4 ions, while CH3COONa and NaNO3 permeated at significantly lower rates. Our experiments with boric acid and sucrose revealed no apparent interaction between solutes when permeated together, and AgNO3 or H[AuCl4] blocked the permeation of all solutes. Docking of sucrose in HvNIP2;1 and spinach water-selective SoPIP2;1 aquaporins revealed the structural basis for sucrose permeation in HvNIP2;1 but not in SoPIP2;1, and defined key residues interacting with this permeant. In a biological context, sucrose transport could constitute a novel element of plant saccharide-transporting machinery. Phylogenomic analyses of 164 Viridiplantae and 2,993 Archaean, bacterial, fungal, and Metazoan aquaporins rationalized solute poly-selectivity in NIP3 sub-clade entries and suggested that they diversified from other sub-clades to acquire a unique specificity of saccharide transporters. Solute specificity definition in NIP aquaporins could inspire developing plants for food production.Akshayaa Venkataraghavan, Julian G. Schwerdt, Stephen D. Tyerman, Maria
Hrmov
Composition and biosynthetic machinery of the Blumeria graminis f. sp. hordei conidia cell wall
Infection of barley with the powdery mildew causal agent, Blumeria graminis f. sp. hordei (Bgh), can lead to devastating damage to barley crops. The recent emergence of fungicide resistance imposes a need to develop new antifungal strategies. The enzymes involved in cell wall biosynthesis are ideal targets for the development of fungicides. However, in order to narrow down any target proteins involved in cell wall formation, a greater understanding of the cell wall structure and composition is required. Here, we present a detailed carbohydrate analysis of the Bgh conidial cell wall, a full annotation of Carbohydrate Active enZymes (CAZy) in the Bgh genome, and a comprehensive expression profile of the genes involved in cell wall metabolism. Glycosidic linkage analysis has revealed that the cell wall polysaccharide fraction of Bgh conidia predominantly consists of glucosyl residues (63.1%) and has a greater proportion of galactopyranosyl residues compared to other species (8.5%). Trace amounts of xylosyl residues were also detected, which is unusual in ascomycetes. Transcripts of the genes involved in cell wall metabolism show high expression of chitin deacetylases, which assist fungi in evading the host defence system by deacetylating chitin to chitosan. The data presented suggest that the cell wall components of the conidia and the corresponding obligate biotrophic CAZy gene profile play a key role in the infection process.Trang A.T. Pham, Julian G. Schwerdt, Neil J. Shirley, Xiaohui Xing, Vincent Bulone, b, Alan Littl
A novel (1,4)-beta-linked glucoxylan is synthesized by members of the cellulose synthase-like F gene family in land plants
As a significant component of monocot cell walls, (1,3;1,4)-β-glucan has conclusively been shown to be synthesized by the cellulose synthase-like F6 protein. In this study, we investigated the synthetic activity of other members of the barley (Hordeum vulgare) CslF gene family using heterologous expression. As expected, the majority of the genes encode proteins that are capable of synthesizing detectable levels of (1,3;1,4)-β-glucan. However, overexpression of HvCslF3 and HvCslF10 genes resulted in the synthesis of a novel linear glucoxylan that consists of (1,4)-β-linked glucose and xylose residues. To demonstrate that this product was not an aberration of the heterologous system, the characteristic (1,4)-β-linkage between glucose and xylose was confirmed to be present in wild type barley tissues known to contain HvCslF3 and HvCslF10 transcripts. This polysaccharide linkage has also been reported in species of Ulva, a marine green alga, and has significant implications for defining the specificity of the cell wall content of many crop species. This finding supports previous observations that members of a single CSL family may not possess the same carbohydrate synthetic activity, with the CSLF family now associated with the formation of not only (1,3)- and (1,4)-β-glucosidic linkages, but also (1,4)-β-glucosidic and (1,4)-β-xylosidic linkages.Alan Little, Jelle Lahnstein, David W. Jeffery, Shi F. Khor, Julian G. Schwerdt, Neil J. Shirley, Michelle Hooi, Xiaohui Xing, Rachel A. Burton, and Vincent Bulon
