The John Innes Centre Publications Repository contains details of all publications resulting from our researchers.
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The creation of this publications repository was funded by BBSRC.
Biochemistry (57) 3387-3401
Publisher's version: 10.1021/acs.biochem.8b00121
ID: 59464read more
The biosynthetic pathway of peptidoglycan is essential for Mycobacterium tuberculosis. We report here the acetyltransferase substrate specificity and catalytic mechanism of the bifunctional N-acetyltransferase/uridylyltransferase from M. tuberculosis (GlmU). This enzyme is responsible for the final two steps of the synthesis of UDP- N-acetylglucosamine, which is an essential precursor of peptidoglycan, from glucosamine 1-phosphate, acetyl-coenzyme A, and uridine 5'-triphosphate. GlmU utilizes ternary complex formation to transfer an acetyl from acetyl-coenzyme A to glucosamine 1-phosphate to form N-acetylglucosamine 1-phosphate. Steady-state kinetic studies and equilibrium binding experiments indicate that GlmU follows a steady-state ordered kinetic mechanism, with acetyl-coenzyme A binding first, which triggers a conformational change in GlmU, followed by glucosamine 1-phosphate binding. Coenzyme A is the last product to dissociate. Chemistry is partially rate-limiting as indicated by pH-rate studies and solvent kinetic isotope effects. A novel crystal structure of a mimic of the Michaelis complex, with glucose 1-phosphate and acetyl-coenzyme A, helps us to propose the residues involved in deprotonation of glucosamine 1-phosphate and the loop movement that likely generates the active site required for glucosamine 1-phosphate to bind. Together, these results pave the way for the rational discovery of improved inhibitors against M. tuberculosis GlmU, some of which might become candidates for antibiotic discovery programs.
Publisher's version: 10.1007/978-1-4939-7874-8_9
ID: 59088read more
An a-1,6-and a-1,3-linked glucan produced by Leuconostoc citreum ABK-1 alternansucrase with nanoparticle and film-forming properties.
Scientific reports (8) 8340
Publisher's version: 10.1038/s41598-018-26721-w
ID: 59139read more
Alternansucrase catalyses the sequential transfer of glucose residues from sucrose onto another sucrose molecule to form a long chain polymer, known as "alternan". The alternansucrase-encoding gene from Leuconostoc citreum ABK-1 (Lcalt) was successfully cloned and expressed in Escherichia coli. Lcalt encoded LcALT of 2,057 amino acid residues; the enzyme possessed an optimum temperature and pH of 40°C and 5.0, respectively, and its' activity was stimulated up to 2.4-fold by the presence of Mn2+. Kinetic studies of LcALT showed a high transglycosylation activity, with Km 32.2 ± 3.2mM and kcat 290±12s-1. Alternan generated by LcALT (Lc-alternan) harbours partially alternating a-1,6 and a- 1,3 glycosidic linkages confirmed by NMR spectroscopy, methylation analysis, and partial hydrolysis of Lc-alternan products. In contrast to previously reported alternans, Lc-alternan can undergo self-assembly, forming nanoparticles with an average size of 90nm in solution. At concentrations above 15% (w/v), Lc-alternan nanoparticles disassemble and form a high viscosity solution, while this polymer forms a transparent film once dried.
Carbohydrate Research (463) 14-23
Publisher's version: 10.1016/j.carres.2018.04.008
ID: 59027read more
A suite of glycosylated glycerol derivatives representing various fragments of the glycosylated ichthyotoxins called prymnesins were chemically synthesised. Glycerol was used to represent a small fragment of the prymnesin backbone, and was glycosylated at the 2° position with the sugars currently reported to be present on prymnesin toxins. Neighbouring group participation was utilised to synthesise 1,2-trans-glycosides. SnCl2-promoted glycosylation with furanosyl fluorides gave 1,2-cis-furanosides with moderate stereocontrol, whilst TMSOTf promoted glycosylation with a furanosyl imidate gave a 1,2-cis-furanoside with good stereocontrol. The chemical synthesis of two larger glyceryl diglycoside fragments of prymnesin-1, glycosylated with a-?-arabinopyranose and a-?-ribofuranose, is also described. As the stereochemistry of the prymnesin backbones at this region is undefined, both the 2R- and 2S- glycerol isomers were synthesised. The separated diastereoisomers were distinguished by comparing NOESY NMR with computational models.
