FIGURE 9
P. aeruginosa wbpM gene can complement R. etli wreV mutant (CE568, wreV). Shown is silver-stained SDS-PAGE (18% gel) of LPS samples from whole-cell lysates. LPS I, O-antigen-containing LPS; LPS II, core oligosaccharide-Lipid A lacking O-antigen. CE3, wild-type R. etli strain; CE568, R. etli strain with a gentamicin resistance cassette insertion in wreV (wreV); CE568/ pTL61, CE568 strain carrying plasmid pTL61, which expresses the full-length WbpM.
Source publication
N-acetyl-D-quinovosamine (QuiNAc) occurs in the polysaccharide structures of many Gram-negative bacteria. In the biosynthesis of QuiNAc-containing polysaccharides, UDP-N-acetyl-D-quinovosamine is the hypothetical donor of the QuiNAc residue. Biosynthesis of UDP-QuiNAc has been proposed to occur by 4,6-dehydration of UDP-N-acetyl-D-glucosamine (UDP-...
Context in source publication
Context 1
... dideoxy-D-xylo-4-hexulose. A soluble truncated version of the WbpM protein from P. aeruginosa was used in this study to catalyze this reaction in vitro (9). The R. etli CE3 wreV gene encodes a protein that is homologous to WbpM. An insertion mutation in wreV abolished O-antigen synthesis in R. etli, and wbpM complemented this mutant (Fig. 9). Hence, WreV is inferred to be the 4,6-dehydratase responsible in R. etli for the first step of UDP-QuiNAc ...
Citations
... Based on this finding, we searched for genes encoding proteins responsible for UDP-d-Qui2N and UDP-d-Fuc2N biosynthesis. Previous studies in bacteria showed that the precursor for both Qui2N and Fuc2N was UDP-d-GlcNAc (Burrows et al. 2000, Li et al. 2014) and that their synthesis involved a two-step reaction: (1) dehydration (UDP-GlcNAc 4,6 dehydratase), resulting in the intermediate UDP-4keto-2-N-Acetyl-d-GlcNAc; and (2) reduction (4-reductase). Depending on the stereospecificity of the reductase, UDP-d-Qui2NAc or its C4-epimer UDP-d-Fuc2NAc are obtained (Fig. 6). ...
... Depending on the stereospecificity of the reductase, UDP-d-Qui2NAc or its C4-epimer UDP-d-Fuc2NAc are obtained (Fig. 6). Using bacte-rial enzymes as queries (Burrows et al. 2000, Li et al. 2014, we were able to identify corresponding enzymes in Moumouvirus maliensis ( Fig. 6 and Table 2). We found two proteins (Mm419, Mm422) homologous to C. jejuni 4,6-dehydratase (Table 2) (Riegert et al. 2017), but only Mm422 appeared to be functional, while Mm419 presented the mutation of the catalytic aspartate with asparagine (Fig. S7), which was reported to induce loss of activity (Riegert et al. 2017). ...
The recent discovery that giant viruses encode proteins related to sugar synthesis and processing paved the way for the study of their glycosylation machinery. We focused on the proposed Megavirinae subfamily for which glycan-related genes were proposed to code for protein involved in glycosylation of the layer of fibrils surrounding their icosahedral capsids. We compared sugar compositions and corresponding biosynthetic pathways among clade members using a combination of chemical and bioinformatics approaches. We first demonstrated that Megavirinae glycosylation differs in many aspects from what was previously reported for viruses, as they have complex glycosylation gene clusters made of six and up to thirty-three genes to synthetize their fibril glycans (biosynthetic pathways for nucleotides sugars and glycosyltransferases). Second, they synthesize rare amino-sugars, usually restricted to bacteria and absent from their eukaryotic host. Finally, we showed that Megavirinae glycosylation is clade-specific and that Moumouvirus australiensis, a B-clade outsider, shares key features with Cotonvirus japonicus (clade E) and Tupanviruses (clade D). The existence of a glycosylation toolbox in this family could represent an advantageous strategy to survive in an environment where members of the same family are competing for the same amoeba host. This study expands the field of viral glycobiology and raises questions on how Megavirinae evolved such versatile glycosylation machinery.
... Meanwhile, enzymatic preparation that based on HPLC or TLC purification only gave a very small amount of product for MS and NMR analysis. [29] The de novo biosynthesis of UDP-D-QuiNAc from UDP-GlcNAc undergoes two steps (C-4,6 dehydration and reduction) catalyzed by UDP-GlcNAc C-4,6 dehydratase and C-4 reductase. UDP-GlcNAc C-4,6 dehydratase that used in this work was cloned from Campylobacter jejuni (PglF) as it doesn't need exogenous NAD + /NADP + . ...
