Elsevier

Methods in Enzymology

Volume 478, 2010, Pages 389-411
Methods in Enzymology

Chapter Nineteen - Methods to Study the Biosynthesis of Bacterial Furanosides

https://doi.org/10.1016/S0076-6879(10)78019-8Get rights and content

Abstract

Carbohydrates in the thermodynamically disfavored furanose ring conformation are not present in mammalian glycoconjugates, but are widespread in the glycans produced by many bacterial pathogens. In bacteria, these furanose sugars are often found in cell surface glycoconjugates, and are essential for the viability or virulence of the organisms. As a result, the enzymes involved in the biosynthesis of bacterial furanosides are attractive targets as potential selective antimicrobial chemotherapeutics. However, before such chemotherapeutics can be designed, synthesized, and evaluated, more information about the activity and specificity of these enzymes is required. This chapter describes assays that have been used to study enzymes involved in the biosynthesis of one of the most abundant naturally occurring furanose residues, galactofuranose (Galf). In particular, the focus is on UDP-galactopyranose mutase and galactofuranosyltransferases. The assays described in this chapter require UDP-galactofuranose (UDP-Galf); therefore, a procedure for the preparation of UDP-Galf, as well as various UDP-Galf derivatives, using a three-enzyme chemoenzymatic procedure, is also described.

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.

References (70)

  • R. Lee et al.

    Enzymatic synthesis of UDP-galactofuranose and an assay for UDP-galactopyranose mutase based on high-performance liquid chromatography

    Anal. Biochem.

    (1996)
  • R.E. Lee et al.

    Inhibition of UDP-Gal mutase and mycobacterial galactan biosynthesis by pyrrolidine analogues of galactofuranose

    Tetrahedron Lett.

    (1997)
  • K. Mikušová et al.

    Biosynthesis of the galactan component of the mycobacterial cell wall

    J. Biol. Chem.

    (2000)
  • P. Peltier et al.

    Recent knowledge and innovations related to hexofuranosides: Structure, synthesis and applications

    Carbohydr. Res.

    (2008)
  • M.B. Poulin et al.

    Characterization of a bifunctional pyranose-furanose mutase from Campylobacter jejuni 11168

    J. Biol. Chem.

    (2010)
  • N.L. Rose et al.

    Development of a coupled spectrophotometric assay for GlfT2, a bifunctional mycobacterial galactofuranosyltransferase

    Carbohydr. Res.

    (2008)
  • M.S. Scherman et al.

    Polyprenylphosphate-pentoses in mycobacteria are synthesized from 5-phosphoribose pyrophosphate

    J. Biol. Chem.

    (1996)
  • P.H. Tam et al.

    Recent advances in mycobacterial cell wall glycan biosynthesis

    Curr. Opin. Chem. Biol.

    (2009)
  • A. Weston et al.

    Biosynthetic origin of mycobacterial cell wall galactofuranosyl residues

    Tuber. Lung Dis.

    (1998)
  • B.A. Wolucka et al.

    The presence of β-D-ribosyl-1-monphosphodecaprenol in mycobacteria

    J. Biol. Chem.

    (1995)
  • M. Belanova et al.

    Galactosyl transferases in mycobacterial cell wall synthesis

    J. Bacteriol.

    (2008)
  • S. Berg et al.

    The glycosyltransferases of Mycobacterium tuberculosis—Roles in the synthesis of arabinogalactan, lipoarabinomannan, and other glycoconjugates

    Glycobiology

    (2007)
  • M. Berst et al.

    Structural investigations on T1 lipopolysaccharides

    Eur. J. Biochem.

    (1969)
  • P.J. Brennan et al.

    The envelope of mycobacteria

    Annu. Rev. Biochem.

    (1995)
  • A. Burton et al.

    Preparation of fluorinated galactosyl nucleoside diphosphates to study the mechanism of the enzyme galactopyranose mutase

    J. Chem. Soc. Perkin Trans.

    (1997)
  • G.C. Completo et al.

    Synthesis of galactofuranose-containing acceptor substrates for mycobacterial galactofuranosyltransferases

    J. Org. Chem.

    (2008)
  • R.M. de Lederkremer et al.

    Synthesis of α-D-galactofuranosyl phosphate

    J. Org. Chem.

    (1994)
  • J.C. Errey et al.

    Flexible enzymatic and chemo-enzymatic approaches to a broad range of uridine-diphospho-sugars

    Chem. Commun.

    (2004)
  • J.C. Errey et al.

    Sugar nucleotide recognition by Klebsiella pneumoniae UDP-D-galactopyranose mutase: Fluorinated substrates, kinetics and equilibria

    Org. Biomol. Chem.

    (2009)
  • S. Guan et al.

    Functional analysis of the galactosyltransferases required for biosynthesis of D-galactan I, a component of the lipopolysaccharide O1 antigen of Klebsiella pneumoniae

    J. Bacteriol.

    (2001)
  • T. Konishi et al.

    A plant mutase that interconverts UDP-arabinofuranose and UDP-arabinopyranose

    Glycobiology

    (2007)
  • M. Li et al.

    Synthesis of glycoconjugates through biosynthesis pathway engineering

    Synthesis of Carbohydrates Through Biotechnology

    (2004)
  • Z.Y. Liu et al.

    Combined biosynthetic pathway for de novo production of UDP-galactose: Catalysis with multiple enzymes immobilized on agarose beads

    Chembiochem

    (2002)
  • T.L. Lowary

    Recent progress towards the identification of inhibitors of mycobacterial cell wall polysaccharide biosynthesis

    Mini Rev. Med. Chem.

    (2003)
  • A.L. Marlow et al.

    Improved chemical synthesis of UDP-galactofuranose

    Org. Lett.

    (2001)
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