Biochemical Society Transactions
Publisher's version: 10.1042/BST20170393
ID: 58114read more
Prymnesium parvum is a toxin-producing microalga that causes harmful algal blooms globally, which often result in large-scale fish kills that have severe ecological and economic implications. Although many toxins have previously been isolated from P. parvum, ambiguity still surrounds the responsible ichthyotoxins in P. parvumblooms and the biotic and abiotic factors that promote bloom toxicity. A major fish kill attributed to P. parvumoccurred in Spring 2015 on the Norfolk Broads, a low-lying set of channels and lakes (Broads) found on the East of England. Here, we discuss how water samples taken during this bloom have led to diverse scientific advances ranging from toxin analysis to discovery of a new lytic virus of P. parvum, P. parvum DNA virus (PpDNAV-BW1). Taking recent literature into account, we propose key roles for sialic acids in this type of viral infection. Finally, we discuss recent practical detection and management strategies for controlling these devastating blooms.
Structural and functional analysis of ErwiniaI amylovora SrlD. The first crystal structure of a sorbitol-6-phosphate 2-dehydrogenase.
Journal of Structural Biology
Publisher's version: 10.1016/j.jsb.2018.03.010
ID: 59254read more
Sorbitol-6-phosphate 2-dehydrogenases (S6PDH) catalyze the interconversion of d-sorbitol 6-phosphate to d-fructose 6-phosphate. In the plant pathogen Erwinia amylovora the S6PDH SrlD is used by the bacterium to utilize sorbitol, which is used for carbohydrate transport in the host plants belonging to the Amygdaloideae subfamily (e.g., apple, pear, and quince). We have determined the crystal structure of S6PDH SrlD at 1.84?Å resolution, which is the first structure of an EC 126.96.36.199 enzyme. Kinetic data show that SrlD is much faster at oxidizing d-sorbitol 6-phosphate than in reducing d-fructose 6-phosphate, however, equilibrium analysis revealed that only part of the d-sorbitol 6-phosphate present in the in vitro environment is converted into d-fructose 6-phosphate. The comparison of the structures of SrlD and Rhodobacter sphaeroides sorbitol dehydrogenase showed that the tetrameric quaternary structure, the catalytic residues and a conserved aspartate residue that confers specificity for NAD+ over NADP+ are preserved. Analysis of the SrlD cofactor and substrate binding sites identified residues important for the formation of the complex with cofactor and substrate and in particular the role of Lys42 in selectivity towards the phospho-substrate. The comparison of SrlD backbone with the backbone of 302 short-chain dehydrogenases/reductases showed the conservation of the protein core and identified the variable parts. The SrlD sequence was compared with 500 S6PDH sequences selected by homology revealing that the C-terminal part is more conserved than the N-terminal, the consensus of the catalytic tetrad (Y[SN]AGXA) and a not previously described consensus for the NAD(H) binding.
Identification ofEuglena gracilisß-1,3-glucan phosphorylase and establishment of a new glycoside hydrolase (GH) family GH149.
