Characterization of Ligand Binding to the Bifunctional Key Enzyme in the Sialic Acid Biosynthesis by NMR (2004)

The bifunctional enzyme UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase is the key enzyme for the biosynthesis of sialic acids. As terminal components of glycoconjugates, sialic acids are associated with a variety of pathological processes such as inflammation and cancer. For the first time, this study reveals characteristics of the interaction of the epimerase site of the enzyme with its natural substrate, UDP-N-acetylglucosamine (UDP-GlcNAc) and derivatives thereof at atomic resolution. Saturation transfer difference NMR experiments were crucial in obtaining ligand binding epitopes and to rank ligands according to their binding affinities. Employing a fragment based approach, it was possible to assign the major component of substrate recognition to the UDP moiety. In particular, the binding epitopes of the uridine moieties of UMP, UDP, UDP-GalNAc, and UDP-GlcNAc are rather similar, suggesting that the binding mode of the UDP moiety is the same in all cases. In contrast, the hexopyranose units of UDP-GlcNAc and UDP-GalNAc display small differences reflecting the inability of the enzyme to process UDP-GalNAc. Surprisingly, saturation transfer difference NMR titrations show that UDP has the largest binding affinity to the epimerase site and that at least one phosphate group is required for binding. Consequently, this study provides important new data for rational drug design. The bifunctional enzyme UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase is the key enzyme for the biosynthesis of sialic acids. As terminal components of glycoconjugates, sialic acids are associated with a variety of pathological processes such as inflammation and cancer. For the first time, this study reveals characteristics of the interaction of the epimerase site of the enzyme with its natural substrate, UDP-N-acetylglucosamine (UDP-GlcNAc) and derivatives thereof at atomic resolution. Saturation transfer difference NMR experiments were crucial in obtaining ligand binding epitopes and to rank ligands according to their binding affinities. Employing a fragment based approach, it was possible to assign the major component of substrate recognition to the UDP moiety. In particular, the binding epitopes of the uridine moieties of UMP, UDP, UDP-GalNAc, and UDP-GlcNAc are rather similar, suggesting that the binding mode of the UDP moiety is the same in all cases. In contrast, the hexopyranose units of UDP-GlcNAc and UDP-GalNAc display small differences reflecting the inability of the enzyme to process UDP-GalNAc. Surprisingly, saturation transfer difference NMR titrations show that UDP has the largest binding affinity to the epimerase site and that at least one phosphate group is required for binding. Consequently, this study provides important new data for rational drug design. Sialylation of glycoproteins and glycolipids on eukaryotic cell surfaces plays an important role during development and regeneration and in the pathogenesis of diseases (1Corfield A.P. Schauer R. Occurrence of sialic acids. Springer, Wien, New York1982: 5-50Google Scholar, 2Suzuki Y. Ito T. Suzuki T. Holland Jr., R.E. Chambers T.M. Kiso M. Ishida H. Kawaoka Y. J. Virol. 2000; 74: 11825-11831Crossref PubMed Scopus (397) Google Scholar, 3Varki A. Glycobiology. 1992; 2: 25-40Crossref PubMed Scopus (478) Google Scholar, 43Ofek I. Sharon N. Curr. Top. Microbiol. Immunol. 1990; 151: 91-113PubMed Google Scholar). Terminal sialic acids are involved in a variety of cellular interactions, such as cell-cell adhesion or cell migration (4Varki A. FASEB J. 1997; 11: 248-255Crossref PubMed Scopus (487) Google Scholar, 5Dennis J.W. Laferte S. Cancer Res. 1985; 45: 6034-6040PubMed Google Scholar), including metastasis formation and progression of a variety of tumors (6Bhavanandan V.P. Glycobiology. 