The biosynthesis of l-rhamnose of plum-leaf polysaccharides.
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ABSTRACT:
SUBMITTER: Andrews P
PROVIDER: S-EPMC1264539 | biostudies-other | 1965 Oct
REPOSITORIES: biostudies-other
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Project description:1. The utilization of specifically labelled d-glucose in the biosynthesis of plum-leaf polysaccharides has been studied. After these precursors had been metabolized in plum leaves, the polysaccharides were isolated from the leaves, and their monosaccharide constituents isolated and purified. 2. Both the specific activities and the distribution of (14)C along the carbon chains of the monosaccharides were determined. Significant (14)C activity was found in units of d-galactose, d-glucose, d-xylose and l-arabinose, but their specific activities varied widely. The labelling patterns suggest that in the leaves the other monosaccharides all arise directly from d-glucose without any skeletal change in the carbon chain, other than the loss of a terminal carbon atom in the synthesis of pentoses. 3. The results indicated that within the leaf there are various precursor pools for polysaccharide synthesis and that these pools are not in equilibrium with one another.
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Project description:Cell wall glycopolymers on the surface of Gram-positive bacteria are fundamental to bacterial physiology and infection biology. Here we identify gacH, a gene in the Streptococcus pyogenes group A carbohydrate (GAC) biosynthetic cluster, in two independent transposon library screens for its ability to confer resistance to zinc and susceptibility to the bactericidal enzyme human group IIA-secreted phospholipase A2. Subsequent structural and phylogenetic analysis of the GacH extracellular domain revealed that GacH represents an alternative class of glycerol phosphate transferase. We detected the presence of glycerol phosphate in the GAC, as well as the serotype c carbohydrate from Streptococcus mutans, which depended on the presence of the respective gacH homologs. Finally, nuclear magnetic resonance analysis of GAC confirmed that glycerol phosphate is attached to approximately 25% of the GAC N-acetylglucosamine side-chains at the C6 hydroxyl group. This previously unrecognized structural modification impacts host-pathogen interaction and has implications for vaccine design.
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Project description:Rhamnose-associated molecules are attracting attention because they are present in bacteria but not mammals, making them potentially useful as antibacterial agents. Additionally, they are also valuable for tumor immunotherapy. Thus, studies on the functions and biosynthetic pathways of rhamnose-containing compounds are in progress. In this paper, studies on the biosynthetic pathways of three rhamnose donors, i.e., deoxythymidinediphosphate-L-rhamnose (dTDP-Rha), uridine diphosphate-rhamnose (UDP-Rha), and guanosine diphosphate rhamnose (GDP-Rha), are firstly reviewed, together with the functions and crystal structures of those associated enzymes. Among them, dTDP-Rha is the most common rhamnose donor, and four enzymes, including glucose-1-phosphate thymidylyltransferase RmlA, dTDP-Glc-4,6-dehydratase RmlB, dTDP-4-keto-6-deoxy-Glc-3,5-epimerase RmlC, and dTDP-4-keto-Rha reductase RmlD, are involved in its biosynthesis. Secondly, several known rhamnosyltransferases from Geobacillus stearothermophilus, Saccharopolyspora spinosa, Mycobacterium tuberculosis, Pseudomonas aeruginosa, and Streptococcus pneumoniae are discussed. In these studies, however, the functions of rhamnosyltransferases were verified by employing gene knockout and radiolabeled substrates, which were almost impossible to obtain and characterize the products of enzymatic reactions. Finally, the application of rhamnose-containing compounds in disease treatments is briefly described.
