Chapter Nineteen - Methods to Study the Biosynthesis of Bacterial Furanosides
Introduction
In addition to thermodynamically favored pyranose sugars, bacteria also produce polysaccharides and glycoconjugates containing saccharides in the five-membered furanose ring conformation. These thermodynamically disfavored furanose sugars are absent in mammalian glycoconjugates, but are widespread in other domains of life ranging from bacteria and archaebacteria, to protozoa, fungi, and plants (Peltier et al., 2008a, Richards & Lowary, 2009). In many pathogenic bacteria, these furanose sugars are found in key cell surface glycoconjugates, including the lipopolysaccharide (LPS) O antigens of Escherichia coli (Stevenson et al., 1994), Klebsiella pneumoniae (Köplin et al., 1997), and Samonella typhimurium (Berst et al., 1969), the capsular polysaccharide (CPS) of Campylobacter jejuni (Hanniffy et al., 1999, McNally et al., 2005, St. Michael et al., 2002), and the mycolyl arabinogalactan (mAG) complex and lipoarabinomannan (LAM) of mycobacteria (Bhamidi et al., 2008, Brennan & Nikaido, 1995), among others. In many of these organisms, the furanose residues in these glycans have been demonstrated to be essential for cell viability, or play a critical role in cell physiology (Lee et al., 1997, Pan et al., 2001). Because these furanosides are absent from mammalian glycoconjugates, there has been a surge of interest in developing inhibitors of furanose biosynthesis as potential selective chemotherapeutics to treat these pathogenic microorganisms (Lowary, 2003, Pedersen & Turco, 2003, Umesiri et al., 2010).
The hexofuranose d-galactofuranose (Galf), the pentofuranose d-arabinofuranose (Araf), and the hexulose d-fructofuranose (Fruf), represent three of the most common furanose sugars found in bacterial glycans, and each of these three furanose sugars employ a different type of activated furanoside donor in their biosynthesis. Microbial fructans are polymers of 20–10,000 Fruf units containing repeating β-(2→1) (inulins), β-(2→6) (levans), or a combination of β-(2→1) and β–(2→6) (mixed levans) glycosidic linkages, which are attached to the Fruf residue of d-sucrose (Velazquez-Hernandez et al., 2009). The fructosyltransferases (FruT) involved in the biosynthesis of microbial fructans are members of the glycoside hydrolase family 68, and catalyze the transglycosylation of Fruf using d-sucrose as the furanose donor (van Hijum et al., 2006) as shown (Fig. 19.1). Fruf residues have also been identified in other microbial glycoconjugates, such as the CPS of C. jejuni HS:1 (McNally et al., 2005); however, it is unclear whether these Fucf residues are also incorporated from d-sucrose.
The only known pathway for the biosynthesis of Araf residues present in the mAG complex and LAM of mycobacterial species utilize the lipid donor decaprenyl-phospho-arabinose (DPA) as the sole Araf source (Wolucka, 2008, Wolucka & Dehoffmann, 1995). A water soluble uridine diphosphate (UDP) derivative of Araf has also been reported in Mycobacterium smegmatis (Singh and Hogan, 1994); however, there have been no subsequent reports to demonstrate a possible biosynthetic role for UDP-Araf. Isotope labeling experiments where M. smegmatis cells were incubated with 14C- and 13C-labeled glucose elegantly show that neither the pentose phosphate pathway, which would result in the loss of C1 of glucose, or the uronic acid pathway, which would result in the loss of C6 of glucose, are involved in the biosynthesis of Araf (Klutts et al., 2002). Instead, a nonoxidative pentose phosphate pathway is utilized. Other isotope labeling experiments with 14C-labeled 5-phosphoribose-1-pyrophosphate (5-P-Rib-1-PP) implicate this species as an intermediate in the biosynthesis of DPA as well as decaprenyl-phospho-ribose (DPR) (Scherman et al., 1996). The subsequent identification of the mycobacterial DPR 5′-phosphate synthetase (Huang et al., 2005), and decaprenyl-phospho-ribose 2′-epimerase (DPRE) (Mikušová et al., 2005) led to the proposed pathway (Fig. 19.2) for the biosynthesis of Araf in mycobacteria. Six arabinofuranosyltransferase (Aft) enzymes involved in the biosynthesis of mycobacterial cell wall arabinan have been identified (Berg et al., 2007, Tam & Lowary, 2009); however, the exact role of many of these proteins in mAG and LAM assembly remain to be determined, and it is likely that other arabinofuranosyltransferases exist. It should be noted that while (myco)bacterial species use lipid linked donors to incorporate d-arabinofuranose residues into their glycoconjugates, plants, which produce large amounts of l-arabinofuranose-containing glycans, do so via enzymes that recognize the sugar nucleotide UDP-l-Araf as the donor species (Konishi et al., 2007, Reiter & Vanzin, 2001).