1991; 1: 493-503Crossref PubMed Scopus (65) Google Scholar). They are also known to be involved in the formation and masking of recognition determinants (7Schauer R. Kelm S. Reuter G. Roggentin P. Shaw L. Rosenberg A. Biology of the Sialic Acids. Plenum Press, New York1995: 7-67Crossref Google Scholar) and the biological stability of glycoproteins (8Rens-Domiano S. Reisine T. J. Biol. Chem. 1991; 266: 20094-20102Abstract Full Text PDF PubMed Google Scholar). N-Acetylneuraminic acid (Neu5Ac) 1The abbreviations used are: Neu5Ac, N-acetylneuraminic acid; GlcNAc, N-acetylglucosamine; STD, saturation transfer difference; ManNAc, N-acetylmannosamine; GalNAc, N-acetylgalactosamine.1The abbreviations used are: Neu5Ac, N-acetylneuraminic acid; GlcNAc, N-acetylglucosamine; STD, saturation transfer difference; ManNAc, N-acetylmannosamine; GalNAc, N-acetylgalactosamine. is the biosynthetic precursor of virtually all of the naturally occurring sialic acids (9Angata T. Varki A. Chem. Rev. 2002; 102: 439-469Crossref PubMed Scopus (1013) Google Scholar). In mammals, Neu5Ac is synthesized in the cytosol from UDP-N-acetylglucosamine by four consecutive reactions. The first two steps in this biosynthesis are catalyzed by the bifunctional enzyme UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (UDP-GlcNAc 2-epimerase/ManNAc kinase, EC 5.1.3.14 and EC 2.7.1.60, respectively) (10Hinderlich S. Stäsche R. Zeitler R. Reutter W. J. Biol. Chem. 1997; 272: 24313-24318Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar, 11Stäsche R. Hinderlich S. Weise C. Effertz K. Lucka L. Moormann P. Reutter W. J. Biol. Chem. 1997; 272: 24319-24324Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar), which catalyzes the conversion of UDP-GlcNAc to ManNAc and the consecutive phosphorylation at the sixth position. Neu5Ac-9-phosphate is then formed by the addition of phosphoenolpyruvate to ManNAc-6-phosphate catalyzed by the Neu5Ac-9-phosphate synthase (12Watson D.R. Jourdian G.W. Roseman S. J. Biol. Chem. 1966; 241: 5627-5636Abstract Full Text PDF PubMed Google Scholar, 13Chen H. Blume A. Zimmermann-Kordmann M. Reutter W. Hinderlich S. Glycobiology. 2002; 12: 65-71Crossref PubMed Scopus (24) Google Scholar). After release of the phosphate group, Neu5Ac is activated by the CMP-Neu5Ac synthase (14Kean E.L. Roseman S. J. Biol. Chem. 1966; 241: 5643-5650Abstract Full Text PDF PubMed Google Scholar) in the cell nucleus. The bifunctional UDP-GlcNAc 2-epimerase/ManNAc kinase was first purified and cloned from rat liver (10Hinderlich S. Stäsche R. Zeitler R. Reutter W. J. Biol. Chem. 1997; 272: 24313-24318Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar, 11Stäsche R. Hinderlich S. Weise C. Effertz K. Lucka L. Moormann P. Reutter W. J. Biol. Chem. 1997; 272: 24319-24324Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). Later, the murine and human enzymes were also cloned and characterized (15Horstkorte R. Nöhring S. Wiechens N. Schwarzkopf M. Danker K. Reutter W. Lucka L. Eur. J. Biochem. 1999; 260: 923-927Crossref PubMed Scopus (45) Google Scholar, 16Lucka L. Krause M. Danker K. Reutter W. Horstkorte R. FEBS Lett. 1999; 454: 341-344Crossref PubMed Scopus (57) Google Scholar). The enzyme consists of 722 amino acids and has a molecular mass of 79 kDa. Natively, it assembles as a homohexamer, possessing both functionalities. Recently, it has been demonstrated that individuals suffering from hereditary inclusion body myopathy and sialuria carry point mutations in the UDP-GlcNAc 2-epimerase part of the gene (17Eisenberg I. Avidan N. Potikha T. Hochner H. Chen M. Olender T. Barash M. Shemesh M. Sadeh M. Grabov-Nardini G. Shmilevich I. Friedmann A. Karpati G. Bradley W.G. Baumbach L. Lancet D. Asher E.B. Beckmann J.S. Mitrani-Rosenbaum S. Nat. Genet. 2001; 29: 83-87Crossref PubMed Scopus (433) Google Scholar, 18Seppala R. Lehto V-P. Gahl W.A. Am. J. Hum. Genet. 1999; 64: 1563-1569Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). The biological importance of the enzyme is further reflected by a drastic reduction of cellular sialylation upon loss of enzyme activity, for example (19Keppler O.T. Hinderlich S. Langner J. Schwartz-Albiez R. Reutter W. Pawlita M. Science. 