Journal of Molecular Biology
Volume 403, Issue 4, 5 November 2010, Pages 578-590
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Chemoenzymatic Synthesis, Inhibition Studies, and X-ray Crystallographic Analysis of the Phosphono Analog of UDP-Galp as an Inhibitor and Mechanistic Probe for UDP-Galactopyranose Mutase

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Abstract

UDP (uridine diphosphate) galactopyranose mutase (UGM) is involved in the cell wall biosynthesis of many pathogenic microorganisms. UGM catalyzes the reversible conversion of UDP-α-d-galactopyranose into UDP-α-d-galactofuranose, with the latter being the precursor of galactofuranose (Galf) residues in cell walls. Glycoconjugates of Galf are essential components in the cell wall of various pathogenic bacteria, including Mycobacterium tuberculosis, the causative agent of tuberculosis. The absence of Galf in humans and its bacterial requirement make UGM a potential target for developing novel antibacterial agents. In this article, we report the synthesis, inhibitory activity, and X-ray crystallographic studies of UDP-phosphono-galactopyranose, a nonhydrolyzable C-glycosidic phosphonate. This is the first report on the synthesis of a phosphonate analog of UDP-α-d-galactopyranose by a chemoenzymatic phosphoryl coupling method. The phosphonate was evaluated against three bacterial UGMs and showed only moderate inhibition. We determined the crystal structure of the phosphonate analog bound to Deinococcus radiodurans UGM at 2.6 Å resolution. The phosphonate analog is bound in a novel conformation not observed in UGM–substrate complex structures or in other enzyme–sugar nucleotide phosphonate complexes. This complex structure provides a structural basis for the observed micromolar inhibition towards UGM. Steric clashes, loss of electrostatic stabilization between an active-site arginine (Arg305) and the phosphonate analog, and a 180° flip of the hexose moiety account for the differences in the binding orientations of the isosteric phosphonate analog and the physiological substrate. This provides new insight into the ability of a sugar-nucleotide-binding enzyme to orient a substrate analog in an unexpected geometry and should be taken into consideration in designing such enzyme inhibitors.

Introduction

Galactose in its furanoside configuration [galactofuranose (Galf)] is present in the glycoconjugates of many species of pathogenic microorganisms, including Mycobacterium,1 Leishmania,2, 3 Trypanosoma,3 and Aspergillus.4 The presence of Galf plays a critical role in the pathogenicity of many microorganisms and, combined with its absence in humans, makes Galf biosynthesis an appealing target for drug discovery.5, 6 UDP (uridine diphosphate) galactopyranose mutase (UGM) is responsible for the reversible isomerization of UDP-α-d-galactopyranose (UDP-Galp) into UDP-α-d-galactofuranose (UDP-Galf; Fig. 1). UDP-Galf is the universal donor substrate for Galf transferases, the enzymes responsible for assembling Galf-containing glycoconjugates.7 Although the mechanism and structure of UGM from different microorganisms have been studied extensively, there is still no unequivocal consensus regarding the catalytic mechanism.8, 9, 10, 11, 12 Positional isotope-exchange experiments revealed that cleavage of the anomeric C–O bond takes place during pyranose–furanose isomerization.13 According to all presently available biochemical and kinetic data, the reaction mechanism of UGM is consistent with the departure of UDP, the formation of an enzyme–galactosyl adduct followed by the ring isomerization of sugar, the reattachment of UDP, and the concomitant release of the product.8, 11 The reduced form of flavin adenine dinucleotide (FAD) coenzyme (FADH) is necessary for this reaction to proceed.9 Recently, we and others reported the crystal structures of UGM in complex with UDP-Galp in oxidized and reduced forms, and these structures are consistent with previous biochemical and mechanistic studies.14, 15 The UGM:UDP-Galp substrate complex structures provide insight into the binding epitope at the active site and reveal the conformational changes that occur upon substrate binding.16, 17, 18 Based on this structural information and other mechanistic studies, we envisioned that a nonhydrolyzable substrate analog could act as a potent/effective inhibitor of UGM.

The phosphonate analog of UDP-Galp [UDP-phosphono-galactopyranose (UDP-CH2-Galp)] (Fig. 1) would be ideal for use in probing the enzymatic mechanism, since the C–C pseudo glycosidic bond cannot be cleaved during the reaction, as suggested by positional isotope-exchange experiments with UDP-Galp as substrate.13 Also, a phosphonate analog would maintain the majority of the interactions through a UDP moiety similar to UDP-Galp, and the sugar moiety would be able to form water-mediated hydrogen bonds in the sugar binding region.14, 19 In addition, phosphonates are well-established isosteric probes of phosphoryl transfer processes in biological systems,19, 20, 21, 22 particularly with respect to the development of antiviral agents23, 24 and maintenance of bone density.24, 25 They have been used as donor substrate analogs for glycosyltransferase studies to probe the enzyme active-site architecture.25 In consideration of these previous studies, access to nonscissile isosteric structural analogs of UDP-Galp may provide new insight into the mechanism of UGM. Chemical synthesis of UDP-CH2-Galp has previously been described.26 However, inhibition studies for evaluating inhibition, namely high (1 mM) substrate concentrations, were carried out under suboptimal conditions.