Glycosylation is catalyzed by glycosyltransferases using sugar nucleotides or occasionally lipid‐linked phosphosugars as donors. However, only very few common sugar nucleotides that occur in humans can be obtained readily, while the majority of sugar nucleotides that exist in bacteria, plants, archaea, or viruses cannot be synthesized in sufficient quantities by either enzymatic or chemical synthesis. The limited availability of such rare sugar nucleotides is one of the major obstacles that has greatly hampered progress in glycoscience. Herein we describe a general cofactor‐driven cascade conversion strategy for the efficient synthesis of sugar nucleotides. The described strategy allows the large‐scale preparation of rare sugar nucleotides from common sugars in high yields and without the need for tedious purification processes.
... IM, inner membrane; OM, outer membrane; PG, peptidoglycan; LPS, lipopolysaccharide. content and changes in the peptidoglycan composition, rendering the bacterium less susceptible to colistin and other AMPs (29)(30)(31)(32). Modifications in the bacterial membrane that reduce or inhibit the AMP-membrane interaction can be achieved indirectly through altered regulators and/or two-component systems. ...
Stenotrophomonas maltophilia is an increasingly relevant multidrug-resistant (MDR) bacterium found, for example, in people with cystic fibrosis and associated with other respiratory infections and underlying pathologies. The infections caused by this nosocomial pathogen are difficult to treat due to the intrinsic resistance of this bacterium against a broad number of antibiotics. Therefore, new treatment options are needed, and considering the growing interest in using AMPs as alternative therapeutic compounds and the restricted number of antibiotics active against S. maltophilia , we addressed the potential for development of AMP resistance, the genetic mechanisms involved, and the physiological effects that acquisition of AMP resistance has on this opportunistic pathogen.
... Due to the difficulty in obtaining purified enzymes and the limited availability of substrates, this initial step in the synthesis of an O-antigen has been demonstrated in only a few cases [16][17][18]. In R. etli CE3 O-antigen (Fig. 1a), the proposed first sugar is 2-acetamido-2,6-dideoxy-D-glucose (D-Qui-NAc, hereafter referred to as QuiNAc) [12,13,19]. Although QuiNAc is found in a number of bacterial polysaccharides, the mechanism of its incorporation into a polysaccharide, in particular as the initiating sugar, has not been reported. ...
... QuiNAc is derived from the central metabolite UDP- [19,20]. Besides WreU, two additional enzyme activities are expected in a pathway from UDP-GlcNAc to BpPP-QuiNAc (Fig. 1c). ...
... The first is a 4,6-dehydratase that catalyses conversion of UDP-GlcNAc to UDP-2-acetamido-2,6-dideoxy-D-xylo-4-hexulose (also known as UDP-4-keto-6-deoxyGlcNAc and hereafter referred to as UDP-KdgNAc). R. etli gene wreV (Fig. 1b) encodes a protein whose predicted sequence aligns with enzymes known to catalyse this reaction in vitro [19,[21][22][23][24][25]. The gene for one of these characterized enzymes, Pseudomonas aeruginosa wbpM, complements R. etli wreV mutants [19]. ...
Bacterial O-antigens are synthesized on lipid carriers before being transferred to lipopolysaccharide core structures. Rhizobium etli CE3 lipopolysaccharide is a model for understanding O-antigen biological function. CE3 O-antigen structure and genetics are known. However, proposed enzymology for CE3 O-antigen synthesis has been examined very little in vitro, and even the sugar added to begin the synthesis is uncertain. A model based on mutagenesis studies predicts that 2-acetamido-2,6-dideoxy-d-glucose (QuiNAc) is the first O-antigen sugar and that genes wreV, wreQ and wreU direct QuiNAc synthesis and O-antigen initiation. Previously, synthesis of UDP-QuiNAc was shown to occur in vitro with a WreV orthologue (4,6-hexose dehydratase) and WreQ (4-reductase), but the WreQ catalysis in this conventional deoxyhexose-synthesis pathway was very slow. This seeming deficiency was explained in the present study after WreU transferase activity was examined in vitro. Results fit the prediction that WreU transfers sugar-1-phosphate to bactoprenyl phosphate (BpP) to initiate O-antigen synthesis. Interestingly, WreU demonstrated much higher activity using the product of the WreV catalysis [UDP-4-keto-6-deoxy-GlcNAc (UDP-KdgNAc)] as the sugar-phosphate donor than using UDP-QuiNAc. Furthermore, the WreQ catalysis with WreU-generated BpPP-KdgNAc as the substrate was orders of magnitude faster than with UDP-KdgNAc. The inferred product BpPP-QuiNAc reacted as an acceptor substrate in an in vitro assay for addition of the second O-antigen sugar, mannose. These results imply a novel pathway for 6-deoxyhexose synthesis that may be commonly utilized by bacteria when QuiNAc is the first sugar of a polysaccharide or oligosaccharide repeat unit: UDP-GlcNAc → UDP-KdgNAc → BpPP-KdgNAc → BpPP-QuiNAc.