1999; 284: 1372-1376Crossref PubMed Scopus (272) Google Scholar), and by the fact that a knockout of the gene in mice is lethal to the embryo at day 8.5 (20Schwarzkopf M. Knobeloch K.P. Rhode E. Hinderlich S. Wiechens N. Lucka L. Horak I. Reutter W. Horstkorte R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5267-5270Crossref PubMed Scopus (279) Google Scholar). Therefore, specific inhibition of UDP-GlcNAc 2-epimerase activity is an important means of regulating cell surface sialylation and therefore in dealing with disease states. Consequently, the characterization of the ligand binding properties of the enzyme at an atomic resolution is key for the design of such inhibitors and also yields insights into the mechanistic aspects of sialic acid biosynthesis. With this aim, the interactions of the enzyme with a number of ligands have been characterized, using saturation transfer difference (STD) NMR spectroscopy (21Mayer M. Meyer B. Angew. Chem. Int. Ed. 1999; 38: 1784-1788Crossref PubMed Scopus (1311) Google Scholar). STD NMR allows one to detect and characterize the binding of ligands to large receptor proteins. The technique discriminates binding from non-binding ligands and furnishes binding epitopes of ligands at an atomic resolution (22Vogtherr M. Peters T. J. Am. Chem. Soc. 2000; 122: 6093-6099Crossref Scopus (95) Google Scholar, 23Mayer M. Meyer B. J. Am. Chem. Soc. 2001; 123: 6108-6117Crossref PubMed Scopus (993) Google Scholar). Recently, it has been shown that STD NMR data may even be used to derive bound conformations of ligands provided that the three-dimensional structure of the receptor protein is known (24Jayalakshmi V. Krishna N.R. J. Magn. Reson. 2002; 155: 106-118Crossref PubMed Scopus (180) Google Scholar, 25Jayalakshmi V. Biet T. Peters T. Krishna N.R. J. Am. Chem. Soc. 2004; 126: 8610-8611Crossref PubMed Scopus (46) Google Scholar). STD NMR has been applied to study rather diverse biological binding reactions (26Peters T. Meyer B. Angew. Chemie. Int. Ed. 2003; 42: 864-890Crossref PubMed Google Scholar). Most noteworthy, there is no size limit for the protein receptor and an assignment of protein resonances is not required. For example, this has rendered possible the investigation of ligands binding to liposome-integrated membrane proteins (27Meinecke R. Meyer B. J. Med. Chem. 2001; 44: 19-3065Crossref Scopus (105) Google Scholar) or to native viruses (28Benie A. Moser R. Bäuml E. Blaas D. Peters T. J. Am. Chem. Soc. 2003; 125: 14-15Crossref PubMed Scopus (83) Google Scholar). Until today, only the monofunctional UDP-GlcNAc 2-epimerase from Escherichia coli, which shows only 22% homology of the amino acid sequence to the mammalian bifunctional enzyme, has been characterized and crystallized (29Campbell R.E. Mosimann S.C. Tanner M.E. Strynadka N.C.J. Biochemistry. 2000; 39: 14993-15001Crossref PubMed Scopus (102) Google Scholar). To date, little is known with regard to the mammalian counterpart of this enzyme. STD NMR has a proven potential to study ligand binding, and here, it is applied to the large and rather fragile bifunctional UDP-GlcNAc 2-epimerase/ManNAc kinase. Materials—All of the chemicals with the exception of 2-acetamidoglucal, which was synthesized according to the method of Pravdic et al. (30Pravdic N. Franjic-Mihalic I. Danilov B. Carbohydr. Res. 1975; 45: 302-306Crossref Scopus (30) Google Scholar), were obtained from Sigma. Expression and Purification of UDP-GlcNAc 2-Epimerase/ManNAc Kinase—Rat UDP-GlcNAc 2-epimerase/ManNAc kinase was recombinantly expressed in Sf-900 insect cells with a His6 tag as previously described (31Blume A. Ghaderi D. Liebich V. Hinderlich S. Donner P. Reutter W. Lucka L. Protein Expression Purif. 2004; 35: 387-396Crossref PubMed Scopus (14) Google Scholar). Cells from a 50-ml culture were harvested, resuspended in 5 ml of 10 mm sodium phosphate, 1 mm EDTA, 1 mm dithiothreitol, pH 7.5, and lysed by passage through a narrow bore needle 20 times. After centrifugation at 20,000 × g at 4 °C for 30 min, UDP-GlcNAc 2-epimerase/ManNAc kinase was purified as described earlier (31Blume A. Ghaderi D. Liebich V. Hinderlich S. Donner P. Reutter W. Lucka L. Protein Expression Purif. 