Design and four step synthesis of simple, readily accessible low-nanomolar divalent LecA ligands with selectivity over human galectin-1.

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. β-1,3-Glucan phosphorylase activities have been reported in the photosynthetic euglenozoan Euglena gracilis, but the cognate protein sequences have not been identified to date. Continuing our efforts to understand the glycobiology of E. gracilis, we identified a candidate phosphorylase sequence, designated EgP1, by proteomic analysis of an enriched cellular protein lysate. We expressed recombinant EgP1 in Escherichia coli and characterized it in vitro as a β-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 in E. coli and confirmed it as a β-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 bacterial GH149 genes 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 Bacteroidetes GH149 genes located to previously identified polysaccharide utilization loci, implicated in the degradation of complex carbohydrates. In summary, we have identified a eukaryotic and a bacterial β-1,3-glucan phosphorylase and uncovered a new family of phosphorylases that we name GH149.

Glycoside phosphorylases catalyse the reversible synthesis of glycosidic bonds by glyco-sylation with concomitant release of inorganic phosphate. The equilibrium position of such reactions can render them of limited synthetic utility, unless coupled with a secondary enzymatic step where the reaction lies heavily in favour of product. This article surveys recent works on the combined use of glycan phosphorylases with other enzymes to achieve synthetically useful processes.

Because of the antimicrobial resistance crisis,lectins are considered novel drug targets.P seudomonas aeruginosa utilizes LecA and LecB in the infection process.I nhibition of both lectins with carbohydrate-derived molecules can reduce biofilm formation to restore antimicrobial susceptibility.Here, we focused on non-carbohydrate inhibitors for LecA to explore new avenues for lectin inhibition. From as creening cascade we obtained one experimentally confirmed hit, ac atechol, belonging to the well-known PAINS compounds. Rigorous analyses validated electron-deficient catechols as millimolar LecA inhibitors.T he first co-crystal structure of anon-carbohydrate inhibitor in complex with abacterial lectin clearly demonstrates the catechol mimicking the binding of natural glycosides with LecA. Importantly,c atechol 3 is the first non-carbohydrate lectin ligand that binds bacterial and mammalian calcium(II)-binding lectins,g iving rise to this fundamentally new class of glycomimetics.

Euglena gracilis is an alga of great biotechnological interest and extensive metabolic capacity, able to make high levels of bioactive compounds, such as polyunsaturated fatty acids, vitamins and β-glucan. Previous work has shown that Euglena expresses a wide range of carbohydrate-active enzymes, suggesting an unexpectedly high capacity for the synthesis of complex carbohydrates for a single-celled organism. Here, we present an analysis of some of the carbohydrates synthesised by Euglena gracilis. Analysis of the sugar nucleotide pool showed that there are the substrates necessary for synthesis of complex polysaccharides, including the unusual sugar galactofuranose. Lectin- and antibody-based profiling of whole cells and extracted carbohydrates revealed a complex galactan, xylan and aminosugar based surface. Protein N-glycan profiling, however, indicated that just simple high mannose-type glycans are present and that they are partially modified with putative aminoethylphosphonate moieties. Together, these data indicate that Euglena possesses a complex glycan surface, unrelated to plant cell walls, while its protein glycosylation is simple. Taken together, these findings suggest that Euglena gracilis may lend itself to the production of pharmaceutical glycoproteins.

