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Generation of pyruvyl neo-human-type complex glycopeptide. (a) Structure of the glycopeptides AGP (left, no pyruvylation) and PvGP (right, both ends of the oligosaccharide chains are pyruvylated). HPLC chromatograms of (b) AGP and (c) PvGP. AGP was treated without (b) or with (c) Pvg1p H168C and reaction mixtures were analyzed by HPLC. Sample collected from the highest peak in (c) indeed contained PvGP, which was confirmed by mass spectrometry analysis (see Supplementary Fig. S5).
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Pyruvylation onto the terminus of oligosaccharide, widely seen from prokaryote to eukaryote, confers negative charges on the cell surface and seems to be functionally similar to sialylation, which is found at the end of human-type complex oligosaccharide. However, detailed molecular mechanisms underlying pyruvylation have not been clarified well. H...
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... and assessment of neo pyruvyl human-type complex glycopeptide. We next determined whether Pvg1p H168C could attach pyruvate onto both terminal β -Gal residues of asialo glycopeptide (AGP), a human-type complex oligosaccharide acceptor substrate (Fig. 5a). HPLC analysis of the reaction mix- ture revealed that the retention time of the resulting reaction product was different from that of AGP (compare Fig. 5b,c), suggesting that the pyruvate moiety became attached to the terminal oligosaccharide chain of AGP. To confirm that biantennary pyruvyl glycopeptide (PvGP) was indeed produced by ...
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... We next determined whether Pvg1p H168C could attach pyruvate onto both terminal β -Gal residues of asialo glycopeptide (AGP), a human-type complex oligosaccharide acceptor substrate (Fig. 5a). HPLC analysis of the reaction mix- ture revealed that the retention time of the resulting reaction product was different from that of AGP (compare Fig. 5b,c), suggesting that the pyruvate moiety became attached to the terminal oligosaccharide chain of AGP. To confirm that biantennary pyruvyl glycopeptide (PvGP) was indeed produced by this reaction, the peak sample obtained from the HPLC analysis was collected and analyzed by MALDI-TOF MS. As can be seen from the mass spectrum, the observed ...
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... [M−N] = 2421.36) ( Supplementary Fig. S5), which confirmed that the peak sample collected from HPLC analysis mainly consisted of PvGP. ...
Citations
... Current pyruvyltransferase structures -The only reported pyruvyltransferase structure to date is of unliganded Pvg1P from Schizosaccharomyces pombe (PDB: 5AXY), which has led to an in silico model (generated using MOE software in combination with ASEDock docking simulations) that attempts to describe Pvg1P substrate binding (Higuchi, Yoshinaga et al., 2016). The Pvg1P structure forms a positively-charged cleft between its N-and C-terminal Rossman-like domains, which is predicted to facilitate binding of the negatively-charged pyruvate moiety (Higuchi, Yoshinaga et al., 2016). ...
... Current pyruvyltransferase structures -The only reported pyruvyltransferase structure to date is of unliganded Pvg1P from Schizosaccharomyces pombe (PDB: 5AXY), which has led to an in silico model (generated using MOE software in combination with ASEDock docking simulations) that attempts to describe Pvg1P substrate binding (Higuchi, Yoshinaga et al., 2016). The Pvg1P structure forms a positively-charged cleft between its N-and C-terminal Rossman-like domains, which is predicted to facilitate binding of the negatively-charged pyruvate moiety (Higuchi, Yoshinaga et al., 2016). Within this cleft, modeled ligands indicate the presence of three candidate catalytic residues, which are conserved in CsaB homologs from bacteria of the phylum Firmicutes. ...
... Pyruvate-ketal modified (henceforth termed "pyruvylated") glycans are found in various kingdoms of life where they have a wide repertoire of biological roles [1]. Pyruvylated galactose, for instance, is an epitope of the N-glycans of Schizosaccharomyces pombe [2], in the exopolysaccharide (EPS) of Xanthomonas campestris [3], and the capsular polysaccharides (CPS) of Bacteroides fragilis [4] and Streptococcus pneumoniae [5]. Pyruvylated N-acetylmannosamine (pyr-ManNAc) is present in peptidoglycan-linked cell wall glycopolymers (CWGPs) of Gram-positive bacteria, where it is an indispensable cell wall ligand for S-layer homology (SLH) domain-containing proteins [6,7]. ...
