Crystallographic snapshots of UDP-glucuronic acid 4-epimerase ligand binding, rotation, and reduction
2020; Elsevier BV; Volume: 295; Issue: 35 Linguagem: Inglês
10.1074/jbc.ra120.014692
ISSN1083-351X
AutoresL.G. Iacovino, Simone Savino, Annika J. E. Borg, Claudia Binda, Bernd Nidetzky, Andrea Mattevi,
Tópico(s)Biochemical and Molecular Research
ResumoUDP-glucuronic acid is converted to UDP-galacturonic acid en route to a variety of sugar-containing metabolites. This reaction is performed by a NAD+-dependent epimerase belonging to the short-chain dehydrogenase/reductase family. We present several high-resolution crystal structures of the UDP-glucuronic acid epimerase from Bacillus cereus. The geometry of the substrate-NAD+ interactions is finely arranged to promote hydride transfer. The exquisite complementarity between glucuronic acid and its binding site is highlighted by the observation that the unligated cavity is occupied by a cluster of ordered waters whose positions overlap the polar groups of the sugar substrate. Co-crystallization experiments led to a structure where substrate- and product-bound enzymes coexist within the same crystal. This equilibrium structure reveals the basis for a “swing and flip” rotation of the pro-chiral 4-keto-hexose-uronic acid intermediate that results from glucuronic acid oxidation, placing the C4′ atom in position for receiving a hydride ion on the opposite side of the sugar ring. The product-bound active site is almost identical to that of the substrate-bound structure and satisfies all hydrogen-bonding requirements of the ligand. The structure of the apoenzyme together with the kinetic isotope effect and mutagenesis experiments further outlines a few flexible loops that exist in discrete conformations, imparting structural malleability required for ligand rotation while avoiding leakage of the catalytic intermediate and/or side reactions. These data highlight the double nature of the enzymatic mechanism: the active site features a high degree of precision in substrate recognition combined with the flexibility required for intermediate rotation. UDP-glucuronic acid is converted to UDP-galacturonic acid en route to a variety of sugar-containing metabolites. This reaction is performed by a NAD+-dependent epimerase belonging to the short-chain dehydrogenase/reductase family. We present several high-resolution crystal structures of the UDP-glucuronic acid epimerase from Bacillus cereus. The geometry of the substrate-NAD+ interactions is finely arranged to promote hydride transfer. The exquisite complementarity between glucuronic acid and its binding site is highlighted by the observation that the unligated cavity is occupied by a cluster of ordered waters whose positions overlap the polar groups of the sugar substrate. Co-crystallization experiments led to a structure where substrate- and product-bound enzymes coexist within the same crystal. This equilibrium structure reveals the basis for a “swing and flip” rotation of the pro-chiral 4-keto-hexose-uronic acid intermediate that results from glucuronic acid oxidation, placing the C4′ atom in position for receiving a hydride ion on the opposite side of the sugar ring. The product-bound active site is almost identical to that of the substrate-bound structure and satisfies all hydrogen-bonding requirements of the ligand. The structure of the apoenzyme together with the kinetic isotope effect and mutagenesis experiments further outlines a few flexible loops that exist in discrete conformations, imparting structural malleability required for ligand rotation while avoiding leakage of the catalytic intermediate and/or side reactions. These data highlight the double nature of the enzymatic mechanism: the active site features a high degree