Journal of Biological Chemistry (293) 2865-2876
Publisher's version: 10.1074/jbc.RA117.000936
ID: 57992read more
Glycoside phosphorylases (EC 2.4.x.x) carry out the reversible phosphorolysis of glucan polymers, producing the corresponding sugar 1-phosphate and a shortened glycan chain. ß-1,3-Glucan phosphorylase activities have been reported in the photosynthetic euglenozoanEuglena gracilis, but the cognate protein sequences have not been identified to date. Continuing our efforts to understand the glycobiology ofE. gracilis, we identified a candidate phosphorylase sequence, designated EgP1, by proteomic analysis of an enriched cellular protein lysate. We expressed recombinant EgP1 inEscherichia coliand characterized itin vitroas a ß-1,3-glucan phosphorylase. BLASTP identified several hundred EgP1 orthologs, most of which were from Gram-negative bacteria and had 37-91% sequence identity to EgP1. We heterologously expressed a bacterial metagenomic sequence, Pro_7066 inE. coliand confirmed it as a ß-1,3-glucan phosphorylase, albeit with kinetics parameters distinct from those of EgP1. EgP1, Pro_7066, and their orthologs are classified as a new glycoside hydrolase (GH) family, designated GH149. Comparisons between GH94, EgP1, and Pro_7066 sequences revealed conservation of key amino acids required for the phosphorylase activity, suggesting a phosphorylase mechanism that is conserved between GH94 and GH149. We found bacterialGH149genes in gene clusters containing sugar transporter and several other GH family genes, suggesting that bacterial GH149 proteins have roles in the degradation of complex carbohydrates. The BacteroidetesGH149genes located to previously identified polysaccharide utilization loci, implicated in the degradation of complex carbohydrates. In summary, we have identified a eukaryotic and a bacterial ß-1,3-glucan phosphorylase and uncovered a new family of phosphorylases that we name GH149.
Carbohydrate Research (451) 110-117
Publisher's version: 10.1016/j.carres.2017.08.008
ID: 57066read more
Naturally occurring 2,7-anhydro-alpha-N-acetylneuraminic acid (2,7-anhydro-Neu5Ac) is a transglycosylation product of bacterial intramolecular trans-sialidases (IT-sialidases). A facile one-pot two-enzyme approach has been established for the synthesis of 2,7-anhydro-sialic acid derivatives including those containing different sialic acid forms such as Neu5Ac and N-glycolylneuraminic acid (Neu5Gc). The approach is based on the use of Ruminoccocus gnavus IT-sialidase for the release of 2,7-anhydro-sialic acid from glycoproteins, and the conversion of free sialic acid by a sialic acid aldolase. This synthetic method, which is based on a membrane-enclosed enzymatic synthesis, can be performed on a preparative scale. Using fetuin as a substrate, high-yield and cost-effective production of 2,7-anhydro-Neu5Ac was obtained to high-purity. This method was also applied to the synthesis of 2,7-anhydro-Neu5Gc. The membrane-enclosed multienzyme (MEME) strategy reported here provides an efficient approach to produce a variety of sialic acid derivatives.
Scientific reports (7) 45341
Publisher's version: 10.1038/srep45341
ID: 56525read more
Extensins are plant cell wall glycoproteins that act as scaffolds for the deposition of the main wall carbohydrate polymers, which are interlocked into the supramolecular wall structure through intra- and inter-molecular iso-di-tyrosine crosslinks within the extensin backbone. In the conserved canonical extensin repeat, Ser-Hyp4, serine and the consecutive C4-hydroxyprolines (Hyps) are substituted with an a-galactose and 1-5 ß- or a-linked arabinofuranoses (Arafs), respectively. These modifications are required for correct extended structure and function of the extensin network. Here, we identified a single Arabidopsis thaliana gene, At3g57630, in clade E of the inverting Glycosyltransferase family GT47 as a candidate for the transfer of Araf to Hyp-arabinofuranotriose (Hyp-ß1,4Araf-ß1,2Araf-ß1,2Araf) side chains in an a-linkage, to yield Hyp-Araf4 which is exclusively found in extensins. T-DNA knock-out mutants of At3g57630 showed a truncated root hair phenotype, as seen for mutants of all hitherto characterized extensin glycosylation enzymes; both root hair and glycan phenotypes were restored upon reintroduction of At3g57630. At3g57630 was named Extensin Arabinose Deficient transferase, ExAD, accordingly. The occurrence of ExAD orthologs within the Viridiplantae along with its' product, Hyp-Araf4, point to ExAD being an evolutionary hallmark of terrestrial plants and charophyte green algae.