... Two strategies were pursued for the generation of β-D-ManNAc-(1→4)-α-D-GlcNAc-PP-UndPh (2) from (1) in sufficient amounts to optimise the CsaB assay. Following strategy A, we performed an in situ coupled reaction with rWecB to epimerise UDP-α-GlcNAc to UDP-α-ManNAc, and rTagA was employed to elongate (1) to the disaccharide state (2). rWecB showed~10% epimerisation efficiency in a 1-h/37 • C reaction, which is comparable to that obtained previously with the UDP-GlcNAc-2-epimerase MnaA from P. alvei [11]. ...
... rWecB showed~10% epimerisation efficiency in a 1-h/37 • C reaction, which is comparable to that obtained previously with the UDP-GlcNAc-2-epimerase MnaA from P. alvei [11]. Prolonged incubation (at 25 • C, overnight) of rWecB, UDP-GlcNAc and rTagA with (1) yielded full glycosylation efficiency to produce (2). Following strategy B, direct provision of an excess of synthesised UDP-α-D-ManNAc in a 1-h/37 • C rTagA reaction, followed by incubation overnight as above, reproducibly yielded (2) to completeness. ...
Ketalpyruvyltransferases belong to a widespread but little investigated class of enzymes, which utilise phosphoenolpyruvate (PEP) for the pyruvylation of saccharides. Pyruvylated saccharides play pivotal biological roles, ranging from protein binding to virulence. Limiting factors for the characterisation of ketalpyruvyltransferases are the availability of cognate acceptor substrates and a straightforward enzyme assay. We report on a fast ketalpyruvyltransferase assay based on the colorimetric detection of phosphate released during pyruvyltransfer from PEP onto the acceptor via complexation with Malachite Green and molybdate. To optimise the assay for the model 4,6-ketalpyruvyl::ManNAc-transferase CsaB from Paenibacillus alvei, a β-d-ManNAc-α-d-GlcNAc-diphosphoryl-11-phenoxyundecyl acceptor mimicking an intermediate of the bacterium’s cell wall glycopolymer biosynthesis pathway, upon which CsaB is naturally active, was produced chemo-enzymatically and used together with recombinant CsaB. Optimal assay conditions were 5 min reaction time at 37 °C and pH 7.5, followed by colour development for 1 h at 37 °C and measurement of absorbance at 620 nm. The structure of the generated pyruvylated product was confirmed by NMR spectroscopy. Using the established assay, the first kinetic constants of a 4,6-ketalpyuvyl::ManNAc-transferase could be determined; upon variation of the acceptor and PEP concentrations, a KM, PEP of 19.50 ± 3.50 µM and kcat, PEP of 0.21 ± 0.01 s−1 as well as a KM, Acceptor of 258 ± 38 µM and a kcat, Acceptor of 0.15 ± 0.01 s−1 were revealed. P. alvei CsaB was inactive on synthetic pNP-β-d-ManNAc and β-d-ManNAc-β-d-GlcNAc-1-OMe, supporting the necessity of a complex acceptor substrate.
... Including WcfO, a total of three pyruvyltransferases have now been functionally characterized. Pvg1b is from an eukaryote, and whose crystal structure has been solved [58,59]. ...
Capsular Polysaccharide A (CPSA) is a polymer of a tetrasaccharide unit found on the surface of the symbiotic gut bacteria Bacteroides fragilis. CPSA has been suggested to be important for maintaining a natural equilibrium between Th1 and Th2 cell levels in the normal immune system of mammals. If this equilibrium is disrupted, the human body can develop different autoimmune disorders. The gene locus responsible for CPSA biosynthesis has been previously identified. The locus was proposed to encode one glycosyl-1-phosphate transferase (WcfS) and three glycosyltransferases (WcfN, -P and -Q), three sugar modifying enzymes (WcfM, WcfR and WcfO), a flippase (Wzx) and a polysaccharide polymerase (Wzy) based on homology tools. A route for the complete biosynthesis of CPSA has been elucidated. The initiating sugar transferase, WcfS has been previously identified and characterized. An in vitro method was used to enzymatically synthesize CPSA, which was assembled on a fluorescent analogue of the native bactoprenyl diphosphate anchor one sugar at a time. Function of the hypothesized pyruvyltransferase WcfO was also determined. This is the first study to characterize a pyruvyltransferase involved in polysaccharide biosynthesis from a prokaryote. The biosynthesis of the polysaccharide was achieved in a single pot, compared to multiple steps involved in chemical synthesis, displaying an enormous leap in the biosynthesis of complex molecules like CPSA.