of precision in substrate recognition combined with the flexibility required for intermediate rotation. Nucleotide sugars are high-energy donor molecules that fulfill many roles, including contributing to cellular and tissue structural integrity (1Bethke G. Thao A. Xiong G. Li B. Soltis N.E. Hatsugai N. Hillmer R.A. Katagiri F. Kliebenstein D.J. Pauly M. Glazebrook J. Pectin biosynthesis is critical for cell wall integrity and immunity in Arabidopsis thaliana.Plant Cell. 2016; 28 (26813622): 537-55610.1105/tpc.15.00404Crossref PubMed Scopus (68) Google Scholar, 2Reboul R. Geserick C. Pabst M. Frey B. Wittmann D. Lütz-Meindl U. Leonard R. Tenhaken R. Down-regulation of UDP-glucuronic acid biosynthesis leads to swollen plant cell walls and severe developmental defects associated with changes in pectic polysaccharides.J. Biol. Chem. 2011; 286 (21949134): 39982-3999210.1074/jbc.M111.255695Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 3Reiter W.D. Biochemical genetics of nucleotide sugar interconversion reactions.Curr. Opin. Plant Biol. 2008; 11 (18486535): 236-24310.1016/j.pbi.2008.03.009Crossref PubMed Scopus (95) Google Scholar), acting as molecular recognition markers for xenobiotic detoxification (4Bowles D. Lim E.K. Poppenberger B. Vaistij F.E. Glycosyltransferases of lipophilic small molecules.Annu. Rev. Plant Biol. 2006; 57 (16669774): 567-59710.1146/annurev.arplant.57.032905.105429Crossref PubMed Scopus (316) Google Scholar, 5Radominska-Pandya A. Little J.M. Czernik P.J. Human UDP-glucuronosyltransferase 2B7.Current drug metabolism. 2001; 2: 283-29810.2174/1389200013338379Crossref PubMed Scopus (53) Google Scholar, 6Ritter J.K. Roles of glucuronidation and UDP-glucuronosyltransferases in xenobiotic bioactivation reactions.Chem. Biol. Interact. 2000; 129 (11154740): 171-19310.1016/S0009-2797(00)00198-8Crossref PubMed Scopus (301) Google Scholar), and providing scaffolds for drug development (7Creuzenet C. Belanger M. Wakarchuk W.W. Lam J.S. Expression, purification, and biochemical characterization of WbpP, a new UDP-GlcNAc C4 epimerase from Pseudomonas aeruginosa serotype O6.J. Biol. Chem. 2000; 275 (10747995): 19060-1906710.1074/jbc.M001171200Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Among these metabolites, UDP-glucuronic acid (UDP-GlcA) represents a very versatile compound that undergoes a variety of enzymatic modifications. UDP-glucuronic acid 4-epimerase (EC 5.1.3.6), UDP-xylose synthase (EC 4.1.1.35), and UDP-apiose/xylose synthase (EC 4.1.1.-) modify UDP-GlcA through NAD+-dependent redox chemistry, producing enzyme-specific sugar products (8Kelleher W.J. Grisebach H. Hydride transfer in the biosynthesis of uridine diphospho-apiose from uridine diphospho-d-glucuronic acid with an enzyme preparation of Lemna minor.Eur. J. Biochem. 1971; 23 (5127378): 136-14210.1111/j.1432-1033.1971.tb01600.xCrossref PubMed Scopus (16) Google Scholar, 9Moriarity J.L. Hurt K.J. Resnick A.C. Storm P.B. Laroy W. Schnaar R.L. Snyder S.H. UDP-glucuronate decarboxylase, a key enzyme in proteoglycan synthesis: cloning, characterization, and localization.J. Biol. Chem. 2002; 277 (11877387): 16968-1697510.1074/jbc.M109316200Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 10Neufeld E.F. Feingold D.S. Hassid W.Z. Enzymatic conversion of uridine diphosphate d-glucuronic acid to uridine diphosphate galacturonic acid, uridine diphosphate xylose, and uridine diphosphate arabinose1,2.J. Am. Chem. Soc. 1958; 80: 4430-443110.1021/ja01549a089Crossref Scopus (17) Google Scholar). These structurally and functionally related enzymes belong to the family of the short-chain dehydrogenase/reductases (SDRs) (11Kavanagh K.L. Jörnvall H. Persson B. Oppermann U. 