The impact of aminopyrene trisulfonate (APTS) label in acceptor glycan substrates for profiling plant pectin beta-galactosyltransferase activities
Carbohydrate Research (433) 97-105
Publisher's version: 10.1016/j.carres.2016.07.017
ID: 53882read more
Aminopyrene trisulfonate (APTS)-labelled disaccharides are demonstrated to serve as readily accessible acceptor substrates for galactosyltransferase activities present in Arabidopsis microsome preparations. The reductive amination procedure used to install the fluorophore results in loss of the ring structure of the reducing terminal sugar unit, such that a single intact sugar ring is present, attached via an alditol tether to the aminopyrene fluorophore. The configuration of the alditol portion of the labelled acceptor, as well as the position of alditol galactosylation, substantially influence the ability of compounds to serve as Arabidopsis galactosyltransferase acceptor substrates. The APTS label exhibits an unexpected reaction-promoting effect that is not evident for structurally similar sulfonated aromatic fluorophores ANDS and ANTS. When APTS-labelled β-(1 → 4)-Gal3 was employed as an acceptor substrate with Arabidopsis microsomes, glycan extension generated β-(1 → 4)-galactan chains running to beyond 60 galactose residues. These studies demonstrate the potential of even very short glycan-APTS probes for assessing plant galactosyltransferase activities and the suitability CE-LIF for CAZyme profiling.
Detection of mSiglec-E, in solution and expressed on the surface of Chinese hamster ovary cells, using sialic acid functionalised gold nanoparticles.
Analyst (141) 5799-5809
Publisher's version: 10.1039/c6an01230b
ID: 55606read more
Sialic acids are widespread in biology, fulfilling a wide range of functions. Their cognate lectin receptors - Siglecs - are equally diverse and widely distributed, with different Siglecs found within distinct populations of cells in the haemopoietic, immune and nervous systems. A convenient way to assay ligand recognition of soluble Siglecs would be useful, as would methods for the concomitant assessment of Siglec distribution on cell surfaces. Here we report the use of gold nanoparticles functionalised with a sialic acid ligand diluted with a polyethylene glycol (PEG) ligand for the plasmonic detection of a soluble form of murine Siglec-E (mSiglec-E-Fc fusion protein) and, importantly, for the specific detection of the same Siglec expressed on the surface of mammalian cells. These sialic acid functionalised nanoparticles are shown to overcome problems such as cellular cis interactions and low Siglec-ligand affinity. The gold nanoparticles were functionalised with various ratios of sialic acid:PEG ligands and the optimum ratio for the detection of murine Siglec-E was established based on the plasmonic detection of the soluble pre-complexed recombinant form of murine Siglec-E (mSiglec-E-Fc fusion protein). The optimum ratio for the detection of the fusion protein was found to be sialic acid:PEG ligands in a 50:50 ratio (glyconanoparticles 1). The optimised glyconanoparticles 1 were used to recognise and bind to the murine Siglec-E expressed on the surface of transfected Chinese hamster ovary cells as determined by transmission electron microscopy.