Glycoside phosphorylases (GPs) catalyze the phosphorolysis of glycans into the corresponding sugar 1-phosphates and shortened glycan chains. Given the diversity of natural β-(1→3)-glucans and their wide range of biotechnological applications, the identification of enzymatic tools that can act on β-(1→3)-glucooligosaccharides is an attractive area of research. GP activities acting on β-(1→3)-glucooligosaccharides have been described in bacteria, the photosynthetic excavate Euglena gracilis , and the heterokont Ochromonas spp. Previously, we characterized β-(1→3)-glucan GPs from bacteria and E. gracilis , leading to their classification in glycoside hydrolase family GH149. Here, we characterized GPs from Gram-positive bacteria and heterokont algae acting on β-(1→3)-glucooligosaccharides. We identified a phosphorylase sequence from Ochromonas spp. (OcP1) together with its orthologs from other species, leading us to propose the establishment of a new GH family, designated GH161. To establish the activity of GH161 members, we recombinantly expressed a bacterial GH161 gene sequence (PapP) from the Gram-positive bacterium Paenibacillus polymyxa ATCC 842 in Escherichia coli . We found that PapP acts on β-(1→3)-glucooligosaccharide acceptors with a degree of polymerization (DP) ≥ 2. This activity was distinct from that of characterized GH149 β-(1→3)-glucan phosphorylases, which operate on acceptors with DP ≥ 1. We also found that bacterial GH161 genes co-localize with genes encoding β-glucosidases and ATP-binding cassette transporters, highlighting a probable involvement of GH161 enzymes in carbohydrate degradation. Importantly, in some species, GH161 and GH94 genes were present in tandem, providing evidence that GPs from different CAZy families may work sequentially to degrade oligosaccharides.

The 6-deoxy sugar l -rhamnose ( l -Rha) is found widely in plant and microbial polysaccharides and natural products. The importance of this and related compounds in host–pathogen interactions often means that l -Rha plays an essential role in many organisms. l -Rha is most commonly biosynthesized as the activated sugar nucleotide uridine 5′-diphospho-β- l -rhamnose (UDP-β- l -Rha) or thymidine 5′-diphospho-β- l -rhamnose (TDP-β- l -Rha). Enzymes involved in the biosynthesis of these sugar nucleotides have been studied in some detail in bacteria and plants, but the activated form of l -Rha and the corresponding biosynthetic enzymes have yet to be explored in algae. Here, using sugar-nucleotide profiling in two representative algae, Euglena gracilis and the toxin-producing microalga Prymnesium parvum , we show that levels of UDP- and TDP-activated l -Rha differ significantly between these two algal species. Using bioinformatics and biochemical methods, we identified and characterized a fusion of the RmlC and RmlD proteins, two bacteria-like enzymes involved in TDP-β- l -Rha biosynthesis, from P. parvum . Using this new sequence and also others, we explored l -Rha biosynthesis among algae, finding that although most algae contain sequences orthologous to plant-like l -Rha biosynthesis machineries, instances of the RmlC-RmlD fusion protein identified here exist across the Haptophyta and Gymnodiniaceae families of microalgae. On the basis of these findings, we propose potential routes for the evolution of nucleoside diphosphate β- l -Rha (NDP-β- l -Rha) pathways among algae.

Glycoside phosphorylases (GPs) carry out a reversible phosphorolysis of carbohydrates into oligosaccharide acceptors and the corresponding sugar 1-phosphates. The reversibility of the reaction enables the use of GPs as biocatalysts for carbohydrate synthesis. Glycosyl hydrolase family 94 (GH94), which only comprises GPs, is one of the most studied GP families that have been used as biocatalysts for carbohydrate synthesis, in academic research and in industrial production. Understanding the mechanism of GH94 enzymes is a crucial step towards enzyme engineering to improve and expand the applications of these enzymes in synthesis. In this work with a GH94 laminaribiose phosphorylase from Paenibacillus sp. YM-1 (PsLBP), we have demonstrated an enzymatic synthesis of disaccharide 1 (β-d-mannopyranosyl-(1→3)-d-glucopyranose) by using a natural acceptor glucose and noncognate donor substrate α-mannose 1-phosphate (Man1P). To investigate how the enzyme recognises different sugar 1-phosphates, the X-ray crystal structures of PsLBP in complex with Glc1P and Man1P have been solved, providing the first molecular detail of the recognition of a noncognate donor substrate by GPs, which revealed the importance of hydrogen bonding between the active site residues and hydroxy groups at C2, C4, and C6 of sugar 1-phosphates. Furthermore, we used saturation transfer difference NMR spectroscopy to support crystallographic studies on the sugar 1-phosphates, as well as to provide further insights into the PsLBP recognition of the acceptors and disaccharide products.