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.Cell Mol. Life Sci. 2008; 65 (19011750): 3895-390610.1007/s00018-008-8588-yCrossref PubMed Scopus (515) Google Scholar). SDRs are characterized by a strictly conserved catalytic triad, Ser/Thr-Tyr-Lys. The tyrosine residue functions as the base that abstracts a proton from the substrate 4′-OH group and promotes C4′ oxidation by NAD+ (12Allard S.T. Giraud M.F. Naismith J.H. Epimerases: structure, function and mechanism.Cell Mol. Life Sci. 2001; 58 (11706991): 1650-166510.1007/PL00000803Crossref PubMed Scopus (77) Google Scholar, 13Eixelsberger T. Sykora S. Egger S. Brunsteiner M. Kavanagh K.L. Oppermann U. Brecker L. Nidetzky B. Structure and mechanism of human UDP-xylose synthase: evidence for a promoting role of sugar ring distortion in a three-step catalytic conversion of UDP-glucuronic acid.J. Biol. Chem. 2012; 287 (22810237): 31349-3135810.1074/jbc.M112.386706Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 14Gatzeva-Topalova P.Z. May A.P. Sousa M.C. Structure and mechanism of ArnA: conformational change implies ordered dehydrogenase mechanism in key enzyme for polymyxin resistance.Structure. 2005; 13 (15939024): 929-94210.1016/j.str.2005.03.018Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 15Samuel J. Tanner M.E. Mechanistic aspects of enzymatic carbohydrate epimerization.Nat. Prod. Rep. 2002; 19 (12137277): 261-27710.1039/b100492lCrossref PubMed Scopus (52) Google Scholar, 16Savino S. Borg A.J.E. Dennig A. Pfeiffer M. de Giorgi F. Weber H. Dubey K.D. Rovira C. Mattevi A. Nidetzky B. Deciphering the enzymatic mechanism of sugar ring contraction in UDP-apiose biosynthesis.Nat. Catal. 2019; 2 (31844840): 1115-112310.1038/s41929-019-0382-8Crossref PubMed Scopus (6) Google Scholar, 17Thibodeaux C.J. Melançon C.E. Liu H.W. Unusual sugar biosynthesis and natural product glycodiversification.Nature. 2007; 446 (17460661): 1008-101610.1038/nature05814Crossref PubMed Scopus (239) Google Scholar, 18Thoden J.B. Henderson J.M. Fridovich-Keil J.L. Holden H.M. Structural analysis of the Y299C mutant of Escherichia coli UDP-galactose 4-epimerase: teaching an old dog new tricks.J. Biol. Chem. 2002; 277 (12019271): 27528-2753410.1074/jbc.M204413200Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). The generated 4-keto-hexose-uronic acid intermediate is unstable and converted into different products, depending on the enzyme (Scheme 1). Because of their catalytic versatility, sugar-modifying SDR enzymes are increasingly recognized as valuable targets for many industrial biocatalytic applications. As can be seen from Scheme 1, the UDP-glucuronic acid 4-epimerase stands out for its ability to prevent decarboxylation at the C5′ position of the 4-keto-hexose-uronic acid intermediate. This enzyme thereby efficiently interconverts UDP-GlcA into UDP-galacturonic acid (UDP-GalA) by inverting the configuration of the 4′-carbon. The strategies used by epimerases to invert the stereochemistry of UDP-sugar carbons have been subject of in-depth investigations (12Allard S.T. Giraud M.F. Naismith J.H. Epimerases: structure, function and mechanism.Cell Mol. Life Sci. 2001; 58 (11706991): 1650-166510.1007/PL00000803Crossref PubMed Scopus (77) Google Scholar). Among these, the structural features underlying the selective C4′ epimerization by UDP-galactose 4-epimerase were extensively studied in the past few years (13Eixelsberger T. Sykora S. Egger S. Brunsteiner M. Kavanagh K.L. Oppermann U. Brecker L. Nidetzky B. Structure and mechanism of human UDP-xylose synthase: evidence for a promoting role of sugar ring distortion in a three-step catalytic conversion of UDP-glucuronic acid.J. Biol. Chem. 2012; 287 (22810237): 31349-3135810.1074/jbc.M112.386706Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 18Thoden J.B. Henderson J.M. Fridovich-Keil J.L. Holden H.M. Structural analysis of the Y299C mutant of Escherichia coli UDP-galactose 4-epimerase: teaching an old dog new tricks.J. Biol. Chem. 2002; 277 (12019271): 27528-2753410.1074/jbc.M204413200Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 19Frirdich E. Whitfield C. Characterization of Gla(KP), a UDP-galacturonic acid C4-epimerase from Klebsiella pneumoniae with extended substrate specificity.J. Bacteriol. 