... In addition to Sia-containing glycans, we explored the synthesis of pyruvalated galactose because this structure displays similar lectin-binding properties to Sia 54 . To build terminally pyruvylated lactose, we selected a pyruvyltransferase from Schizosaccharomyces pombe (SpPvg1) 54 . ...
Glycosylation plays important roles in cellular function and endows protein therapeutics with beneficial properties. However, constructing biosynthetic pathways to study and engineer precise glycan structures on proteins remains a bottleneck. Here, we report a modular, versatile cell-free platform for glycosylation pathway assembly by rapid in vitro mixing and expression (GlycoPRIME). In GlycoPRIME, glycosylation pathways are assembled by mixing-and-matching cell-free synthesized glycosyltransferases that can elaborate a glucose primer installed onto protein targets by an N-glycosyltransferase. We demonstrate GlycoPRIME by constructing 37 putative protein glycosylation pathways, creating 23 unique glycan motifs, 18 of which have not yet been synthesized on proteins. We use selected pathways to synthesize a protein vaccine candidate with an α-galactose adjuvant motif in a one-pot cell-free system and human antibody constant regions with minimal sialic acid motifs in glycoengineered Escherichia coli. We anticipate that these methods and pathways will facilitate glycoscience and make possible new glycoengineering applications. Constructing biosynthetic pathways to study and engineer glycoprotein structures is difficult. Here, the authors use GlycoPRIME, a cell-free workflow for mixing-and-matching glycosylation enzymes, to evaluate 37 putative glycosylation pathways and discover routes to 18 new glycoprotein structures
... One constraint is that glycosylation structures are difficult to control within complex cellular environments because glycan biosynthesis is not template driven 1,13,14 . Glycans are synthesized by the coordinated activities of many glycosyltransferases (GTs) across several subcellular compartments 1 , leading to heterogeneity and complicating engineering efforts [13][14][15] . Another challenge is the shortage of methods for modular assembly of biosynthetic pathways to rapidly access a diversity of glycan structures. ...
... New cell lines must be developed to test each new glycosylation pathway 11,12 , and essential biosynthetic pathways in eukaryotic organisms constrain the ability to modularly build synthetic pathways towards any user-defined glycosylation structure 9,16 . Furthermore, the efficient and controlled conjugation of glycans onto proteins outside of natural systems remains challenging 13,15 . A key issue for biochemical approaches is that many of the most important components of protein glycosylation pathways are associated with cellular membranes 13 . ...
... OSTs are integral membrane proteins that often contain multiple subunits 17 and LLOs are difficult to synthesize and manipulate in vitro 13 . Despite recent advances enabling the production, characterization, and use of OSTs in cell-free systems [18][19][20] , the complexity of these membraneassociated components still presents a major barrier for glycoengineering and the facile construction of multienzyme glycosylation pathways in vitro 13,15 . ...
Glycosylation plays important roles in cellular function and endows protein therapeutics with beneficial properties. However, constructing biosynthetic pathways to study and engineer protein glycosylation remains a bottleneck. To address this limitation, we describe a modular, versatile cell-free platform for glycosylation pathway assembly by rapid in vitro mixing and expression (GlycoPRIME). In GlycoPRIME, crude cell lysates are enriched with glycosyltransferases by cell-free protein synthesis and then glycosylation pathways are assembled in a mix-and-match fashion to elaborate a single glucose priming handle installed by an N-linked glycosyltransferase. We demonstrate GlycoPRIME by constructing 37 putative protein glycosylation pathways, creating 23 unique glycan motifs. We then use selected pathways to design a one-pot cell-free system to synthesize a vaccine protein with an α-galactose motif and engineered Escherichia coli strains to produce human antibody constant regions with minimal sialic acid motifs. We anticipate that our work will facilitate glycoscience and make possible new glycoengineering applications.
... Charged surface representation analysis revealed a positively charged cleft situated between the N-and Cterminal halves of Pvg1p, which suggests a possible mode of binding that may accommodate the negatively charged PEP donor substrate. Since neither PEP-nor pNP-β-Gal-co-crystal structures with the enzyme could be obtained, the empty substrate-binding cleft was used as a scaffold for computational substrate modelling using PEP [198]. In the proposed computational model, residues R217, R337, L338, and H339 form direct hydrogen bond contacts with PEP. ...
... The crystallization study indicated that the pyruvylation process mimics sialyation; interestingly, Pvg1p shows resistance to sialidase digestion. Thus, a better characterization of the effects of pyruvylation might facilitate the development of pharmaceutical glycoproteins [198]. From the same research group, an enzyme was characterised as a 4,6Pyr-β-D-Gal-releasing enzyme (PyrGal-ase) with specificity for the (1→3) yeast linkage; mammalian (1→4)-linked PyrGal could not be hydrolysed. ...
