Michele R. Richards

Chemistry and biology of galactofuranose-containing polysaccharides.

The thermodynamically less stable form of galactose—galactofuranose (Galf)—is essential for the viability of several pathogenic species of bacteria and protozoa but absent in this form in mammals, so the biochemical pathways by which Galf‐containing glycans are assembled and catabolysed are attractive sites for drug action. This potential has led to increasing interest in the synthesis of molecules containing Galf residues, their subsequent use in studies directed towards understanding the enzymes that process these residues and the identification of potential inhibitors of these pathways. Major achievements of the past several years have included an in‐depth understanding of the mechanism of UDP‐galactopyranose mutase (UGM), the enzyme that produces UDP‐Galf, which is the donor species for galactofuranosyltransferases. A number of methods for the synthesis of galactofuranosides have also been developed, and practitioners in the field now have many options for the initiation of a synthesis of glycoconjugates containing either α‐ or β‐Galf residues. UDP‐Galf has also been prepared by a number of approaches, and it appears that a chemoenzymatic approach is currently the most viable method for producing multi‐milligram amounts of this important intermediate. Recent advances both in the understanding of the mechanism of UGM and in the synthesis of galactofuranose and its derivatives are highlighted in this review.

Conserved glycolipid termini in capsular polysaccharides synthesized by ATP-binding cassette transporter-dependent pathways in Gram-negative pathogens

Bacterial capsules are surface layers made of long-chain polysaccharides. They are anchored to the outer membrane of many Gram-negative bacteria, including pathogens such as Escherichia coli, Neisseria meningitidis, Haemophilus influenzae, and Pasteurella multocida. Capsules protect pathogens from host defenses including complement-mediated killing and phagocytosis and therefore represent a major virulence factor. Capsular polysaccharides are synthesized by enzymes located in the inner (cytoplasmic) membrane and are then translocated to the cell surface. Whereas the enzymes that synthesize the polysaccharides have been studied in detail, the structure and biosynthesis of the anchoring elements have not been definitively resolved. Here we determine the structure of the glycolipid attached to the reducing terminus of the polysialic acid capsular polysaccharides from E. coli K1 and N. meningitidis group B and the heparosan-like capsular polysaccharide from E. coli K5. All possess the same unique glycolipid terminus consisting of a lyso-phosphatidylglycerol moiety with a β-linked poly-(3-deoxy-d-manno-oct-2-ulosonic acid) (poly-Kdo) linker attached to the reducing terminus of the capsular polysaccharide.

Biological roles of the O-methyl phosphoramidate capsule modification in Campylobacter jejuni.

Campylobacter jejuni is a major cause of bacterial gastroenteritis worldwide, and the capsular polysaccharide (CPS) of this organism is required for persistence and disease. C. jejuni produces over 47 different capsular structures, including a unique O-methyl phosphoramidate (MeOPN) modification present on most C. jejuni isolates. Although the MeOPN structure is rare in nature it has structural similarity to some synthetic pesticides. In this study, we have demonstrated, by whole genome comparisons and high resolution magic angle spinning NMR, that MeOPN modifications are common to several Campylobacter species. Using MeOPN biosynthesis and transferase mutants generated in C. jejuni strain 81–176, we observed that loss of MeOPN from the cell surface correlated with increased invasion of Caco-2 epithelial cells and reduced resistance to killing by human serum. In C. jejuni, the observed serum mediated killing was determined to result primarily from activation of the classical complement pathway. The C. jejuni MeOPN transferase mutant showed similar levels of colonization relative to the wild-type in chickens, but showed a five-fold drop in colonization when co-infected with the wild-type in piglets. In Galleria mellonella waxmoth larvae, the MeOPN transferase mutant was able to kill the insects at wild-type levels. Furthermore, injection of the larvae with MeOPN-linked monosaccharides or CPS purified from the wild-type strain did not result in larval killing, indicating that MeOPN does not have inherent insecticidal activity.