2005; 187 (15937173): 4104-411510.1128/JB.187.12.4104-4115.2005Crossref PubMed Scopus (20) Google Scholar). This enzyme is part of the Leloir pathway and interconverts UDP-galactose into UDP-glucose (20Holden H.M. Rayment I. Thoden J.B. Structure and function of enzymes of the Leloir pathway for galactose metabolism.J. Biol. Chem. 2003; 278 (12923184): 43885-4388810.1074/jbc.R300025200Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar). It belongs to the SDR family, featuring the same Ser/Thr-Tyr-Lys catalytic triad as UDP-glucuronic acid 4-epimerase. The substrate is first oxidized at the C4′, and the resulting 4-keto-hexose intermediate is thought to undergo a rotational movement within the active site. The intermediate is then reduced by NADH, leading to the inversion of the C4′ configuration (18Thoden J.B. Henderson J.M. Fridovich-Keil J.L. Holden H.M. Structural analysis of the Y299C mutant of Escherichia coli UDP-galactose 4-epimerase: teaching an old dog new tricks.J. Biol. Chem. 2002; 277 (12019271): 27528-2753410.1074/jbc.M204413200Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 21Thoden J.B. Wohlers T.M. Fridovich-Keil J.L. Holden H.M. Human UDP-galactose 4-epimerase: accommodation of UDP-N-acetylglucosamine within the active site.J. Biol. Chem. 2001; 276 (11279032): 15131-1513610.1074/jbc.M100220200Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). The same catalytic strategy has been hypothesized also for the epimerases acting on UDP-GlcA (22Sun H. Ko T.P. Liu W. Liu W. Zheng Y. Chen C.C. Guo R.T. Structure of an antibiotic-synthesizing UDP-glucuronate 4-epimerase MoeE5 in complex with substrate.Biochem. Biophys. Res. Commun. 2020; 521 (31653344): 31-3610.1016/j.bbrc.2019.10.035Crossref PubMed Scopus (6) Google Scholar). As demonstrated by recent work, these enzymes fine-tune the rates of the individual redox steps to prevent the accumulation of the 4-keto-hexose-uronic acid, minimizing the undesired decarboxylation and/or release of this reactive intermediate (23Annika J.E. Borg A.D. Weber H. Nidetzky B. Mechanistic characterization of UDP-glucuronic acid 4-epimerase.FEBS J. 2020; (32645249)10.1111/febs.15478Google Scholar). Whereas the catalytic mechanisms of UDP-xylose synthase and UDP-apiose/xylose synthase have been thoroughly elucidated (13Eixelsberger T. Sykora S. Egger S. Brunsteiner M. Kavanagh K.L. Oppermann U. Brecker L. Nidetzky B. Structure and mechanism of human UDP-xylose synthase: evidence for a promoting role of sugar ring distortion in a three-step catalytic conversion of UDP-glucuronic acid.J. Biol. Chem. 2012; 287 (22810237): 31349-3135810.1074/jbc.M112.386706Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 16Savino S. Borg A.J.E. Dennig A. Pfeiffer M. de Giorgi F. Weber H. Dubey K.D. Rovira C. Mattevi A. Nidetzky B. Deciphering the enzymatic mechanism of sugar ring contraction in UDP-apiose biosynthesis.Nat. Catal. 