Galf residues are incorporated into bacterial glycans using nucleotide-activated sugar donors (Fig. 19.3). Early studies established that the Galf in S. typhimurium is synthesized from a derivative of galactopyranose and that the ring contraction does not occur at the level of either free galactose or galactose-1-phosphate (Nikaido & Sarvas, 1971, Sarvas & Nikaido, 1971). The enzyme uridine 5′-diphospho-galactopyranose mutase (UGM), which was first identified in E. coli (Nassau et al., 1996) and subsequently in K. pneumonia (Köplin et al., 1997), Mycobacterium tuberculosis (Weston et al., 1998), and Deinococcus radiodurans (Partha et al., 2009b), was found to carry out the ring contraction of uridine 5′-diphospho-d-galactopyranose (UDP-Galp) to uridine 5′-diphospho-d-galactofuranose (UDP-Galf), the biosynthetic precursor of bacterial Galf residues. The UDP-Galp originates either from glucopyranose-1-phosphate by de novo biosynthesis, or from free galactose via the galactose salvage pathway (Thibodeaux et al., 2008). Enzymes known as galactofuranosyltransferases (GlfTs) catalyze the final coupling of UDP-Galf to the appropriate Galf-containing glycans.
In this chapter, we describe assays used to study the enzymes involved in the biosynthesis of Galf-containing bacterial glycoconjugates; specifically, the UGM and GlfT proteins. We also provide a procedure for the chemoenzymatic preparation of UDP-Galf (and derivatives of UDP-Galf) that takes advantage of the reduced substrate specificity of enzymes from the galactose salvage pathway.
Section snippets
Discussion
The assays described in this chapter are used to study the biosynthesis of Galf-containing glycoconjugates; as a result, these assays require the use of the nucleotide-activated galactofuranose donor, UDP-Galf. Multiple chemical syntheses of this activated Galf donor have been reported using standard pyrophosphate bond formation reactions between galactofuranose-1-phosphate (Galf-1-P) and UMP-morpholidate (Zhang and Liu, 2000), UMP-N-methylimidazolide (Marlow and Kiessling, 2001), or N,N
Discussion
Pyranose–furanose mutase enzymes are flavoproteins that catalyze the ring contraction involved in the biosynthesis of furanose sugar nucleotides from the corresponding pyranose sugar nucleotides (Scheme 19.2) using a unique catalytic mechanism (Sanders et al., 2001, Soltero-Higgin et al., 2004a). The pyranose–furanose interconversion favors the pyranose ring conformation in a ratio of approximately 9:1 (Richards and Lowary, 2009); as a result, assays are typically run in the reverse direction
Discussion
GlfTs are the final enzymes involved in the biosynthesis of Galf-containing glycoconjugates. These enzymes catalyze glycosidic bond formation between the activated UDP-Galf donor and an acceptor species, typically a glycan. Two bifunctional GlfTs, encoded by the Rv3808c and Rv3782 genes, from M. tuberculosis are reported to be required for the biosynthesis of the galactan portion of the mycobacterial mAG complex (Belanova et al., 2008, Mikušová et al., 2000, Mikušová et al., 2006). The first,
Acknowledgments
This work was supported by the Alberta Ingenuity Centre for Carbohydrate Science and the Natural Sciences and Engineering Research Council of Canada. M.B.P. is supported by a Ph.D. studentship from Alberta Innovates–Technology Futures.
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