RESUMO
KpsC is a dual-module glycosyltransferase (GT) essential for "group 2" capsular polysaccharide biosynthesis in Escherichia coli and other Gram-negative pathogens. Capsules are vital virulence determinants in high-profile pathogens, making KpsC a viable target for intervention with small-molecule therapeutic inhibitors. Inhibitor development can be facilitated by understanding the mechanism of the target enzyme. Two separate GT modules in KpsC transfer 3-deoxy-ß-d-manno-oct-2-ulosonic acid (ß-Kdo) from cytidine-5'-monophospho-ß-Kdo donor to a glycolipid acceptor. The N-terminal and C-terminal modules add alternating Kdo residues with ß-(2â4) and ß-(2â7) linkages, respectively, generating a conserved oligosaccharide core that is further glycosylated to produce diverse capsule structures. KpsC is a retaining GT, which retains the donor anomeric carbon stereochemistry. Retaining GTs typically use an SNi (substitution nucleophilic internal return) mechanism, but recent studies with WbbB, a retaining ß-Kdo GT distantly related to KpsC, strongly suggest that this enzyme uses an alternative double-displacement mechanism. Based on the formation of covalent adducts with Kdo identified here by mass spectrometry and X-ray crystallography, we determined that catalytically important active site residues are conserved in WbbB and KpsC, suggesting a shared double-displacement mechanism. Additional crystal structures and biochemical experiments revealed the acceptor binding mode of the ß-(2â4)-Kdo transferase module and demonstrated that acceptor recognition (and therefore linkage specificity) is conferred solely by the N-terminal α/ß domain of each GT module. Finally, an Alphafold model provided insight into organization of the modules and a C-terminal membrane-anchoring region. Altogether, we identified key structural and mechanistic elements providing a foundation for targeting KpsC.
Assuntos
Cápsulas Bacterianas , Glicosiltransferases , Cápsulas Bacterianas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Glicolipídeos/metabolismo , Glicosiltransferases/genética , Glicosiltransferases/química , Lipopolissacarídeos/metabolismo , Açúcares Ácidos/metabolismo , Transferases/metabolismo , Polissacarídeos Bacterianos/metabolismoRESUMO
We developed an efficient method to achieve the regioselective acyl migration of benzoyl ester. In all the cases, the reactions required only the commercially available organic acid catalyst TsOH·H2O. This method enables the benzoyl group to migrate from secondary groups to primary hydroxyl groups, or from equatorial secondary hydroxyl groups to axial hydroxyl groups. The 1,2 or 1,3 acyl migration would potentially occur via five- and six-membered cyclic ortho acid intermediates. A wide range of orthogonally protected monosaccharides, which are useful intermediates for the synthesis of natural oligosaccharides, were synthesized. Finally, to demonstrate the utility of the method, a tetrasaccharide portion from a mycobacterial cell wall polysaccharide was assembled.
RESUMO
Several important Gram-negative bacterial pathogens possess surface capsular layers composed of hypervariable long-chain polysaccharides linked via a conserved 3-deoxy-ß-D-manno-oct-2-ulosonic acid (ß-Kdo) oligosaccharide to a phosphatidylglycerol residue. The pathway for synthesis of the terminal glycolipid was elucidated by determining the structures of reaction intermediates. In Escherichia coli, KpsS transfers a single Kdo residue to phosphatidylglycerol; this primer is extended using a single enzyme (KpsC), possessing two cytidine 5'-monophosphate (CMP)-Kdo-dependent glycosyltransferase catalytic centers with different linkage specificities. The structure of the N-terminal ß-(2â4) Kdo transferase from KpsC reveals two α/ß domains, supplemented by several helices. The N-terminal Rossmann-like domain, typically responsible for acceptor binding, is severely reduced in size compared with canonical GT-B folds in glycosyltransferases. The similar structure of the C-terminal ß-(2â7) Kdo transferase indicates a past gene duplication event. Both Kdo transferases have a narrow active site tunnel, lined with key residues shared with GT99 ß-Kdo transferases. This enzyme provides the prototype for the GT107 family.
Assuntos
Cápsulas Bacterianas/metabolismo , Glicolipídeos/biossíntese , Bactérias Gram-Negativas/metabolismo , Transferases/metabolismo , Modelos Moleculares , Estrutura Molecular , Transferases/químicaRESUMO
We report a siloxane-protected donor (7) for the highly stereoselective formation of ß-(2,3-cis)-xylulofuranosyl bonds. Glycosylation reactions with 7 gave >80% yields, and only ß-xylulofuranosides were isolated in all cases. The utility of 7 for the synthesis of complex glycans was shown by its successful application to the preparation of the repeating unit from the lipopolysaccharide O-antigen of Yersinia enterocolitica serovars O:5/O:5,27. This structure is a pentasaccharide with two ß-xylulofuranose residues; using 7, both were introduced simultaneously with excellent stereocontrol.
RESUMO
Reported is the first stereoselective method for ß-xylulofuranosylation, which employs 3,4-O-xylylene-protected thioglycoside donors. For most acceptors, the best results were observed with a donor (8) that possesses both the xylylene group and a benzoate ester at O-1. To demonstrate its utility, the methodology was applied to the first synthesis of the pentasaccharide repeating unit from the lipopolysaccharide O-antigen of Yersinia enterocolitica serovars O:5/O:5,27, a structure containing two ß-xylulofuranose residues.