Another building block in the plant cell wall: Barley xyloglucan xyloglucosyl transferases link covalently xyloglucan and anionic oligosaccharides derived from pectin
Published online 16 August 2020.We report on the homo‐ and hetero‐transglycosylation activities of the HvXET3 and HvXET4 xyloglucan xyloglucosyl transferases (XET; EC 2.4.1.207) from barley (Hordeum vulgare L.), and the visualisation of these activities in young barley roots using Alexa Fluor 488‐labelled oligosaccharides. We discover that these isozymes catalyse the transglycosylation reactions with the chemically defined donor and acceptor substrates, specifically with the xyloglucan donor and the penta‐galacturonide [α(1‐4)GalAp]5 acceptor – the homogalacturonan (pectin) fragment. This activity is supported by 3D molecular models of HvXET3 and HvXET4 with the docked XXXG donor and [α(1‐4)GalAp]5 acceptor substrates at the ‐4 to +5 subsites in the active sites. Comparative sequence analyses of barley isoforms and seed‐localised TmXET6.3 from nasturtium (Tropaeolum majus L.) permitted the engineering of mutants of TmXET6.3 that could catalyse the hetero‐transglycosylation reaction with the xyloglucan/[α(1‐4)GalAp]5 substrate pair, while wild‐type TmXET6.3 lacked this activity. Expression data obtained by real‐time quantitative PCR of HvXET transcripts and a clustered heatmap of expression profiles of the gene family revealed that HvXET3 and HvXET6 co‐expressed but did not share the monophyletic origin. Conversely, HvXET3 and HvXET4 shared this relationship, when we examined the evolutionary history of 419 glycoside hydrolase 16 family members, spanning monocots, eudicots, and a basal Angiosperm. The discovered hetero‐transglycosylation activity in HvXET3 and HvXET4 with the xyloglucan/[α(1‐4)GalAp]5 substrate pair is discussed against the background of roles of xyloglucan‐pectin heteropolymers and how they may participate in spatial patterns of cell wall formation and re‐modelling, and affect the structural features of walls.Barbora Stratilová, Sergej Šesták, Jozef Mravec, Soňa Garajová, Zuzana Pakanová, Kristína Vadinová, Danica Kučerová, Stanislav Kozmon, Julian G. Schwerdt, Neil Shirley Eva Stratilová and Maria Hrmov
A chromosome-level genome assembly of Plantago ovata
Plantago ovata is cultivated for production of its seed husk (psyllium). When wet, the husk transforms into a mucilage with properties suitable for pharmaceutical industries, utilised in supplements for controlling blood cholesterol levels, and food industries for making gluten-free products. There has been limited success in improving husk quantity and quality through breeding approaches, partly due to the lack of a reference genome. Here we constructed the frst chromosome-scale reference assembly of P. ovata using a combination of 5.98 million PacBio and 636.5 million Hi-C reads. We also used corrected PacBio reads to estimate genome size and transcripts to generate gene models. The fnal assembly covers ~ 500 Mb with 99.3% gene set completeness. A total of 97% of the sequences are anchored to four chromosomes with an N50 of~ 128.87 Mb. The P. ovata genome contains 61.90% repeats, where 40.04% are long terminal repeats. We identifed 41,820 protein-coding genes, 411 non-coding RNAs, 108 ribosomal RNAs, and 1295 transfer RNAs. This genome will provide a resource for plant breeding programs to, for example, reduce agronomic constraints such as seed shattering, increase psyllium yield and quality, and overcome crop disease susceptibility.Lina Herliana, Julian G. Schwerdt, Tycho R. Neumann, Anita Severn-Ellis, Jana L. Phan, James M. Cowley, Neil J. Shirley, Matthew R.Tucker, Tina Bianco, Miotto, Jacqueline Batley, Nathan S. Watson, Haigh, Rachel A. Burto
Identification and spatio-temporal expression analysis of barley genes that encode putative modular xylanolytic enzymes
