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Molecular structures of the S124A, S124T, and S124V site-directed mutants of UDP-galactose 4-epimerase from Escherichia coli.

Thoden JB, Gulick AM, Holden HM
Biochemistry 36 10685-95 (1997)
Related entries: 1kvq, 1kvr, 1kvt

Cited: 26 times
EuropePMC logo PMID: 9271499

Abstract

UDP-galactose 4-epimerase plays a critical role in sugar metabolism by catalyzing the interconversion of UDP-galactose and UDP-glucose. Originally, it was assumed that the enzyme contained a "traditional" catalytic base that served to abstract a proton from the 4'-hydroxyl group of the UDP-glucose or UDP-galactose substrates during the course of the reaction. However, recent high-resolution X-ray crystallographic analyses of the protein from Escherichia coli have demonstrated the lack of an aspartate, a glutamate, or a histidine residue properly oriented within the active site cleft for serving such a functional role. Rather, the X-ray crystallographic investigation of the epimerase.NADH.UDP-glucose abortive complex from this laboratory has shown that both Ser 124 and Tyr 149 are located within hydrogen bonding distance to the 4'- and 3'-hydroxyl groups of the sugar, respectively. To test the structural role of Ser 124 in the reaction mechanism of epimerase, three site-directed mutant proteins, namely S124A, S124T, and S124V, were constructed and crystals of the S124A.NADH.UDP, S124A.NADH.UDP-glucose, S124T. NADH.UDP-glucose, and S124V.NADH.UDP-glucose complexes were grown. All of the crystals employed in this investigation belonged to the space group P3221 with the following unit cell dimensions: a = b = 83.8 A, c = 108.4 A, and one subunit per asymmetric unit. X-ray data sets were collected to at least 2.15 A resolution, and each protein model was subsequently refined to an R value of lower than 19.0% for all measured X-ray data. The investigations described here demonstrate that the decreases in enzymatic activities observed for these mutant proteins are due to the loss of a properly positioned hydroxyl group at position 124 and not to major tertiary and quaternary structural perturbations. In addition, these structures demonstrate the importance of a hydroxyl group at position 124 in stabilizing the anti conformation of the nicotinamide ring as observed in the previous structural analysis of the epimerase.NADH. UDP complex.

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  1. Medium- and short-chain dehydrogenase/reductase gene and protein families : the SDR superfamily: functional and structural diversity within a family of metabolic and regulatory enzymes. Kavanagh KL, Jörnvall H, Persson B, Oppermann U. Cell Mol Life Sci 65 3895-3906 (2008)
  2. Structure and function of enzymes of the Leloir pathway for galactose metabolism. Holden HM, Rayment I, Thoden JB. J Biol Chem 278 43885-43888 (2003)
  3. The rhamnose pathway. Giraud MF, Naismith JH. Curr Opin Struct Biol 10 687-696 (2000)