... We also note that these assignments correspond closely with those recently published for the D-QuiNAc sugar of UDP-D-QuiNAc (Supplementary information, Table S1). 26,27 Hence, the NMR data confirm that the QVMs contain an α1′′, β11′-N-acetylquinovosamine ring in place of the GlcNAc residue for TUNs. Other NMR assignments are essentially identical for the QVMs and TUNs, with the uracil motif confirmed by the characteristic H5 and H-6 signals, the 11-carbon tunicamine dialdose by the two anomeric signals H-1′/C-1′ and H11′/C11′, and the 2,3-unsaturation in the N-acyl chain from the H-2′′′/C-2′′′ and H3′′′/C3′′′ signals (Supplementary information, Table S1). ...
Tunicamycins (TUN) are potent inhibitors of polyprenyl phosphate N-acetylhexosamine 1-phosphate transferases (PPHP), including essential eukaryotic GPT enzymes and bacterial HexNAc 1-P translocases. Hence, TUN blocks the formation of eukaryotic N-glycoproteins and the assembly of bacterial call wall polysaccharides. The genetic requirement for TUN production is well-established. Using two genes unique to the TUN pathway (tunB and tunD) as probes we identified four new prospective TUN-producing strains. Chemical analysis showed that one strain, Streptomyces niger NRRL B-3857, produces TUN plus new compounds, named quinovosamycins (QVMs). QVMs are structurally akin to TUN, but uniquely in the 1″,11'-HexNAc sugar head group, which is invariably d-GlcNAc for the known TUN, but is d-QuiNAc for the QVM. Surprisingly, this modification has only a minor effect on either the inhibitory or antimicrobial properties of QVM and TUN. These findings have unexpected consequences for TUN/QVM biosynthesis, and for the specificity of the PPHP enzyme family.The Journal of Antibiotics advance online publication, 18 May 2016; doi:10.1038/ja.2016.49.
... As shown in Table 1B, Preq shares, for example, 33% amino acid sequence identity with functional GDP-4-keto-6-deoxy-D-mannose 4-reductase from Aneurinibacillus thermoaerophilus [30] and lower sequence homology (24%) with dTDP-glucose 4,6-dehydratase (Rmlb) from Salmonella Enterica serovar Typhimurium [31]. Also, it shares lower sequence homology (29%) with functional UDP-2-acetamido-2,6-dideoxy-D-xylo-4-hexulose-4-reductase from Rhizobium etili [32] even though its function, as we will described, is the same with Bacillus protein Preq. Below we provide biochemical evidences of Pdeg (Bc3750) and Preq (Bc3749) proteins for their sequential ability to convert UDP-GlcNAc to UDP-4-keto-6-deoxy-GlcNAc and to UDP-QuiNAc. ...
... While this work was in progress, a study in the gram-negative, Rhizobium etili, discovered for the first time that this bacterium has two enzyme activities [32] identical to those as described here for the gram-positive bacterium, Bacillus. Both the rhizobium and Bacillus proteins have the same 4,6-dehydratase and 4-reductase activities to make UDP-QuiNAc. ...
N-acetylquinovosamine (2-acetamido-2,6-di-deoxy-D-glucose, QuiNAc) is a relatively rare amino sugar residue found in glycans of few pathogenic gram-negative bacteria where it can play a role in infection. However, little is known about QuiNAc-related polysaccharides in gram-positive bacteria. In a routine screen for bacillus glycan grown at defined medium, it was surprising to identify a QuiNAc residue in polysaccharides isolated from this gram-positive bacterium. To gain insight into the biosynthesis of these glycans, we report the identification of an operon in Bacillus cereus ATCC 14579 that contains two genes encoding activities not previously described in gram-positive bacteria. One gene encodes a UDP-N-acetylglucosamine C4,6-dehydratase, (abbreviated Pdeg) that converts UDP-GlcNAc to UDP-4-keto-4,6-D-deoxy-GlcNAc (UDP-2-acetamido-2,6-dideoxy-α-D-xylo-4-hexulose); and the second encodes a UDP-4-reductase (abbr. Preq) that converts UDP-4-keto-4,6-D-deoxy-GlcNAc to UDP-N-acetyl-quinovosamine in the presence of NADPH. Biochemical studies established that the sequential Pdeg and Preq reaction product is UDP-D-QuiNAc as determined by mass spectrometry and one- and two-dimensional NMR experiments. Also, unambiguous evidence for the conversions of the dehydratase product, UDP-α-D-4-keto-4,6-deoxy-GlcNAc, to UDP-α-D-QuiNAc was obtained using real-time 1H-NMR spectroscopy and mass spectrometry. The two genes overlap by 4 nucleotides and similar operon organization and identical gene sequences were also identified in a few other Bacillus species suggesting they may have similar roles in the lifecycle of this class of bacteria important to human health. Our results provide new information about the ability of Bacilli to form UDP-QuiNAc and will provide insight to evaluate their role in the biology of Bacillus.