Tetrameric Structure of the GlfT2 Galactofuranosyltransferase Reveals a Scaffold for the Assembly of Mycobacterial Arabinogalactan

Background: GlfT2 is a bifunctional galactofuranosyltransferase essential for mycobacterial cell wall biosynthesis. Results: Crystal structures of free and UDP-bound GlfT2, along with mutagenesis and kinetic studies, reveal novel details underlying substrate binding and catalysis. Conclusion: The homotetrameric architecture, distinctive nucleotide-binding site, and unprecedented structural features underlying the bifunctional polymerase activity of GlfT2 are revealed. Significance: Novel insights into polymerizing glycosyltransferases and the design of anti-mycobacterial therapeutics are obtained. Biosynthesis of the mycobacterial cell wall relies on the activities of many enzymes, including several glycosyltransferases (GTs). The polymerizing galactofuranosyltransferase GlfT2 (Rv3808c) synthesizes the bulk of the galactan portion of the mycolyl-arabinogalactan complex, which is the largest component of the mycobacterial cell wall. We used x-ray crystallography to determine the 2.45-Å resolution crystal structure of GlfT2, revealing an unprecedented multidomain structure in which an N-terminal β-barrel domain and two primarily α-helical C-terminal domains flank a central GT-A domain. The kidney-shaped protomers assemble into a C4-symmetric homotetramer with an open central core and a surface containing exposed hydrophobic and positively charged residues likely involved with membrane binding. The structure of a 3.1-Å resolution complex of GlfT2 with UDP reveals a distinctive mode of nucleotide recognition. In addition, models for the binding of UDP-galactofuranose and acceptor substrates in combination with site-directed mutagenesis and kinetic studies suggest a mechanism that explains the unique ability of GlfT2 to generate alternating β-(1→5) and β-(1→6) glycosidic linkages using a single active site. The topology imposed by docking a tetrameric assembly onto a membrane bilayer also provides novel insights into aspects of processivity and chain length regulation in this and possibly other polymerizing GTs.

In Vitro Reconstruction of the Chain Termination Reaction in Biosynthesis of the Escherichia coli O9a O-Polysaccharide THE CHAIN-LENGTH REGULATOR, WbdD, CATALYZES THE ADDITION OF METHYL PHOSPHATE TO THE NON-REDUCING TERMINUS OF THE GROWING GLYCAN

Background: WbdD is a chain-length regulator that modifies the non-reducing terminus of the Escherichia coli O9a glycan. Results: WbdD phosphorylates and methylates a synthetic O9a repeating unit acceptor. Conclusion: The acceptor is modified with a terminal methyl phosphate. Significance: Determining the terminal structure is crucial to understanding the quality control processes of chain-length regulation and export of this prototypical glycan. The Escherichia coli O9a O-polysaccharide (O-PS) represents a model system for glycan biosynthesis and export by the ATP-binding cassette (ABC) transporter-dependent pathway. The polymannose O9a O-PS is synthesized using an undecaprenol-diphosphate-linked acceptor by mannosyltransferases located at the cytoplasmic membrane. An ABC-transporter subsequently exports the polymer to the periplasm where it is assembled onto lipopolysaccharide prior to translocation to the cell surface. The chain length of the O9a O-PS is regulated by the dual kinase/methyltransferase activity of the WbdD enzyme and modification of the polymer is crucial for binding and export by the ABC-transporter. Previous biochemical data provided evidence for phosphorylation/methylation at the non-reducing end of the O9a O-PS but the structure of the terminus has not been determined. Here, we describe the exploitation of a synthetic O9a O-PS repeating unit carrying a fluorescent tag as an acceptor for in vitro phosphorylation and methylation by a purified soluble form of WbdD. Phosphorylation of the acceptor was evident by both a mobility shift in thin layer chromatography and radiolabeling of the acceptor using [γ-33P]ATP. Methylation of the acceptor was dependent on phosphorylation and was demonstrated by radiolabeling using S-[methyl-3H]adenosyl-methionine as a substrate, in the presence of ATP. NMR spectroscopic and mass spectrometric methods were used to determine the precise structure of the terminal modification, leading to the conclusion that WbdD catalyzes the addition of a novel methyl phosphate group to the 3-position of the non-reducing terminal mannose of the O9a O-PS repeating unit.