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  1. Human UDP-galactose 4-epimerase. Accommodation of UDP-N-acetylglucosamine within the active site. Thoden JB, Wohlers TM, Fridovich-Keil JL, Holden HM. J Biol Chem 276 15131-15136 (2001)
  2. The crystal structure of dTDP-D-Glucose 4,6-dehydratase (RmlB) from Salmonella enterica serovar Typhimurium, the second enzyme in the dTDP-l-rhamnose pathway. Allard ST, Giraud MF, Whitfield C, Graninger M, Messner P, Naismith JH. J Mol Biol 307 283-295 (2001)
  3. Toward a structural understanding of the dehydratase mechanism. Allard ST, Beis K, Giraud MF, Hegeman AD, Gross JW, Wilmouth RC, Whitfield C, Graninger M, Messner P, Allen AG, Maskell DJ, Naismith JH. Structure 10 81-92 (2002)
  4. Structure and mechanism of ArnA: conformational change implies ordered dehydrogenase mechanism in key enzyme for polymyxin resistance. Gatzeva-Topalova PZ, May AP, Sousa MC. Structure 13 929-942 (2005)
  5. Widespread head-to-head hydrocarbon biosynthesis in bacteria and role of OleA. Sukovich DJ, Seffernick JL, Richman JE, Gralnick JA, Wackett LP. Appl Environ Microbiol 76 3850-3862 (2010)
  6. Variation on a theme of SDR. dTDP-6-deoxy-L- lyxo-4-hexulose reductase (RmlD) shows a new Mg2+-dependent dimerization mode. Blankenfeldt W, Kerr ID, Giraud MF, McMiken HJ, Leonard G, Whitfield C, Messner P, Graninger M, Naismith JH. Structure 10 773-786 (2002)
  7. Identification and characterization of a mutation, in the human UDP-galactose-4-epimerase gene, associated with generalized epimerase-deficiency galactosemia. Wohlers TM, Christacos NC, Harreman MT, Fridovich-Keil JL. Am J Hum Genet 64 462-470 (1999)
  8. Determinants of function and substrate specificity in human UDP-galactose 4'-epimerase. Schulz JM, Watson AL, Sanders R, Ross KL, Thoden JB, Holden HM, Fridovich-Keil JL. J Biol Chem 279 32796-32803 (2004)
  9. Crystal structure of isoflavone reductase from alfalfa (Medicago sativa L.). Wang X, He X, Lin J, Shao H, Chang Z, Dixon RA. J Mol Biol 358 1341-1352 (2006)
  10. Crystal structure of SQD1, an enzyme involved in the biosynthesis of the plant sulfolipid headgroup donor UDP-sulfoquinovose. Mulichak AM, Theisen MJ, Essigmann B, Benning C, Garavito RM. Proc Natl Acad Sci U S A 96 13097-13102 (1999)
  11. Crystal structure of a tetrameric GDP-D-mannose 4,6-dehydratase from a bacterial GDP-D-rhamnose biosynthetic pathway. Webb NA, Mulichak AM, Lam JS, Rocchetta HL, Garavito RM. Protein Sci 13 529-539 (2004)
  12. First molecular characterization of a uridine diphosphate galacturonate 4-epimerase: an enzyme required for capsular biosynthesis in Streptococcus pneumoniae type 1. Muñoz R, López R, de Frutos M, García E. Mol Microbiol 31 703-713 (1999)
  13. GDP-4-keto-6-deoxy-D-mannose epimerase/reductase from Escherichia coli, a key enzyme in the biosynthesis of GDP-L-fucose, displays the structural characteristics of the RED protein homology superfamily. Rizzi M, Tonetti M, Vigevani P, Sturla L, Bisso A, Flora AD, Bordo D, Bolognesi M. Structure 6 1453-1465 (1998)
  14. Crystal structure of UDP-galactose 4-epimerase from the hyperthermophilic archaeon Pyrobaculum calidifontis. Sakuraba H, Kawai T, Yoneda K, Ohshima T. Arch Biochem Biophys 512 126-134 (2011)
  15. The Hypocrea jecorina gal10 (uridine 5'-diphosphate-glucose 4-epimerase-encoding) gene differs from yeast homologues in structure, genomic organization and expression. Seiboth B, Karaffa L, Sándor E, Kubicek C. Gene 295 143-149 (2002)
  16. Prediction of the active-site structure and NAD(+) binding in SQD1, a protein essential for sulfolipid biosynthesis in Arabidopsis. Essigmann B, Hespenheide BM, Kuhn LA, Benning C. Arch Biochem Biophys 369 30-41 (1999)
  17. Towards a better understanding of the substrate specificity of the UDP-N-acetylglucosamine C4 epimerase WbpP. Demendi M, Ishiyama N, Lam JS, Berghuis AM, Creuzenet C. Biochem J 389 173-180 (2005)
  18. Functional characterization and transcriptional analysis of galE gene encoding a UDP-galactose 4-epimerase in Xanthomonas campestris pv. campestris. Li CT, Liao CT, Du SC, Hsiao YP, Lo HH, Hsiao YM. Microbiol Res 169 441-452 (2014)
  19. Insights into role of the hydrogen bond networks in substrate recognition by UDP-GalNAc 4-epimerases. Bhatt VS, Guan W, Xue M, Yuan H, Wang PG. Biochem Biophys Res Commun 412 232-237 (2011)
  20. Structural basis for broad substrate specificity of UDP-glucose 4-epimerase in the human milk oligosaccharide catabolic pathway of Bifidobacterium longum. Nam YW, Nishimoto M, Arakawa T, Kitaoka M, Fushinobu S. Sci Rep 9 11081 (2019)
  21. Elucidation of substrate specificity in Aspergillus nidulans UDP-galactose-4-epimerase. Dalrymple SA, Ko J, Sheoran I, Kaminskyj SG, Sanders DA. PLoS One 8 e76803 (2013)
  22. UDP-Glucose 4-Epimerase and β-1,4-Galactosyltransferase from the Oyster Magallana gigas as Valuable Biocatalysts for the Production of Galactosylated Products. Song HB, He M, Cai ZP, Huang K, Flitsch SL, Liu L, Voglmeir J. Int J Mol Sci 19 E1600 (2018)


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