Chemoenzymatic Preparation of a Campylobacter jejuni Lipid-Linked Heptasaccharide on an Azide-Linked Polyisoprenoid.

Complex poly- and oligosaccharides on the surface of bacteria provide a unique fingerprint to different strains of pathogenic and symbiotic microbes that could be exploited for therapeutics or sensors selective for specific glycans. To discover reagents that can selectively interact with specific bacterial glycans, a system for both the chemoenzymatic preparation and immobilization of these materials would be ideal. Bacterial glycans are typically synthesized in nature on the C55 polyisoprenoid bactoprenyl (or undecaprenyl) phosphate. However, this long-chain isoprenoid can be difficult to work with in vitro. Here, we describe the addition of a chemically functional benzylazide tag to polyisoprenoids. We have found that both the organic-soluble and water-soluble benzylazide isoprenoid can serve as a substrate for the well-characterized system responsible for Campylobacter jejuni N-linked heptasaccharide assembly. Using the organic-soluble analogue, we demonstrate the use of an N-acetyl-glucosamine epimerase that can be used to lower the cost of glycan assembly, and using the water-soluble analogue, we demonstrate the immobilization of the C. jejuni heptasaccharide on magnetic beads. These conjugated beads are then shown to interact with soybean agglutinin, a lectin known to interact with N-acetyl-galactosamine in the C. jejuni heptasaccharide. The methods provided could be used for a wide variety of applications including the discovery of new glycan-interacting partners.

Identification of the Functional Roles of Six Key Proteins in the Biosynthesis of Enterobacteriaceae Colanic Acid.

When subjected to harsh conditions such as low pH, pathogenic Escherichia coli can secrete colanic acid to establish a protective barrier between the organism and the acidic environment. The colanic acid consists of a six-sugar repeating unit polymer comprised of glucose, fucose, galactose, and glucuronic acid. The region of the E. coli genome that encodes colanic acid biosynthesis has been reported, and the first enzyme in the biosynthesis pathway has been biochemically characterized. However, the specific roles of the remaining genes required for colanic acid biosynthesis have not been identified. Here we report the in vitro reconstitution of the next six steps in the assembly of the colanic acid repeating unit. To do this, we have cloned and overexpressed each gene within the colanic acid biosynthesis operon. We then tested the activity of the protein product of these genes using high-performance liquid chromatography analysis and a fluorescent analogue of the isoprenoid anchor bactoprenyl diphospho-glucose as a starting substrate. To ensure that retention time changes were associated with varying sugar additions or modifications, we developed a liquid chromatography-mass spectrometry method for analysis of the products produced by each enzyme. We have identified the function of all but one encoded glycosyltransferase and have identified the function of two acetyltransferases. This work demonstrates the centrality of acetylation in the biosynthesis of colanic acid and provides insight into the activity of key proteins involved in the production of an important and highly conserved bacterial glycopolymer.

Complete Tetrasaccharide Repeat Unit Biosynthesis of the Immunomodulatory Bacteroides fragilis Capsular Polysaccharide A.

Capsular polysaccharide A (CPSA) is a four-sugar repeating unit polymer found on the surface of the gut symbiont Bacteroides fragilis that has therapeutic potential in animal models of autoimmune disorders. This therapeutic potential has been credited to its zwitterionic character derived from a positively charged N-acetyl-4-aminogalactosamine (AADGal) and a negatively charged 4,6-O-pyruvylated galactose (PyrGal). In this report, using a fluorescent polyisoprenoid chemical probe, the complete enzymatic assembly of the CPSA tetrasaccharide repeat unit is achieved. The proposed pyruvyltransferase, WcfO; galactopyranose mutase, WcfM; and glycosyltransferases, WcfP and WcfN, encoded by the CPSA biosynthesis gene cluster were heterologously expressed and functionally characterized. Pyruvate modification, catalyzed by WcfO, was found to occur on galactose of the polyisoprenoid-linked disaccharide (AADGal-Gal), and did not occur on galactose linked to uridine diphosphate (UDP) or a set of nitrophenyl-galactose analogues. This pyruvate modification was also found to be required for the incorporation of the next sugar in the pathway N-acetylgalactosamine (GalNAc) by the glycosyltransferase WcfP. The pyruvate acetal modification of a galactose has not been previously explored in the context of a polysaccharide biosynthesis pathway, and this work demonstrates the importance of this modification to repeat unit assembly. Upon production of the polyisoprenoid-linked AADGal-PyrGal-GalNAc, the proteins WcfM and WcfN were found to work in concert to form the final tetrasaccharide, where WcfM formed UDP-galactofuranose (Galf) and WcfN transfers Galf to the AADGal-PyrGal-GalNAc. This work demonstrates the first enzymatic assembly of the tetrasaccharide repeat unit of CPSA in a sequential single pot reaction.