Scientific reports (6) 33215
Publisher's version: 10.1038/srep33215
ID: 55092read more
Starch degradation in barley endosperm provides carbon for early seedling growth, but the control of this process is poorly understood. We investigated whether endosperm cell wall degradation is an important determinant of the rate of starch degradation. We identified iminosugar inhibitors of enzymes that degrade the cell wall component arabinoxylan. The iminosugar 1,4-dideoxy-1, 4-imino-l-arabinitol (LAB) inhibits arabinoxylan arabinofuranohydrolase (AXAH) but does not inhibit the main starch-degrading enzymes a- and ß-amylase and limit dextrinase. AXAH activity in the endosperm appears soon after the onset of germination and resides in dimers putatively containing two isoforms, AXAH1 and AXAH2. Upon grain imbibition, mobilisation of arabinoxylan and starch spreads across the endosperm from the aleurone towards the crease. The front of arabinoxylan degradation precedes that of starch degradation. Incubation of grains with LAB decreases the rate of loss of both arabinoxylan and starch, and retards the spread of both degradation processes across the endosperm. We propose that starch degradation in the endosperm is dependent on cell wall degradation, which permeabilises the walls and thus permits rapid diffusion of amylolytic enzymes. AXAH may be of particular importance in this respect. These results provide new insights into the mobilization of endosperm reserves to support early seedling growth.
Iminosugar inhibitors of carbohydrate-active enzymes that underpin cereal grain germination and endosperm metabolism.
Biochemical Society Transactions (44) 159-165
Publisher's version: 10.1042/BST20150222
ID: 52586read more
Starch is a major energy store in plants. It provides most of the calories in the human diet and, as a bulk commodity, it is used across broad industry sectors. Starch synthesis and degradation are not fully understood, owing to challenging biochemistry at the liquid/solid interface and relatively limited knowledge about the nature and control of starch degradation in plants. Increased societal and commercial demand for enhanced yield and quality in starch crops requires a better understanding of starch metabolism as a whole. Here we review recent advances in understanding the roles of carbohydrate-active enzymes in starch degradation in cereal grains through complementary chemical and molecular genetics. These approaches have allowed us to start dissecting aspects of starch degradation and the interplay with cell-wall polysaccharide hydrolysis during germination. With a view to improving and diversifying the properties and uses of cereal grains, it is possible that starch degradation may be amenable to manipulation through genetic or chemical intervention at the level of cell wall metabolism, rather than simply in the starch degradation pathway per se.
Structural Dissection of the Maltodextrin Disproportionation Cycle of the Arabidopsis Plastidial Enzyme DPE1.
Journal of Biological Chemistry (290) 2983429853
Publisher's version: 10.1074/jbc.M115.682245
ID: 52112read more
The degradation of transitory starch in the chloroplast to provide fuel for the plant during the night requires a suite of enzymes that generate a series of short chain linear glucans. However, glucans of less than four glucose units are no longer substrates for these enzymes, whilst export from the plastid is only possible in the form of either maltose or glucose. In order to make use of maltotriose, which would otherwise accumulate, disproportionating enzyme 1 (DPE1; a 4-a-glucanotransferase) converts two molecules of maltotriose to a molecule of maltopentaose, which can now be acted on by the degradative enzymes, and one molecule of glucose that can be exported. We have determined the structure of the Arabidopsis plastidial DPE1 (AtDPE1) and, through ligand soaking experiments, we have trapped the enzyme in a variety of conformational states. AtDPE1 forms a homodimer with a deep, long and open-ended active site canyon contained within each subunit. The canyon is divided into donor and acceptor sites with the catalytic residues at their junction; a number of loops around the active site adopt different conformations dependent on the occupancy of these sites. The ?gate? is the most dynamic loop, and appears to play a role in substrate capture, in particular, in the binding of the acceptor molecule. Subtle changes in the configuration of the active site residues may prevent undesirable reactions or abortive hydrolysis of the covalently bound enzyme-substrate intermediate. Together, these observations allow us to delineate the complete AtDPE1 disproportionation cycle in structural terms.
Crystal structure of a novel two domain GH78 family a-rhamnosidase from Klebsiella oxytoca with rhamnose bound.