2019; 2 (31844840): 1115-112310.1038/s41929-019-0382-8Crossref PubMed Scopus (6) Google Scholar, 24Eixelsberger T. Horvat D. Gutmann A. Weber H. Nidetzky B. Isotope probing of the UDP-apiose/UDP-xylose synthase reaction: evidence of a mechanism via a coupled oxidation and aldol cleavage.Angew. Chem. Int. Ed. Engl. 2017; 56 (28102965): 2503-250710.1002/anie.201609288Crossref PubMed Scopus (9) Google Scholar), the epimerization reaction by UDP-glucuronic acid 4-epimerase remains partly unexplored. In this work, we carried out a comprehensive structural analysis of UDP-glucuronic acid 4-epimerase from Bacillus cereus (BcUGAepi) by solving six high-resolution crystal structures that elucidate different steps along the reaction. The main aspects of these structures were compared with other SDR epimerases and decarboxylases. We also isolated a remarkable complex showing an equilibrium mixture of both substrate and product in approximately 1:1 ratio. We further used structural, kinetic isotope effect and site-directed mutagenesis experiments to inspect the fine roles of catalytic residues. Our studies provide an overview of catalysis with critical insight into the mechanism of 4-ketose-uronic acid intermediate rotation and protection against intermediate decarboxylation. They further demonstrate that the UDP moiety is not only an accessory part of the molecule, but it is fundamental for substrate recognition and active-site configuration. The crystal structure of BcUGAepi co-purified with NAD+ was initially solved at 2.2 Å resolution by molecular replacement (Table 1). The enzyme is composed of nine β-strands and eight α-helices assembled in two domains (13Eixelsberger T. Sykora S. Egger S. Brunsteiner M. Kavanagh K.L. Oppermann U. Brecker L. Nidetzky B. Structure and mechanism of human UDP-xylose synthase: evidence for a promoting role of sugar ring distortion in a three-step catalytic conversion of UDP-glucuronic acid.J. Biol. Chem. 2012; 287 (22810237): 31349-3135810.1074/jbc.M112.386706Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 27Gatzeva-Topalova P.Z. May A.P. Sousa M.C. Crystal structure of Escherichia coli ArnA (PmrI) decarboxylase domain: a key enzyme for lipid A modification with 4-amino-4-deoxy-l-arabinose and polymyxin resistance.Biochemistry. 2004; 43 (15491143): 13370-1337910.1021/bi048551fCrossref PubMed Scopus (44) Google Scholar, 28Polizzi S.J. Walsh Jr., R.M. Peeples W.B. Lim J.M. Wells L. Wood Z.A. Human UDP-α-d-xylose synthase and Escherichia coli ArnA conserve a conformational shunt that controls whether xylose or 4-keto-xylose is produced.Biochemistry. 2012; 51 (23072385): 8844-885510.1021/bi301135bCrossref PubMed Scopus (15) Google Scholar) (Fig. 1). The N-terminal domain is characterized by a Rossmann fold motif comprising a core of seven β-strands surrounded by six α-helices. The NAD+ cofactor is fully embedded within this domain, whereas the smaller C-terminal domain provides the binding site for the UDP-GlcA substrate. The crevice between the two domains encloses the active site (29Thoden J. Frey P. Holden H. High-resolution X-ray structure of UDP-galactose 4-epimerase complexed with UDP-phenol.Protein Sci. 1996; 5 (8931134): 2149-216110.1002/pro.5560051102Crossref PubMed Scopus (82) Google Scholar, 30Thoden J. Frey P. Holden H. Molecular structure of the NADH/UDP-glucose abortive complex of UDP-galactose 4-epimerase from Escherichia coli: implications for the catalytic mechanism.Biochemistry. 1996; 35 (8611497): 5137-514410.1021/bi9601114Crossref PubMed Scopus (145) Google Scholar, 31Thoden J. Frey P. Holden H. Crystal structures of the oxidized and reduced forms of UDP-galactose 4-epimerase isolated from Escherichia coli.Biochemistry. 1996; 35 (8611559): 2557-256610.1021/bi952715yCrossref PubMed Scopus (123) Google Scholar). As for other SDR family enzymes, two protein chains are arranged to form a tight homodimer (11Kavanagh K.L. Jörnvall H. Persson B. Oppermann U. 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.Cell Mol. Life Sci. 2008; 65 (19011750): 3895-390610.1007/s00018-008-8588-yCrossref PubMed Scopus (515) Google Scholar, 32Jörnvall H. Persson B. Krook M. Atrian S. Gonzàlez-Duarte R. Jeffery J. Ghosh D. Short-chain dehydrogenases/reductases (SDR).Biochemistry. 