Journal of Molecular Biology
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
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.
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S.K.P. and A.S.-K. contributed equally to this work.