Proteins (Volume 83 Issue 9) 17421749
Publisher's version: 10.1002/prot.24807
ID: 49408read more
The crystal structure of the GH78 family a-rhamnosidase from Klebsiella oxytoca (KoRha) has been determined at 2.7 Å resolution with rhamnose bound in the active site of the catalytic domain. Curiously, the putative catalytic acid, Asp 222, is preceded by an unusual non-proline cis-peptide bond which helps to project the carboxyl group into the active centre. This KoRha homodimeric structure is significantly smaller than those of the other previously determined GH78 structures. Nevertheless, the enzyme displays a-rhamnosidase activity when assayed in vitro, suggesting that the additional structural domains found in the related enzymes are dispensible for function. This article is protected by copyright. All rights reserved.
The transcriptome of Euglena gracilis reveals unexpected metabolic capabilities for carbohydrate and natural product biochemistry.
Molecular Biosystems (11) 2808-20
Publisher's version: 10.1039/c5mb00319a
ID: 51984read more
Euglena gracilis is a highly complex alga belonging to the green plant line that shows characteristics of both plants and animals, while in evolutionary terms it is most closely related to the protozoan parasites Trypanosoma and Leishmania. This well-studied organism has long been known as a rich source of vitamins A, C and E, as well as amino acids that are essential for the human diet. Here we present de novo transcriptome sequencing and preliminary analysis, providing a basis for the molecular and functional genomics studies that will be required to direct metabolic engineering efforts aimed at enhancing the quality and quantity of high value products from E. gracilis. The transcriptome contains over 30000 protein-encoding genes, supporting metabolic pathways for lipids, amino acids, carbohydrates and vitamins, along with capabilities for polyketide and non-ribosomal peptide biosynthesis. The metabolic and environmental robustness of Euglena is supported by a substantial capacity for responding to biotic and abiotic stress: it has the capacity to deploy three separate pathways for vitamin C (ascorbate) production, as well as producing vitamin E (alpha-tocopherol) and, in addition to glutathione, the redox-active thiols nor-trypanothione and ovothiol.
Different ROS-Scavenging Properties of Flavonoids Determine Their Abilities to Extend Shelf Life of Tomato.
Plant Physiology (169(3)) 1568-1583
Publisher's version: 10.1104/pp.15.00346
ID: 51528read more
The shelf-life of tomato (Solanum lycopersicum) fruit is determined by the processes of over-ripening and susceptibility to pathogens. Post-harvest shelf life is one of the most important traits for commercially grown tomatoes. We compared the shelf life of tomato fruit that accumulate different flavonoids and found that delayed over-ripening is associated with increased total antioxidant capacity caused by the accumulation of flavonoids in the fruit. However, reduced susceptibility to Botrytis cinerea, a major post-harvest fungal pathogen of tomato, is conferred by specific flavonoids only. We demonstrate an association between flavonoid structure, selective scavenging ability for different free radicals and reduced susceptibility to B. cinerea. Our study provides mechanistic insight into how flavonoids influence shelf life of tomato, information which could be used to improve the shelf life of tomato, and potentially of other soft fruit.
Carbohydrate Research (404) 17-25
Publisher's version: 10.1016/j.carres.2014.12.005
ID: 48902read more
Glucose-1-phosphate uridylyltransferase in conjunction with UDP-glucose pyrophosphorylase was found to catalyse the conversion of a range of 5-substituted UTP derivatives into the corresponding UDP-galactose derivatives in poor yield. Notably the 5-iodo derivative was not converted to UDP-sugar. In contrast, UDP-glucose pyrophosphorylase in conjunction with inorganic pyrophosphatase was particularly effective at converting 5-substituted UTP derivatives, including the iodo compound, into a range of gluco-configured 5-substituted UDP-sugar derivatives in good yields. Attempts to effect 4?-epimerization of these 5-substituted UDP-glucose with UDP-glucose 4?-epimerase from yeast were unsuccessful, while use of the corresponding enzyme from Erwinia amylovora resulted in efficient epimerization of only 5-iodo-UDP-Glc, but not the corresponding 5-aryl derivatives, to give 5-iodo-UDP-Gal. Given the established potential for Pd-mediated cross-coupling of 5-iodo-UDP-sugars, this provides convenient access to the galacto-configured 5-substituted-UDP-sugars from gluco-configured substrates and 5-iodo-UTP.