Biosynthesis of the Polymannose Lipopolysaccharide O-antigens from Escherichia coli Serotypes O8 and O9a Requires a Unique Combination of Single- and Multiple-active Site Mannosyltransferases

Background: The Escherichia coli O8 and O9a antigens are influential models for bacterial glycan assembly. Results: O8 and O9a glycan biosynthesis requires three mannosyltransferases, whose activities were defined. Conclusion: Two conserved mannosyltransferases (WbdCB) produce an adaptor trisaccharide on which each WbdA polymerizes a serotype-specific repeat unit polysaccharide. Significance: These systems require multiple mannosyltransferase modules in a flexible arrangement. The Escherichia coli O9a and O8 O-antigen serotypes represent model systems for the ABC transporter-dependent synthesis of bacterial polysaccharides. The O9a and O8 antigens are linear mannose homopolymers containing conserved reducing termini (the primer-adaptor), a serotype-specific repeat unit domain, and a terminator. Synthesis of these glycans occurs on the polyisoprenoid lipid-linked primer, undecaprenol pyrophosphoryl-GlcpNAc, by two conserved mannosyltransferases, WbdC and WbdB, and a serotype-specific mannosyltransferase, WbdA. The glycan structure and pattern of conservation in the O9a and O8 mannosyltransferases are not consistent with the existing model of O9a biosynthesis. Here we establish a revised pathway using a combination of in vivo (mutant complementation) experiments and in vitro strategies with purified enzymes and synthetic acceptors. WbdC and WbdB synthesize the adaptor region, where they transfer one and two α-(1→3)-linked mannose residues, respectively. The WbdA enzymes are solely responsible for forming the repeat unit domains of these O-antigens. WbdAO9a has two predicted active sites and polymerizes a tetrasaccharide repeat unit containing two α-(1→3)- and two α-(1→2)-linked mannopyranose residues. In contrast, WbdAO8 polymerizes trisaccharide repeat units containing single α-(1→3)-, α-(1→2)-, and β-(1→2)-mannopyranoses. These studies illustrate assembly systems exploiting several mannosyltransferases with flexible active sites, arranged in single- and multiple-domain formats.

Kinetic Stability of the Streptavidin–Biotin Interaction Enhanced in the Gas Phase

Results of the first detailed study of the structure and kinetic stability of the model high-affinity protein-ligand interaction between biotin (B) and the homotetrameric protein complex streptavidin (S(4)) in the gas phase are described. Collision cross sections (Ω) measured for protonated gaseous ions of free and ligand-bound truncated (residues 13-139) wild-type (WT) streptavidin, i.e., S(4)(n+) and (S(4)+4B)(n+) at charge states n = 12-16, were found to be independent of charge state and in agreement (within 10%) with values estimated for crystal structures reported for S(4) and (S(4)+4B). These results suggest that significant structural changes do not occur upon transfer of the complexes from solution to the gas phase by electrospray ionization. Temperature-dependent rate constants were measured for the loss of B from the protonated (S(4)+4B)(n+) ions. Over the temperature range investigated, the kinetic stability increases with decreasing charge state, from n = 16 to 13, but is indistinguishable for n = 12 and 13. A comparison of the activation energies (E(a)) measured for the loss of B from the (S(4)+4B)(13+) ions composed of WT streptavidin and five binding site mutants (Trp79Phe, Trp108Phe, Trp120Phe, Ser27Ala, and Tyr43Ala) suggests that at least some of the specific intermolecular interactions are preserved in the gas phase. The results of molecular dynamics simulations performed on WT (S(4)+4B)(12+) ions with different charge configurations support this conclusion. The most significant finding of this study is that the gaseous WT (S(4)+4B)(n+) ions at n = 12-14, owing to a much larger E(a) (by as much as 13 kcal mol(-1)) for the loss of B, are dramatically more stable kinetically at 25 °C than the (S(4)+4B) complex in aqueous neutral solution. The differences in E(a) values measured for the gaseous (S(4)+4B)(n+) ions and solvated (S(4)+4B) complex can be largely accounted for by a late dissociative transition state and the rehydration of B and the protein binding cavity in solution.