RESUMO
Bacterial capsular polysaccharides are important virulence factors. Capsular polysaccharides from several important Gram-negative pathogens share a conserved glycolipid terminus containing 3-deoxy-ß-d- manno-oct-2-ulosonic acid (ß-Kdo). The ß-Kdo glycosyltransferases responsible for synthesis of this conserved glycolipid belong to a new family of glycosyltransferases that shares little homology with other such enzymes, thereby representing an attractive antivirulence target. Here, we report the development of a fluorescence polarization-based, high-throughput screening assay (FP-tag) for ß-Kdo glycosyltransferases, and use it to identify a class of marine natural products as lead inhibitors. This "FP-tag" assay should be readily adaptable to high-throughput screens of other glycosyltransferases.
Assuntos
Inibidores Enzimáticos/farmacologia , Glicosiltransferases/antagonistas & inibidores , Química Click , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/química , Ensaios de Triagem em Larga Escala , Concentração Inibidora 50 , Açúcares Ácidos/química , Açúcares Ácidos/farmacologiaRESUMO
3-Deoxy-d-manno-oct-2-ulosonic acid (Kdo) is an essential component of bacterial lipopolysaccharides, where it provides the linkage between lipid and carbohydrate moieties. In all known LPS structures, Kdo residues possess α-anomeric configurations, and the corresponding inverting α-Kdo transferases are well characterized. Recently, it has been shown that a large group of capsular polysaccharides from Gram-negative bacteria, produced by ATP-binding cassette transporter-dependent pathways, are also attached to a lipid anchor through a conserved Kdo oligosaccharide. In the study reported here, the structure of this Kdo linker was determined by NMR spectroscopy, revealing alternating ß-(2â4)- and ß-(2â7)-linked Kdo residues. KpsC contains two retaining ß-Kdo glycosyltransferase domains belonging to family GT99 that are responsible for polymerizing the ß-Kdo linker on its glycolipid acceptor. Full-length Escherichia coli KpsC was expressed and purified, together with the isolated N-terminal domain and a mutant protein (KpsC D160A) containing a catalytically inactivated N-terminal domain. The Kdo transferase activities of these proteins were determined in vitro using synthetic acceptors, and the reaction products were characterized using TLC, mass spectrometry, and NMR spectroscopy. The N- and C-terminal domains were found to catalyze formation of ß-(2â4) and ß-(2â7) linkages, respectively. Based on phylogenetic analyses, we propose the linkage specificities of the glycosyltransferase domains are conserved in KpsC homologs from other bacterial species.
Assuntos
Cápsulas Bacterianas , Escherichia coli , Filogenia , Açúcares Ácidos , Transferases , Cápsulas Bacterianas/química , Cápsulas Bacterianas/genética , Cápsulas Bacterianas/metabolismo , Catálise , Escherichia coli/enzimologia , Escherichia coli/genética , Lipopolissacarídeos/biossíntese , Lipopolissacarídeos/química , Lipopolissacarídeos/genética , Ressonância Magnética Nuclear Biomolecular , Oligossacarídeos/química , Oligossacarídeos/genética , Oligossacarídeos/metabolismo , Domínios Proteicos , Açúcares Ácidos/química , Açúcares Ácidos/metabolismo , Transferases/química , Transferases/genética , Transferases/metabolismoRESUMO
Glycosaminoglycans (GAGs) are abundant, ubiquitous carbohydrates in biology, yet their structural complexity has limited an understanding of their biological roles and structure-function relationships. Synthetic access to large collections of well defined, structurally diverse GAG oligosaccharides would provide critical insights into this important class of biomolecules and represent a major advance in glycoscience. Here we report a new platform for synthesizing large heparan sulfate (HS) oligosaccharide libraries displaying comprehensive arrays of sulfation patterns. Library synthesis is made possible by improving the overall synthetic efficiency through universal building blocks derived from natural heparin and a traceless fluorous tagging method for rapid purification with minimal manual manipulation. Using this approach, we generated a complete library of 64 HS oligosaccharides displaying all possible 2-O-, 6-O- and N-sulfation sequences in the tetrasaccharide GlcN-IdoA-GlcN-IdoA. These diverse structures provide an unprecedented view into the sulfation code of GAGs and identify sequences for modulating the activities of important growth factors and chemokines.
Assuntos
Glicosaminoglicanos , Heparitina Sulfato , Glicosaminoglicanos/química , Heparitina Sulfato/química , Heparitina Sulfato/metabolismo , Oligossacarídeos/químicaRESUMO
Galectins represent ß-galactoside-binding proteins and are known to bind Galß1-3/4GlcNAc disaccharides (abbreviated as LN1 and LN2, respectively). Despite high sequence and structural homology shared by the carbohydrate recognition domain (CRD) of all galectin members, how each galectin displays different sugar-binding specificity still remains ambiguous. Herein we provided the first structural evidence of human galectins-1, 3-CRD and 7 in complex with LN1. Galectins-1 and 3 were shown to have higher affinity for LN2 than for LN1, while galectin-7 displayed the reversed specificity. In comparison with the previous LN2-complexed structures, the results indicated that the average glycosidic torsion angle of galectin-bound LN1 (ψ(LN1) ≈ 135°) was significantly differed from that of galectin-bound LN2 (ψ(LN2 )≈ -108°), i.e. the GlcNAc moiety adopted a different orientation to maintain essential interactions. Furthermore, we also identified an Arg-Asp/Glu-Glu-Arg salt-bridge network and the corresponding loop (to position the second Asp/Glu residue) critical for the LN1/2-binding preference.