Addendum: Discrimination of epimeric glycans and glycopeptides using IM-MS and its potential for carbohydrate sequencing.
Nature: Chemistry (6) 368
Publisher's version: 10.1038/nchem.1901
ID: 46879read more
Expression and characterization of 4-alpha-glucanotransferase genes from Manihot esculenta Crantz and Arabidopsis thaliana and their use for the production of cycloamyloses.
Process Biochemistry (49) 84-89
Publisher's version: 10.1016/j.procbio.2013.10.009
ID: 46884read more
4-alpha-Glucanotransferase or disproportionating enzyme (D-enzyme, DPE) catalyzes the -1.4 glycosyl transfer between oligosaccharides. Type I D-enzyme (DPE1) can transfer maltosyl unit from one 1.4-alpha-D-glucan to an acceptor mono- or oligo-saccharide, which reflects the physiological role of DPE1 in plant starch metabolism. In this study, the genes encoding DPE1 from Arabidopsis thaliana (AtDPE1 ) and Manihot esculenta Crantz cultivar KU50 (MeDPE1) were cloned and expressed in Escherichia coli and purified to homogeneity. Me DPE1 encoded 585 amino acid residues, including a 56 residue signal peptide, while At DPE1 encoded 576 amino acid residues with a 45 residue signal peptide. The molecular mass of both mature enzymes, estimated from deduced amino acid sequence, were the same at 59.4 kDa, with a p I of 5.13. The predicted structures of both enzymes showed the conserved 250’s loop and three catalytic amino acid residues, characteristics of disproportionating enzymes in the GH77 glycoside hydrolase family. Biochemical characterization showed that both purified recombinant enzymes were homodimers in solution, with similar optimum pH and temperature for disproportionating activity at pH 6–8 and 37 ◦ C. Using potato amylose as a substrate, AtDPE1 can produce cycloamyloses in the range 16–50 glucose residues, while products from the action of MeDPE1 on the same substrate were in a wider range of 16 to DP > 60. These recombinant enzymes are useful tools for elucidation of their functional roles in starch metabolism and for applications in the starch industry.
Discrimination of epimeric glycans and glycopeptides using IM-MS and its potential for carbohydrate sequencing.
Nature: Chemistry (6) 65-74
Publisher's version: 10.1038/nchem.1817
ID: 46880read more
Mass spectrometry is the primary analytical technique used to characterize the complex oligosaccharides that decorate cell surfaces. Monosaccharide building blocks are often simple epimers, which when combined produce diastereomeric glycoconjugates indistinguishable by mass spectrometry. Structure elucidation frequently relies on assumptions that biosynthetic pathways are highly conserved. Here, we show that biosynthetic enzymes can display unexpected promiscuity, with human glycosyltransferase pp-a-GanT2 able to utilize both uridine diphosphate N-acetylglucosamine and uridine diphosphate N-acetylgalactosamine, leading to the synthesis of epimeric glycopeptides in vitro. Ion-mobility mass spectrometry (IM-MS) was used to separate these structures and, significantly, enabled characterization of the attached glycan based on the drift times of the monosaccharide product ions generated following collision-induced dissociation. Finally, ion-mobility mass spectrometry following fragmentation was used to determine the nature of both the reducing and non-reducing glycans of a series of epimeric disaccharides and the branched pentasaccharide Man3 glycan, demonstrating that this technique may prove useful for the sequencing of complex oligosaccharides.
Sugar-coated sensor chip and nanoparticle surfaces for the in vitro enzymatic synthesis of starch-like materials.