... Importantly, pyruvylation imparts an anionic character to the glycoconjugates, which is pivotal to many biological functions. Described functions include the influence on the viscosity of the EPS, bacterial symbiosis with plants [18,28,46], immunostimulatory effects (mostly of CPSs [7,93]), employment of sialylation-like properties in human-type oligosaccharides [198], and cell wall anchoring relying on the Pyr-β-D-ManNAc epitope [14,99,104,195], to name a few. However, learning more about the biological significance of pyruvylated glycoconjugates and delineating a possible association between the position of pyruvylation and functionality are remaining challenges for future research. ...
Glycoconjugates are the most diverse biomolecules of life. Mostly located at the cell surface, they translate into cell-specific “barcodes” and offer a vast repertoire of functions, including support of cellular physiology, lifestyle, and pathogenicity. Functions can be fine-tuned by non-carbohydrate modifications on the constituting monosaccharides. Among these modifications is pyruvylation, which is present either in enol or ketal form. The most commonly best-understood example of pyruvylation is enol-pyruvylation of N-acetylglucosamine, which occurs at an early stage in the biosynthesis of the bacterial cell wall component peptidoglycan. Ketal-pyruvylation, in contrast, is present in diverse classes of glycoconjugates, from bacteria to algae to yeast—but not in humans. Mild purification strategies preventing the loss of the acid-labile ketal-pyruvyl group have led to a collection of elucidated pyruvylated glycan structures. However, knowledge of involved pyruvyltransferases creating a ring structure on various monosaccharides is scarce, mainly due to the lack of knowledge of fingerprint motifs of these enzymes and the unavailability of genome sequences of the organisms undergoing pyruvylation. This review compiles the current information on the widespread but under-investigated ketal-pyruvylation of monosaccharides, starting with different classes of pyruvylated glycoconjugates and associated functions, leading to pyruvyltransferases, their specificity and sequence space, and insight into pyruvate analytics.
... This pyruvylation confers a negative charge and has versatile physiological roles, for example, in intercellular interactions (3). Although several enzymes involved in the biosynthesis of Pv-containing sugar chains have been reported, enzymes that metabolize Pv-attached oligosaccharides are not well understood (4)(5)(6). We previously screened soil samples and found a strain that exhibits -D-galactosidase activity to release Pv-attached galactose (PvGal) (7). ...
The genome sequence of the Bacillus sp. strain HMA207, the culture supernatant of which exhibited β- d -galactosidase activity to release pyruvylated galactose (PvGal), was examined to identify a PvGal-ase-encoding gene. We report here the result of whole-genome shotgun sequencing, which revealed putative PvGal-ase genes.
... Therefore, it is currently hard to predict how S. pombe galactomannan is metabolized. Previously, we generated pyruvylated human-type complex glycopeptides by using a Pvg1p H168C mutant that can attach Pv to the terminal β-1,4-linked Gal residue of human-type complex glycopeptide 20 . Thus, we also tested whether ORF1119 PvGal-ase can cleave PvGal from pyruvylated human-type complex glycopeptide; in this case, however, we did not detect enzymatic activity (data not shown). ...
Pyruvyl modification of oligosaccharides is widely seen in both prokaryotes and eukaryotes. Although the biosynthetic mechanisms of pyruvylation have been investigated, enzymes that metabolize and degrade pyruvylated oligosaccharides are not well known. Here, we searched for a pyruvylated galactose (PvGal)-releasing enzyme by screening soil samples. We identified a Bacillus strain, as confirmed by the 16S ribosomal RNA gene analysis, that exhibited PvGal-ase activity toward p-nitrophenyl-β-D-pyruvylated galactopyranose (pNP-β-D-PvGal). Draft genome sequencing of this strain, named HMA207, identified three candidate genes encoding potential PvGal-ases, among which only the recombinant protein encoded by ORF1119 exhibited PvGal-ase activity. Although ORF1119 protein displayed broad substrate specificity for pNP sugars, pNP-β-D-PvGal was the most favorable substrate. The optimum pH for the ORF1119 PvGal-ase was determined as 7.5. A BLAST search suggested that ORF1119 homologs exist widely in bacteria. Among two homologs tested, BglC from Clostridium but not BglH from Bacillus showed PvGal-ase activity. Crystal structural analysis together with point mutation analysis revealed crucial amino acids for PvGal-ase activity. Moreover, ORF1119 protein catalyzed the hydrolysis of PvGal from galactomannan of Schizosaccharomyces pombe, suggesting that natural polysaccharides might be substrates of the PvGal-ase. This novel PvGal-catalyzing enzyme might be useful for glycoengineering projects to produce new oligosaccharide structures.