... Signals for keto or hydrated keto groups as well as signals for deuterated UDP-d-glucose in the product mixture could not be detected within the detection limit of the NMR instrument. Also, in situ monitoring of the enzymatic reaction in an NMR tube for 5 h at 333 K (60 °C) revealed a slow formation of the UDP-d-glucose-5,6-ene product but did not provide direct evidence of a 4-keto intermediate (Li et al. 2014). ...
The UDP-sulfoquinovose synthase Agl3 from Sulfolobus acidocaldarius converts UDP-d-glucose and sulfite to UDP-sulfoquinovose, the activated form of sulfoquinovose required for its incorporation into glycoconjugates. Based on the amino acid sequence, Agl3 belongs to the short-chain dehydrogenase/reductase enzyme superfamily, together with SQD1 from Arabidopsis thaliana, the only UDP-sulfoquinovose synthase with known crystal structure. By comparison of sequence and structure of Agl3 and SQD1, putative catalytic amino acids of Agl3 were selected for mutational analysis. The obtained data suggest for Agl3 a modified dehydratase reaction mechanism. We propose that in vitro biosynthesis of UDP-sulfoquinovose occurs through an NAD+-dependent oxidation/dehydration/enolization/sulfite addition process. In the absence of a sulfur donor, UDP-d-glucose is converted via UDP-4-keto-d-glucose to UDP-d-glucose-5,6-ene, the structure of which was determined by 1H and 13C-NMR spectroscopy. During the redox reaction the cofactor remains tightly bound to Agl3 and participates in the reaction in a concentration-dependent manner. For the first time, the rapid initial electron transfer between UDP-d-glucose and NAD+ could be monitored in a UDP-sulfoquinovose synthase. Deuterium labeling confirmed that dehydration of UDP-d-glucose occurs only from the enol form of UDP-4-keto-glucose. The obtained functional data are compared with those from other UDP-sulfoquinovose synthases. A divergent evolution of Agl3 from S.
acidocaldarius is suggested.
... It would be difficult to extract the natural donor and acceptor substrates from bacteria in the pure form. Therefore, syntheses for bacteria-specific donor substrate analogs have been developed, e.g., for UDP-QuiNAc (UDP-6-deoxy-GlcNAc) found in E. coli and Pseudomonas aeruginosan (PA) (53) or for GDPd-Rha found in PA (54). Oligosaccharides linked to a synthetic aglycone group may be suitable acceptors for both, mammalian GTs and bacterial GTs. ...
Bacterial glycosyltransferases (GT) often synthesize the same glycan linkages as mammalian GT; yet, they usually have very little sequence identity. Nevertheless, enzymatic properties, folding, substrate specificities, and catalytic mechanisms of these enzyme proteins may have significant similarity. Thus, bacterial GT can be utilized for the enzymatic synthesis of both bacterial and mammalian types of complex glycan structures. A comparison is made here between mammalian and bacterial enzymes that synthesize epitopes found in mammalian glycoproteins, and those found in the O antigens of Gram-negative bacteria. These epitopes include Thomsen–Friedenreich (TF or T) antigen, blood group O, A, and B, type 1 and 2 chains, Lewis antigens, sialylated and fucosylated structures, and polysialic acids. Many different approaches can be taken to investigate the substrate binding and catalytic mechanisms of GT, including crystal structure analyses, mutations, comparison of amino acid sequences, NMR, and mass spectrometry. Knowledge of the protein structures and functions helps to design GT for specific glycan synthesis and to develop inhibitors. The goals are to develop new strategies to reduce bacterial virulence and to synthesize vaccines and other biologically active glycan structures.
Glycosylation is catalyzed by glycosyltransferases using sugar nucleotides or occasionally lipid‐linked phosphosugars as donors. However, only very few common sugar nucleotides that occur in humans can be obtained readily, while the majority of sugar nucleotides that exist in bacteria, plants, archaea, or viruses cannot be synthesized in sufficient quantities by either enzymatic or chemical synthesis. The limited availability of such rare sugar nucleotides is one of the major obstacles that has greatly hampered progress in glycoscience. Herein we describe a general cofactor‐driven cascade conversion strategy for the efficient synthesis of sugar nucleotides. The described strategy allows the large‐scale preparation of rare sugar nucleotides from common sugars in high yields and without the need for tedious purification processes.