Chemical Science (5) 341-350
Publisher's version: 10.1039/c3sc51829a
ID: 46881read more
The insoluble glucan polymer starch is a major player in the human diet; it is also an important bulk commodity. Nonetheless, our understanding of its biochemistry remains poor, not least because of the challenge of analysing enzymes that operate across the solid–liquid interface. In the present study, the enzymatic polymerisation of glucans immobilised on gold sensor chip and nanoparticle surfaces was achieved with Arabidopsis phosphorylase AtPHS2. The basis of the action of AtPHS2 on surface glucans could be rationalised through consideration of the X-ray crystal structure of this enzyme, which identified a previously unreported enzyme surface binding site for glucans. Extension of the glucan-coated sensor chip surfaces could be monitored in real time by SPR, enabling kinetic analysis of AtPHS2-mediated glucan synthesis, which showed similar efficiency to in solution analyses. Extension of both sensor and nanoparticles surfaces coated with glucan was analysed by TEM, which confirmed glucan polymerisation. The arrangement of newly formed glucan chains into ordered helical arrangements was evident from iodine staining, as well as from enzyme response characteristics that proved typical of starch-like material. As such, the glucan-modified sensor chip and nanoparticle surfaces represent novel tools with which to analyse starch-active enzymes.
Biomolecular characterization of the levansucrase of Erwinia amylovora, a promising biocatalyst for the synthesis of fructooligosaccharides.
Journal of Agricultural and Food Chemistry (61) 12265-73
Publisher's version: 10.1021/jf4023178
ID: 46882read more
Erwinia amylovora is a plant pathogen that affects Rosaceae, such as apple and pear. In E. amylovora the fructans, produced by the action of a levansucrase (EaLsc), play a role in virulence and biofilm formation. Fructans are bioactive compounds, displaying health-promoting properties in their own right. Their use as food and feed supplements is increasing. In this study, we investigated the biomolecular properties of EaLsc using HPAEC-PAD, MALDI-TOF MS, and spectrophotometric assays. The enzyme, which was heterologously expressed in Escherichia coli in high yield, was shown to produce mainly fructooligosaccharides (FOSs) with a degree of polymerization between 3 and 6. The kinetic properties of EaLsc were similar to those of other phylogenetically related Gram-negative bacteria, but the good yield of FOSs, the product spectrum, and the straightforward production of the enzyme suggest that EaLsc is an interesting biocatalyst for future studies aimed at producing tailor-made fructans.
Glyconanoparticles for the plasmonic detection and discrimination between human and avian influenza virus.
Organic and Biomolecular Chemistry (11) 7101-7
Publisher's version: 10.1039/c3ob41703d
ID: 43863read more
A plasmonic bioassay for the specific detection of human influenza virus has been developed based on gold nanoparticles functionalised with a designed and synthesised thiolated trivalent a2,6-thio-linked sialic acid derivative. The glyconanoparticles consist of the thiolated trivalent a2,6-thio-linked sialic acid derivative and a thiolated polyethylene glycol (PEG) derivative self-assembled onto the gold surface. Varying ratios of the trivalent a2,6-thio-linked sialic acid ligand and the PEG ligand were used; a ratio of 25?:?75 was found to be optimum for the detection of human influenza virus X31 (H3N2). In the presence of the influenza virus a solution of the glyconanoparticles aggregate following the binding of the trivalent a2,6-thio-linked sialic acid ligand to the haemagglutinin on the surface of the virus. The aggregation of the glycoparticles with the influenza virus induces a colour change of the solution within 30 min. Non-purified influenza virus in allantoic fluid was successfully detected using the functionalised glyconanoparticles. A comparison between the trivalent and a monovalent a2,6-thio-linked sialic acid functionalised nanoparticles confirmed that more rapid results, with greater sensitivity, were achieved using the trivalent ligand for the detection of the X31 virus. Importantly, the glyconanoparticles were able to discriminate between human (a2,6 binding) and avian (a2,3 binding) RG14 (H5N1) influenza virus highlighting the binding specificity of the trivalent a2,6-thio-linked sialic acid ligand.