... The recombinant Pvg1p enzyme transfered pyruvyl residues from PEP specifically to β-linked galactose as present in p-nitrophenyl-β-Gal (pNPβ-Gal) or pNP-β-lactose (Yoritsune et al., 2013). The crystal structure of the pyruvyltransferase Pvg1p was obtained at a resolution of 2.46 Å, which served as a basis for enzyme/substrate modeling (Higuchi et al., 2016). PvGal biosynthesis was also studied in vitro in the context of the therapeutic potential of the Bacteroides fragilis capsular polysaccharide A (Sharma et al., 2017). ...
Various mechanisms of protein cell surface display have evolved during bacterial evolution. Several Gram-positive bacteria employ S-layer homology (SLH) domain-mediated sorting of cell-surface proteins and concomitantly engage a pyruvylated secondary cell-wall polymer as a cell-wall ligand. Specifically, pyruvate ketal linked to β-D-ManNAc is regarded as an indispensable epitope in this cell-surface display mechanism. That secondary cell wall polymer (SCWP) pyruvylation and SLH domain-containing proteins are functionally coupled is supported by the presence of an ortholog of the predicted pyruvyltransferase CsaB in bacterial genomes, such as those of Bacillus anthracis and Paenibacillus alvei. The P. alvei SCWP, consisting of pyruvylated disaccharide repeats [→4)-β-D-GlcNAc-(1→3)-4,6-Pyr-β-D-ManNAc-(1→] serves as a model to investigate the widely unexplored pyruvylation reaction. Here, we reconstituted the underlying enzymatic pathway in vitro in combination with synthesized compounds, used mass spectrometry, and nuclear magnetic resonance spectroscopy for product characterization, and found that CsaB-catalyzed pyruvylation of β-D-ManNAc occurs at the stage of the lipid-linked repeat. We produced the P. alvei TagA (PAV_RS07420) and CsaB (PAV_RS07425) enzymes as recombinant, tagged proteins, and using a synthetic 11-phenoxyundecyl-diphosphoryl-α-GlcNAc acceptor, we uncovered that TagA is an inverting UDP-α-D-ManNAc:GlcNAc-lipid carrier transferase, and that CsaB is a pyruvyltransferase, with synthetic UDP-α-D-ManNAc and phosphoenolpyruvate serving as donor substrates. Next, to substitute for the UDP-α-D-ManNAc substrate, the recombinant UDP-GlcNAc-2-epimerase MnaA (PAV_RS07610) of P. alvei was included in this in vitro reconstitution system. When all three enzymes, their substrates and the lipid-linked GlcNAc primer were combined in a one-pot reaction, a lipid-linked SCWP repeat precursor analog was obtained. This work highlights the biochemical basis of SCWP biosynthesis and bacterial pyruvyl transfer.
Pyruvylation is a biologically versatile but mechanistically unexplored saccharide modification. 4,6-Ketal pyruvylated N-acetylmannosamine within bacterial secondary cell wall polymers serves as a cell wall anchoring epitope for proteins possessing a terminal S-layer homology domain trimer. The pyruvyltransferase CsaB from Paenibacillus alvei served as a model to investigate the structural basis of the pyruvyltransfer reaction by a combination of molecular modelling and site-directed mutagenesis together with an enzyme assay using phosphoenolpyruvate (PEP; donor) and synthetic β-D-ManNAc-(1 → 4)-α-D-GlcNAc-diphosphoryl-11-phenoxyundecyl (acceptor). CsaB protein structure modelling was done using Phyre2 and I-Tasser based on the partial crystal structure of the Schizosaccharomyces pombe pyruvyltransferase Pvg1p and by AlphaFold. The models informed the construction of twelve CsaB mutants targeted at plausible PEP and acceptor binding sites and KM and kcat values were determined to evaluate the mutants, indicating the importance of a loop region for catalysis. R148, H308 and K328 were found to be critical to PEP binding and insight into acceptor binding was obtained from an analysis of Y14 and F16 mutants, confirming the modelled binding sites and interactions predicted using Molecular Operating Environment. These data lay the basis for future mechanistic studies of saccharide pyruvylation as a novel target for interference with bacterial cell wall assembly.