Tuning the production of variable length, fluorescent polyisoprenoids using surfactant-controlled enzymatic synthesis.

Bactoprenyl diphosphate (BPP), a two-E eight-Z configuration C55 isoprenoid, serves as a critical anchor for the biosynthesis of complex glycans central to bacterial survival and pathogenesis. BPP is formed by the polymerase undecaprenyl pyrophosphate synthase (UppS), which catalyzes the elongation of a single farnesyl diphosphate (FPP) with eight Z-configuration isoprene units from eight isopentenyl diphosphates. In vitro analysis of UppS and other polyprenyl diphosphate synthases requires the addition of a surfactant such as Triton X-100 to stimulate the release of the hydrophobic product from the enzyme for effective and efficient turnover. Here using a fluorescent 2-nitrileanilinogeranyl diphosphate analogue of FPP, we have found that a wide range of surfactants can stimulate release of product from UppS and that the structure of the surfactant has a major impact on the lengths of products produced by the protein. Of particular importance, shorter chain surfactants promote the release of isoprenoids with four to six Z-configuration isoprene additions, while larger chain surfactants promote the formation of natural isoprenoid lengths (8Z) and larger. We have found that the product chain lengths can be readily controlled and coarsely tuned by adjusting surfactant identity, concentration, and reaction time. We have also found that binary mixtures of just two surfactants can be used to fine-tune isoprenoid lengths. The surfactant effects discovered do not appear to be significantly altered with an alternative isoprenoid substrate. However, the surfactant effects do appear to be dependent on differences in UppS between bacterial species. This work provides new insights into surfactant effects in enzymology and highlights how these effects can be leveraged for the chemoenzymatic synthesis of otherwise difficult to obtain glycan biosynthesis probes. This work also provides key reagents for the systematic analysis of structure-activity relationships between glycan biosynthesis enzymes and isoprenoid structure.

Functional identification of a galactosyltransferase critical to Bacteroides fragilis Capsular Polysaccharide A biosynthesis.

Capsular Polysaccharide A (CPSA), a polymer of a four-sugar repeating unit that coats the surface of the mammalian symbiont Bacteroides fragilis, has therapeutic potential in animal models of Multiple Sclerosis and other autoinflammatory diseases. Genetic studies have demonstrated that CPSA biosynthesis is dependent primarily on a single gene cluster within the B. fragilis genome. However, the precise functions of the individual glycosyltransferases encoded by this cluster have not been identified. In this report each of these glycosyltransferases (WcfQ, WcfP, and WcfN) have been expressed and tested for their function in vitro. Using a reverse phase high performance liquid chromatography (HPLC) assay, WcfQ and WcfP were found to transfer galactose from uridine diphosphate (UDP)-linked galactose (Gal) to N-acetyl-4-amino-6-deoxy-galactosamine (AADGal) linked to a fluorescent mimic of bactoprenyl diphosphate, the native isoprenoid anchor for bacterial polysaccharide biosynthesis. The incorporation of galactose to form a bactoprenyl-linked disaccharide was confirmed by radiolabel incorporation and mass spectrometry (MS) of purified product. Using varying concentrations of UDP-Gal and enzyme, WcfQ was found to be the most effective protein at transferring galactose, and is the most likely candidate for in vivo incorporation of the sugar. WcfQ also cooperated in the presence of three preceding biosynthetic enzymes to form an isoprenoid-linked disaccharide in a single-pot reaction. This work represents a critical step in understanding the biosynthetic pathway responsible for the formation of CPSA, an unusual and potentially therapeutic biopolymer.

Fluorescent probes for investigation of isoprenoid configuration and size discrimination by bactoprenol-utilizing enzymes.

Undecaprenyl Pyrophosphate Synthase (UPPS) is an enzyme critical to the production of complex polysaccharides in bacteria, as it produces the crucial bactoprenol scaffold on which these materials are assembled. Methods to characterize the systems associated with polysaccharide production are non-trivial, in part due to the lack of chemical tools to investigate their assembly. In this report, we develop a new fluorescent tool using UPPS to incorporate a powerful fluorescent anthranilamide moiety into bactoprenol. The activity of this analogue in polysaccharide biosynthesis is then tested with the initiating hexose-1-phosphate transferases involved in Capsular Polysaccharide A biosynthesis in the symbiont Bacteroides fragilis and the asparagine-linked glycosylation system of the pathogenic Campylobacter jejuni. In addition, it is shown that the UPPS used to make this probe is not specific for E-configured isoprenoid substrates and that elongation by UPPS is required for activity with the downstream enzymes.