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Project description:The composition of the Gram-positive cell wall is typically described as containing peptidoglycan, proteins and essential secondary cell wall structures called teichoic acids, which comprise approximately half of the cell wall mass. The cell walls of many species within the genera Streptococcus, Enterococcus and Lactococcus contain large amounts of the sugar rhamnose, which is incorporated in cell wall-anchored polysaccharides (CWP) that possibly function as homologues of well-studied wall teichoic acids (WTA). The presence and chemical structure of many rhamnose-containing cell wall polysaccharides (RhaCWP) has sometimes been known for decades. In contrast to WTA, insight into the biosynthesis and functional role of RhaCWP has been lacking. Recent studies in human streptococcal and enterococcal pathogens have highlighted critical roles for these complex polysaccharides in bacterial cell wall architecture and pathogenesis. In this review, we provide an overview of the RhaCWP with regards to their biosynthesis, genetics and biological function in species most relevant to human health. We also briefly discuss how increased knowledge in this field can provide interesting leads for new therapeutic compounds and improve biotechnological applications.
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Project description:The presence of L-rhamnose (Rha) branches in the coaggregation receptor polysaccharides (RPS) of Streptococcus gordonii 38 and Streptococcus oralis J22 was eliminated by replacement of wefB with ermAM in these strains. The expression of this gene in S. oralis 34 did not, however, result in the addition of Rha branches to the linear RPS of this strain, which is identical to that produced by the wefB-deficient mutant of S. gordonii 38. This paradoxical finding was explained by a subtle difference in acceptor specificity of the galactose-1-phosphotransferases encoded by downstream wefC in S. gordonii 38 and wefH in S. oralis 34. These genes were distinguished by the unique ability of WefC to act on the branched acceptor formed by the action of WefB.
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Project description:The cell wall of Gram-positive bacteria has been shown to mediate environmental stress tolerance, antibiotic susceptibility, host immune evasion and overall virulence. The majority of these traits have been demonstrated for the well-studied system of wall teichoic acid (WTA) synthesis, a common cell wall polysaccharide among Gram-positive organisms. Streptococcus mutans, a Gram-positive odontopathogen that contributes to the enamel-destructive disease dental caries, lacks the capabilities to generate WTA. Instead, the cell wall of S. mutans is highly decorated with rhamnose-glucose polysaccharides (RGP), for which functional roles are poorly defined. Here, we demonstrate that the RGP has a distinct role in protecting S. mutans from a variety of stress conditions pertinent to pathogenic capability. Mutant strains with disrupted RGP synthesis failed to properly localize cell division complexes, suffered from aberrant septum formation and exhibited enhanced cellular autolysis. Surprisingly, mutant strains of S. mutans with impairment in RGP side chain modification grew into elongated chains and also failed to properly localize the presumed cell wall hydrolase, GbpB. Our results indicate that fully mature RGP has distinct protective and morphogenic roles for S. mutans, and these structures are functionally homologous to the WTA of other Gram-positive bacteria.
| S-EPMC6736739 | biostudies-literature
Project description:In Gram-positive bacteria, cell wall polysaccharides (CWPS) play critical roles in bacterial cell wall homeostasis and bacterial interactions with their immediate surroundings. In lactococci, CWPS consist of two components: a conserved rhamnan embedded in the peptidoglycan layer and a surface-exposed polysaccharide pellicle (PSP), which are linked together to form a large rhamnose-rich CWPS (Rha-CWPS). PSP, whose structure varies from strain to strain, is a receptor for many bacteriophages infecting lactococci. Here, we examined the first two steps of PSP biosynthesis, using in vitro enzymatic tests with lipid acceptor substrates combined with LC-MS analysis, AlfaFold2 modeling of protein 3D-structure, complementation experiments, and phage assays. We show that the PSP repeat unit is assembled on an undecaprenyl-monophosphate (C55P) lipid intermediate. Synthesis is initiated by the WpsA/WpsB complex with GlcNAc-P-C55 synthase activity and the PSP precursor GlcNAc-P-C55 is then elongated by specific glycosyltransferases that vary among lactococcal strains, resulting in PSPs with diverse structures. Also, we engineered the PSP biosynthesis pathway in lactococci to obtain a chimeric PSP structure, confirming the predicted glycosyltransferase specificities. This enabled us to highlight the importance of a single sugar residue of the PSP repeat unit in phage recognition. In conclusion, our results support a novel pathway for PSP biosynthesis on a lipid-monophosphate intermediate as an extracellular modification of rhamnan, unveiling an assembly machinery for complex Rha-CWPS with structural diversity in lactococci.