In this report, we describe in detail a novel chemoenzymatic synthesis of UDP-CH2-Galp, inhibition towards three bacterial UGMs, and the first crystal structure of a UGM:UDP-CH2-Galp complex. The structure offers a clear insight into why UDP-CH2-Galp is only a moderate inhibitor of UGM.

Section snippets

Enzymatic synthesis of UDP-CH2-Galp

Our earlier studies aimed at the enzymatic preparation of UDP-CH2-Galp had been unsuccessful due to a lack of turnover of the phosphono analog of galactose 1-phosphate by several recombinant bacterial enzymes in our laboratory.27, 28 We next explored the substrate specificity of a recombinant Arabidopsis thaliana UDP-sugar pyrophosphorylase (AtUSP) with an apparently broad substrate specificity toward monosaccharide 1-phosphates29 to determine whether it would accept UTP and the phosphonate

General procedures and instrumentations

High-quality deionized water (> 17 MΩ) prepared with Nanopure II Sybron/Barnstead was used in all purification steps. Ion-exchange resins were prewashed several times with deionized water before use. Evaporation was achieved using a Büchi rotary evaporator, followed by drying at < 1 mmHg using an Edward rotary vacuum pump. A Heto PowerDry LL300 Freeze dryer was used for lyophilization of samples. Except where specified, all reagents were purchased from commercial sources and used without further

Acknowledgements

This research was supported by the Mizutani Foundation of Glycoscience of Japan, the Natural Sciences and Engineering Research Council of Canada, and the Canadian Institutes of Health Research.

References (50)

  • de LederkremerR. et al.

    Galactofuranose-containing glycoconjugates in trypanosomatids

    Glycobiology

    (1995)
  • KremerL. et al.

    Current status and future development of antitubercular chemotherapy

    Expert Opin. Invest. Drugs

    (2002)
  • RichardsM.R. et al.

    Chemistry and biology of galactofuranose-containing polysaccharides

    ChemBioChem

    (2009)
  • RoseN. et al.

    Expression, purification, and characterization of a galactofuranosyltransferase involved in Mycobacterium tuberculosis arabinogalactan biosynthesis

    J. Am. Chem. Soc.

    (2006)
  • Soltero-HigginM. et al.

    A unique catalytic mechanism for UDP-galactopyranose mutase

    Nat. Struct. Mol. Biol.

    (2004)
  • SandersD. et al.

    UDP-galactopyranose mutase has a novel structure and mechanism

    Nat. Struct. Biol.

    (2001)
  • CaravanoA. et al.

    Efficient synthesis of a nucleoside-diphospho-exo-glycal displaying time-dependent inactivation of UDP-galactopyranose mutase

    Chem. Commun. (Cambridge)

    (2004)
  • ItohK. et al.

    Synthesis and analysis of substrate analogues for UDP-galactopyranose mutase: implication for an oxocarbenium ion intermediate in the catalytic mechanism

    Org. Lett.

    (2007)
  • FullertonS. et al.

    Potentiometric analysis of UDP-galactopyranose mutase: stabilization of the flavosemiquinone by substrate

    Biochemistry

    (2003)
  • BarlowJ. et al.

    Positional isotope exchange catalyzed by UDP-galactopyranose mutase

    J. Am. Chem. Soc.

    (1999)
  • ParthaS. et al.

    Structural basis of substrate binding to UDP-galactopyranose mutase: crystal structures in the reduced and oxidized state complexed with UDP-galactopyranose and UDP

    J. Mol. Biol.

    (2009)
  • GruberT. et al.

    X-ray crystallography reveals a reduced substrate complex of UDP-galactopyranose mutase poised for covalent catalysis by flavin

    Biochemistry

    (2009)
  • ChadJ. et al.

    Site-directed mutagenesis of UDP-galactopyranose mutase reveals a critical role for the active-site, conserved arginine residues

    Biochemistry

    (2007)
  • YuanY. et al.

    Investigation of binding of UDP-Galf and UDP-[3-F]Galf to UDP-galactopyranose mutase by STD-NMR spectroscopy, molecular dynamics, and CORCEMA-ST calculations

    J. Am. Chem. Soc.

    (2008)
  • YaoX. et al.

    Substrate directs enzyme dynamics by bridging distal sites: UDP-galactopyranose mutase

    Proteins

    (2009)
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