2004; 35: 387-396Crossref PubMed Scopus (14) Google Scholar). The resulting supernatant was applied to a 1-ml nickel-nitrilotriacetic acid-agarose (Qiagen) column, which had been equilibrated with 10 ml of 50 mm sodium phosphate, 300 mm sodium chloride, 20 mm imidazole, pH 8.0. After with the nickel-nitrilotriacetic acid for 1 at the was with ml of 50 mm sodium phosphate, 300 mm sodium chloride, 20 mm imidazole, pH by 4 ml of the same with in for the of The of the protein was with ml of 50 mm sodium phosphate, 300 mm sodium chloride, mm in of were and the of protein was by the Protein were and applied to a For inhibition of the 2-epimerase activity, the enzyme was with A. Chen H. Reutter Hinderlich S. FEBS Lett. 2002; PubMed Scopus Google Scholar) for 1 to the The protein was from the using 5 ml of 50 mm sodium phosphate, mm sodium in of were and protein was by using the activity was by the UDP-GlcNAc 2-epimerase A. Chen H. Reutter Hinderlich S. FEBS Lett. 2002; PubMed Scopus Google Scholar), and was by NMR NMR were obtained at 5 °C using a with a in the 10 and 20 protein and a ligand and 4 For the of STD NMR a sequence a was used (21Mayer M. Meyer B. Angew. Chem. Int. Ed. 1999; 38: 1784-1788Crossref PubMed Scopus (1311) Google Scholar). was at and at was using with a and a and by a of 1 to a saturation of The of the was STD NMR were with a of in addition to to the to to The were with a of and data were using the same with only STD NMR and for the epimerase substrate, were obtained with and of the ligands were the same as the STD NMR through the of and To the size of the STD the were obtained with to the in the Therefore, a STD of is as the in both have the same For group were then further the largest STD and to the with the largest STD to binding were the described The titrations were by the of the STD in the of a ligand at of and 4 with to the first The STD were then the to which of the ligands had the study of ligands binding to UDP-GlcNAc 2-epimerase/ManNAc kinase is based on STD NMR experiments (21Mayer M. Meyer B. Angew. Chem. Int. Ed. 1999; 38: 1784-1788Crossref PubMed Scopus (1311) Google Scholar) to detect binding (22Vogtherr M. Peters T. J. Am. Chem. Soc. 2000; 122: 6093-6099Crossref Scopus (95) Google Scholar) and to characterize binding epitopes at atomic resolution M. Meyer B. J. Am. Chem. Soc. 2001; 123: 6108-6117Crossref PubMed Scopus (993) Google Scholar). The experiments were a of the enzyme on the of to As one the STD NMR technique the of or on the design of the Therefore, the data obtained reflected the enzyme and for NMR the and of is the natural substrate of the The enzyme UDP-GlcNAc to ManNAc and In the of the at this point the kinase activity that ManNAc into ManNAc-6-phosphate in a In the of UDP-GlcNAc 2-epimerase/ManNAc kinase, STD were obtained for that UDP-GlcNAc is bound to the enzyme The STD also of ManNAc and UDP, which were by the the same time, this that the enzyme is the The epimerase was by NMR that the of ManNAc to be the of the The was formed by is in with that have been Hinderlich S. Reutter W. Tanner M.E. J. Am. Chem. Soc. 2003; 125: PubMed Scopus Google Scholar). of with STD the STD shown in STD were data reflected the of saturation from the protein the and the with a STD were to be in with on the protein surface with STD it is that the part of UDP-GlcNAc is the largest saturation transfer and therefore is bound to the To the role of the and the moiety in binding, STD NMR experiments were using a The experiments that UDP, UMP, GlcNAc, and all to the UDP-GlcNAc 2-epimerase/ManNAc kinase as for UDP in The binding epitopes of the moieties in UMP, UDP, and UDP-GalNAc a suggesting that the a binding in all cases. The binding epitopes of and and from with the of to the moiety of is from that the binding epitopes of and to the one of The in with the enzyme were and of GlcNAc, the of the group and the largest saturation transfer in the of and the small differences and UDP-GlcNAc may be to binding to the or of the a binding a from the binding mode of and or binding to a binding has been shown that be by the of the bifunctional enzyme, the ManNAc kinase, to A. Blume A. Reutter W. Hinderlich S. Peters T. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). Therefore, it is that to the ManNAc kinase of the UDP-GlcNAc To this the enzyme was with the A. Chen H. Reutter Hinderlich S. FEBS Lett. 2002; PubMed Scopus Google Scholar). STD NMR experiments with the ligands no STD with the exception of the of an STD was Therefore, to the ManNAc kinase site of the bifunctional enzyme. was also obtained for ManNAc A. Blume A. Reutter W. Hinderlich S. Peters T. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). STD NMR further that it is the part that the binding of ligands to the epimerase site of UDP-GlcNAc 2-epimerase/ManNAc kinase, STD titrations were as has been described previously M. Meyer B. J. Am. Chem. Soc. 2001; 123: 6108-6117Crossref PubMed Scopus (993) Google Scholar, A. Moser R. Bäuml E. Blaas D. Peters T. J. Am. Chem. Soc. 2003; 125: 14-15Crossref PubMed Scopus (83) Google Scholar). The experiments a of the binding of the ligands as shown in UDP had the affinity of the ligands the substrate UDP-GalNAc that was not by the enzyme had the same affinity as it has been that the of UDP-GlcNAc as an PubMed Scopus Google Scholar), also this ligand to STD The STD were that had a large or a small binding In both the of STD NMR were and were (26Peters T. Meyer B. Angew. Chemie. Int. Ed. 2003; 42: 864-890Crossref PubMed Google Scholar). To the two titrations with UDP-GalNAc were as a ligand with a rather binding have STD NMR experiments to detect and characterize the binding of ligands to receptor proteins (26Peters T. Meyer B. Angew. Chemie. Int. Ed. 2003; 42: 864-890Crossref PubMed Google Scholar). the of this to the key enzyme for the biosynthesis of sialic acids in this enzyme, UDP-GlcNAc 2-epimerase/ManNAc kinase, is rather large and data are and little is known the atomic of ligand binding to the of the enzyme, the epimerase and the kinase Employing a approach, were to the binding of ligands to the epimerase site at atomic resolution. of the binding of the natural substrate, bound to the enzyme that the moiety is the binding In particular, the of the uridine and of the moiety the largest of saturation is to the binding of bound to UDP-GlcNAc 2-epimerase/ManNAc kinase to the binding of bound to T. Peters T. Angew. Chem. Int. Ed. 2001; PubMed Scopus Google Scholar) the UDP moiety was also to the largest saturation transfer and therefore the ligand in the binding In the it was possible to a of STD (24Jayalakshmi V. Krishna N.R. J. Magn. Reson. 2002; 155: 106-118Crossref PubMed Scopus (180) Google Scholar) and the of bound to at atomic resolution in V. Biet T. Peters T. Krishna N.R. J. Am. Chem. Soc. 2004; 126: 8610-8611Crossref PubMed Scopus (46) Google Scholar). also be to ligands binding to UDP-GlcNAc 2-epimerase/ManNAc kinase data on the protein are The binding of UDP-GlcNAc to UDP-GlcNAc 2-epimerase and of to are rather For a counterpart of UDP-GlcNAc 2-epimerase/ManNAc kinase, a monofunctional UDP-GlcNAc the structure at a has been (29Campbell R.E. Mosimann S.C. Tanner M.E. Strynadka N.C.J. Biochemistry. 2000; 39: 14993-15001Crossref PubMed Scopus (102) Google Scholar). In this a interaction was the and interaction of with was in the structure of bound to B. J. Biol. 2002; PubMed Scopus Google Scholar). that such a interaction with an amino acid is also for the ligand into the epimerase site of UDP-GlcNAc 2-epimerase/ManNAc kinase. key role of the moiety during the binding process has been by inhibition experiments with UDP-GlcNAc 2-epimerase/ManNAc kinase no difference in enzyme inhibition was the inhibitors and A. Chen H. Reutter Hinderlich S. FEBS Lett. 2002; PubMed Scopus Google Scholar). that the moiety is not a in binding or the The that