| S-EPMC10821137 | biostudies-literature
Project description:Despite the apparent absence of genes coding for the known pathways for biosynthesis, the monosaccharide rhamnose was detected in the d configuration in Mycoplasma pneumoniae and Mycoplasma pulmonis, and in both the d and l configurations in Mycoplasma arthritidis. Surprisingly, the monosaccharide glucose was not a precursor for rhamnose biosynthesis and was not incorporated at detectable levels in glucose-containing polysaccharides or glycoconjugates. In contrast, carbon atoms from starch, a polymer of glucose, were incorporated into rhamnose in each of the three species examined. When grown in a serum-free medium supplemented with starch, M.?arthritidis synthesized higher levels of rhamnose, with a shift in the relative amounts of the d and l configurations. Our findings suggest the presence of a novel pathway for rhamnose synthesis that is widespread in the genus Mycoplasma.
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Project description:In the halophilic archaea Haloferax volcanii, the surface (S)-layer glycoprotein can be modified by two distinct N-linked glycans. The tetrasaccharide attached to S-layer glycoprotein Asn-498 comprises a sulfated hexose, two hexoses and a rhamnose. While Agl11-14 have been implicated in the appearance of the terminal rhamnose subunit, the precise roles of these proteins have yet to be defined. Accordingly, a series of in vitro assays conducted with purified Agl11-Agl14 showed these proteins to catalyze the stepwise conversion of glucose-1-phosphate to dTDP-rhamnose, the final sugar of the tetrasaccharide glycan. Specifically, Agl11 is a glucose-1-phosphate thymidylyltransferase, Agl12 is a dTDP-glucose-4,6-dehydratase and Agl13 is a dTDP-4-dehydro-6-deoxy-glucose-3,5-epimerase, while Agl14 is a dTDP-4-dehydrorhamnose reductase. Archaea thus synthesize nucleotide-activated rhamnose by a pathway similar to that employed by Bacteria and distinct from that used by Eukarya and viruses. Moreover, a bioinformatics screen identified homologues of agl11-14 clustered in other archaeal genomes, often as part of an extended gene cluster also containing aglB, encoding the archaeal oligosaccharyltransferase. This points to rhamnose as being a component of N-linked glycans in Archaea other than Hfx. volcanii.
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Project description:Biosynthesis of the nucleotide sugar precursor dTDP-L-rhamnose is critical for the viability and virulence of many human pathogenic bacteria, including Streptococcus pyogenes (Group A Streptococcus; GAS), Streptococcus mutans and Mycobacterium tuberculosis. Streptococcal pathogens require dTDP-L-rhamnose for the production of structurally similar rhamnose polysaccharides in their cell wall. Via heterologous expression in S. mutans, we confirmed that GAS RmlB and RmlC are critical for dTDP-L-rhamnose biosynthesis through their action as dTDP-glucose-4,6-dehydratase and dTDP-4-keto-6-deoxyglucose-3,5-epimerase enzymes respectively. Complementation with GAS RmlB and RmlC containing specific point mutations corroborated the conservation of previous identified catalytic residues. Bio-layer interferometry was used to identify and confirm inhibitory lead compounds that bind to GAS dTDP-rhamnose biosynthesis enzymes RmlB, RmlC and GacA. One of the identified compounds, Ri03, inhibited growth of GAS, other rhamnose-dependent streptococcal pathogens as well as M. tuberculosis with an IC50 of 120-410 µM. Importantly, we confirmed that Ri03 inhibited dTDP-L-rhamnose formation in a concentration-dependent manner through a biochemical assay with recombinant rhamnose biosynthesis enzymes. We therefore conclude that inhibitors of dTDP-L-rhamnose biosynthesis, such as Ri03, affect streptococcal and mycobacterial viability and can serve as lead compounds for the development of a new class of antibiotics that targets dTDP-rhamnose biosynthesis in pathogenic bacteria.
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