Domain Organization of the Polymerizing Mannosyltransferases Involved in Synthesis of the Escherichia coli O8 and O9a Lipopolysaccharide O-antigens

Background: Escherichia coli O8 and O9a polysaccharide repeat units are synthesized by serotype-specific multidomain (WbdA) mannosyltransferases. Results: The various WbdA domains are functional when expressed individually. Conclusion: The number of domains identified in each WbdA protein correlates with the different linkage types formed by the enzyme. Significance: Modular glycosyltransferases could be exploited for synthesis of precise glycoconjugates with medical and industrial applications. The Escherichia coli O9a and O8 polymannose O-polysaccharides (O-PSs) serve as model systems for the biosynthesis of bacterial polysaccharides by ATP-binding cassette transporter-dependent pathways. Both O-PSs contain a conserved primer-adaptor domain at the reducing terminus and a serotype-specific repeat unit domain. The repeat unit domain is polymerized by the serotype-specific WbdA mannosyltransferase. In serotype O9a, WbdA is a bifunctional α-(1→2)-, α-(1→3)-mannosyltransferase, and its counterpart in serotype O8 is trifunctional (α-(1→2), α-(1→3), and β-(1→2)). Little is known about the detailed structures or mechanisms of action of the WbdA polymerases, and here we establish that they are multidomain enzymes. WbdAO9a contains two separable and functionally active domains, whereas WbdAO8 possesses three. In WbdCO9a and WbdBO9a, substitution of the first Glu of the EX7E motif had detrimental effects on the enzyme activity, whereas substitution of the second had no significant effect on activity in vivo. Mutation of the Glu residues in the EX7E motif of the N-terminal WbdAO9a domain resulted in WbdA variants unable to synthesize O-PS. In contrast, mutation of the Glu residues in the motif of the C-terminal WbdAO9a domain generated an enzyme capable of synthesizing an altered O-PS repeat unit consisting of only α-(1→2) linkages. In vitro assays with synthetic acceptors unequivocally confirmed that the N-terminal domain of WbdAO9a possesses α-(1→2)-mannosyltransferase activity. Together, these studies form a framework for detailed structure-function studies on individual domains and a strategy applicable for dissection and analysis of other multidomain glycosyltransferases.

Comparison between DFT- and NMR-based conformational analysis of methyl galactofuranosides

Galactofuranose (Galf) residues are found in a number of microbial polysaccharides, and knowledge of their conformation is key for developing a molecular-level understanding of their biological roles. To this end, we studied 180 conformations of methyl α- and β-Galf in aqueous solution (COSMO solvation model) using density functional theory (DFT). We compare the calculated low energy conformations to those determined from the program PSEUROT using (1)H NMR data. The lowest energy ring conformation for methyl α-Galf is (2)E, and this conformer is also the major solution conformation obtained by NMR spectroscopy. For methyl β-Galf, (4)E is the lowest energy ring conformation; however, DFT results do not agree with the solution NMR spectroscopic results. Additionally, we developed Galf-specific Karplus-like equations from these conformations.

Conformational Analysis of Oligoarabinofuranosides: Overcoming Torsional Barriers with Umbrella Sampling.

In this report, the conformations of a series of mono- and oligoarabinofuranosides were probed through the use of umbrella sampling simulations with the AMBER force field and the GLYCAM carbohydrate parameter set. The rotamer population distribution about the exocyclic C4-C5 bonds and the puckering distributions of the rings obtained from these umbrella sampling simulations were found to be in excellent agreement with those obtained from conventional long MD simulations for small monosaccharide fragments. For larger systems, the conventional MD approach becomes impractical, and we propose the use of umbrella sampling to circumvent poor sampling of certain conformations. The same umbrella sampling simulations were used to calculate the distributions about the vicinal protons and ensemble-averaged vicinal proton-proton coupling constants ((3)JH,H). The distributions about the vicinal protons of a monomer, methyl-α-l-arabinofuranoside (1), were found to be very similar to those obtained from direct umbrella sampling simulations about the vicinal protons. We calculated (3)JH,H based on DFT-based Karplus-like relationships for l-arabinofuranosides. The (3)JH,H values were found to be very similar to those obtained with the conventional MD simulations. For 1, the (3)JH,H values obtained with the DFT-based Karplus equations agree very well with experimental results; the agreement is, however, not as good for the larger oligomers. An approach to determine the experimental rotamer populations from the simulations is also discussed.