Arabinoxylans are cell wall polysaccharides whose re-modelling and degradation during plant development are mediated by several classes of xylanolytic enzymes. Here, we present the identification and new annotation of twelve putative (1,4)-β-xylanase and six β-xylosidase genes, and their spatio-temporal expression patterns during vegetative and reproductive growth of barley (Hordeum vulgare cv. Navigator). The encoded xylanase proteins are all predicted to contain a conserved carbohydrate-binding module (CBM) and a catalytic glycoside hydrolase (GH) 10 domain. Additional domains in some xylanases define three discrete phylogenetic clades: one clade contains proteins with an additional N-terminal signal sequence, while another clade contains proteins with multiple CBMs. Homology modelling revealed that all fifteen xylanases likely contain a third domain, a β-sandwich folded from two non-contiguous sequence segments that bracket the catalytic GH domain, which may explain why the full length protein is required for correct folding of the active enzyme. Similarly, predicted xylosidase proteins share a highly conserved domain structure, each with an N-terminal signal peptide, a split GH 3 domain, and a C-terminal fibronectin-like domain. Several genes appear to be ubiquitously expressed during barley growth and development, while four newly annotated xylanase and xylosidase genes are expressed at extremely high levels, which may be of broader interest for industrial applications where cell wall degradation is necessary.Natalie S. Betts, Helen M. Collins, Neil J. Shirley, Jose A. Cuesta-Seijo, Julian G. Schwerdt, Renee J. Phillipsa ... et al
A randomised, blinded, trial of clopidogrel versus aspirin in patients at risk of ischaemic events (CAPRIE). CAPRIE Steering Committee
Many clinical trials have evaluated the benefit of long-term use of antiplatelet drugs in reducing the risk of clinical thrombotic events. Aspirin and ticlopidine have been shown to be effective, but both have potentially serious adverse effects. Clopidogrel, a new thienopyridine derivative similar to ticlopidine, is an inhibitor of platelet aggregation induced by adenosine diphosphate. METHODS: CAPRIE was a randomised, blinded, international trial designed to assess the relative efficacy of clopidogrel (75 mg once daily) and aspirin (325 mg once daily) in reducing the risk of a composite outcome cluster of ischaemic stroke, myocardial infarction, or vascular death; their relative safety was also assessed. The population studied comprised subgroups of patients with atherosclerotic vascular disease manifested as either recent ischaemic stroke, recent myocardial infarction, or symptomatic peripheral arterial disease. Patients were followed for 1 to 3 years. FINDINGS: 19,185 patients, with more than 6300 in each of the clinical subgroups, were recruited over 3 years, with a mean follow-up of 1.91 years. There were 1960 first events included in the outcome cluster on which an intention-to-treat analysis showed that patients treated with clopidogrel had an annual 5.32% risk of ischaemic stroke, myocardial infarction, or vascular death compared with 5.83% with aspirin. These rates reflect a statistically significant (p = 0.043) relative-risk reduction of 8.7% in favour of clopidogrel (95% Cl 0.3-16.5). Corresponding on-treatment analysis yielded a relative-risk reduction of 9.4%. There were no major differences in terms of safety. Reported adverse experiences in the clopidogrel and aspirin groups judged to be severe included rash (0.26% vs 0.10%), diarrhoea (0.23% vs 0.11%), upper gastrointestinal discomfort (0.97% vs 1.22%), intracranial haemorrhage (0.33% vs 0.47%), and gastrointestinal haemorrhage (0.52% vs 0.72%), respectively. There were ten (0.10%) patients in the clopidogrel group with significant reductions in neutrophils (< 1.2 x 10(9)/L) and 16 (0.17%) in the aspirin group. INTERPRETATION: Long-term administration of clopidogrel to patients with atherosclerotic vascular disease is more effective than aspirin in reducing the combined risk of ischaemic stroke, myocardial infarction, or vascular death. The overall safety profile of clopidogrel is at least as good as that of medium-dose aspirin