the binding epitopes of the hexopyranose moieties of UDP-GlcNAc and UDP-GalNAc are and that binding is also for UDP and further to the that UDP the ligand into the epimerase in with this is the that all of the ligands with for binding, that to the same the epimerase titrations with UDP, and UDP to have the that the moiety in UDP-GlcNAc even binding by on the binding of the protein or by the of a as with the the of UDP-GlcNAc is the of UDP E. Eur. J. Biochem. PubMed Scopus Google Scholar). this it is the enzyme is to a by The experiments further show that the affinity of a ligand to the enzyme on the of phosphate the binding affinity of is with one phosphate is to into the epimerase to the kinase site A. Blume A. Reutter W. Hinderlich S. Peters T. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar) and has virtually no binding affinity for the epimerase as was from STD experiments with UDP-GlcNAc experiments with have shown that this UDP-GlcNAc 2-epimerase UDP A. Chen H. Reutter Hinderlich S. FEBS Lett. 2002; PubMed Scopus Google Scholar). Therefore, in with the here, it is to that two or even as provided by the moiety are for binding to the epimerase in enzyme reactions the is to to date, the has been in in the of the sialuria G. L. J. PubMed Scopus Google Scholar). The UDP-GlcNAc 2-epimerase of ManNAc 2-acetamidoglucal, and that the enzyme a of the enzyme (29Campbell R.E. Mosimann S.C. Tanner M.E. Strynadka N.C.J. Biochemistry. 2000; 39: 14993-15001Crossref PubMed Scopus (102) Google Scholar). is required to the the site of the enzyme to the of the to the addition of UDP, and a to is that the mammalian UDP-GlcNAc 2-epimerase a with the exception of the addition of of UDP in the Consequently, the affinity of the enzyme the is it is formed in a of the enzyme. In with this is the that only of the is to ManNAc at °C Hinderlich S. Reutter W. Tanner M.E. J. Am. Chem. Soc. 2003; 125: PubMed Scopus Google Scholar), the natural substrate UDP-GlcNAc is at In no conversion of be at 5 STD for as In this it is to that a protein from E. has been as an UDP-GlcNAc 2-epimerase Tanner M.E. J. J. 2004; PubMed Scopus Google Scholar). As the mammalian this enzyme also UDP-GlcNAc the mechanistic the a of the enzyme also UDP with a affinity the natural substrate In for the to the enzyme for a specific The binding of the UDP moiety is to and the of the enzyme, the to has been that the enzyme UDP to ManNAc PubMed Scopus Google Scholar), further the that the binding of UDP the The that UDP-GlcNAc and UDP-GalNAc are bound with and display binding epitopes that both ligands are bound to the same of the enzyme, the The inability of the enzyme to UDP-GalNAc may be to the fact that UDP-GalNAc has an group to of the and is in to according to the Hinderlich S. Reutter W. Tanner M.E. J. Am. Chem. Soc. 2003; 125: PubMed Scopus Google Scholar), is by a of the enzyme to the In the of UDP-GalNAc, the of this and therefore the formation of a is also possible that a is formed that further to it is that for the design of and inhibitors into the importance of the UDP moiety. In the of new it is not that derivatives of UDP M. Blume A. Reutter W. J. Chem. 2004; PubMed Scopus Google Scholar, Blume A. Hinderlich S. Reutter W. Eur. J. Chem. 2004; Scopus Google Scholar) have been to the enzyme. to for a is the of two or a for potential inhibitors be as in also that and have to and process activated and therefore the that enzymes may have a as has been previously (29Campbell R.E. Mosimann S.C. Tanner M.E. Strynadka N.C.J. Biochemistry. 2000; 39: 14993-15001Crossref PubMed Scopus (102) Google Scholar). study provides new insights into the epimerase activity of UDP-GlcNAc 2-epimerase/ManNAc kinase, the key enzyme of mammalian sialic acid biosynthesis. For the first time, the binding of ligands including the natural substrate UDP-GlcNAc has been described at atomic the for the rational design of inhibitors of the epimerase activity of UDP-GlcNAc 2-epimerase/ManNAc kinase that have a potential in the of or for for insect cell