1995; 34 (7742302): 6003-601310.1021/bi00018a001Crossref PubMed Scopus (1121) Google Scholar, 33Persson B. Kallberg Y. Bray J.E. Bruford E. Dellaporta S.L. Favia A.D. Duarte R.G. Jörnvall H. Kavanagh K.L. Kedishvili N. Kisiela M. Maser E. Mindnich R. Orchard S. Penning T.M. et al.The SDR (short-chain dehydrogenase/reductase and related enzymes) nomenclature initiative.Chem. Biol. Interact. 2009; 178 (19027726): 94-9810.1016/j.cbi.2008.10.040Crossref PubMed Scopus (256) Google Scholar) where each subunit (37 kDa) interacts with two adjacent α-helices, generating an extensively intermolecular hydrophobic core of four α-helices (14Gatzeva-Topalova P.Z. May A.P. Sousa M.C. Structure and mechanism of ArnA: conformational change implies ordered dehydrogenase mechanism in key enzyme for polymyxin resistance.Structure. 2005; 13 (15939024): 929-94210.1016/j.str.2005.03.018Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 16Savino S. Borg A.J.E. Dennig A. Pfeiffer M. de Giorgi F. Weber H. Dubey K.D. Rovira C. Mattevi A. Nidetzky B. Deciphering the enzymatic mechanism of sugar ring contraction in UDP-apiose biosynthesis.Nat. Catal. 2019; 2 (31844840): 1115-112310.1038/s41929-019-0382-8Crossref PubMed Scopus (6) Google Scholar) (Fig. 1). Structural comparisons using the Dali server (34Holm L. Benchmarking fold detection by DaliLite v.5.Bioinformatics. 2019; 35 (31263867): 5326-532710.1093/bioinformatics/btz536Crossref PubMed Scopus (161) Google Scholar) indicate that the overall structure of BcUGAepi is similar to the structures of UDP-galactose 4-epimerase from Escherichia coli (PDB entry 1UDA) (29Thoden J. Frey P. Holden H. High-resolution X-ray structure of UDP-galactose 4-epimerase complexed with UDP-phenol.Protein Sci. 1996; 5 (8931134): 2149-216110.1002/pro.5560051102Crossref PubMed Scopus (82) Google Scholar, 30Thoden J. Frey P. Holden H. Molecular structure of the NADH/UDP-glucose abortive complex of UDP-galactose 4-epimerase from Escherichia coli: implications for the catalytic mechanism.Biochemistry. 1996; 35 (8611497): 5137-514410.1021/bi9601114Crossref PubMed Scopus (145) Google Scholar, 31Thoden J. Frey P. Holden H. Crystal structures of the oxidized and reduced forms of UDP-galactose 4-epimerase isolated from Escherichia coli.Biochemistry. 1996; 35 (8611559): 2557-256610.1021/bi952715yCrossref PubMed Scopus (123) Google Scholar) and MoeE5 (a UDP-glucuronic acid epimerase from Streptomyces viridosporus; PDB entry 6KV9) (22Sun H. Ko T.P. Liu W. Liu W. Zheng Y. Chen C.C. Guo R.T. Structure of an antibiotic-synthesizing UDP-glucuronate 4-epimerase MoeE5 in complex with substrate.Biochem. Biophys. Res. Commun. 2020; 521 (31653344): 31-3610.1016/j.bbrc.2019.10.035Crossref PubMed Scopus (6) Google Scholar) with Z scores of 37.2 and 42.8 and sequence identities of 30 and 38%, respectively.Table 1Data collection and refinement statistics for the UGAepi crystal structuresNAD+NAD+/UDPNAD+/UDP-GlcANAD+/UDP-GlcA/UDP-GalANAD+/4F-UDP-GlcANAD+/UDP-GalASpace groupC2P1P1P21P1P1Unit cell axes (Å)214.5, 78.5, 87.942.5, 58.6, 64.942.4, 58.4, 64.753.7, 124.3, 98.442.2, 58.2, 64.456.8, 62.7, 105.8Unit cell angles (degrees)90.0, 91.0, 90.096.8, 98.4, 110.396.8, 98.4, 110.690.0, 90.6, 90.097.2, 98.2, 109.991.9, 99.9, 92.0No. of chains/asymmetrical unit422424Resolution (Å)45.6–2.20 (2.25–2.20)aValues in parentheses are for reflections in the highest-resolution shell.45.3–1.70 (1.74–1.70)45.2–1.80 (1.84–1.80)49.4–1.50 (1.53–1.50)45.2–1.70 (1.74–1.70)46.1–1.85 (1.89–1.85)PDB code6ZLA6ZL66ZLD6ZLK6ZLJ6ZLLRsymbRsym = ∑|Ii − 〈I〉|/∑Ii, where Ii is the intensity of the ith observation and is the mean intensity of the reflection. (%)4.6 (20.8)6.5 (55.7)8.4 (25.9)8.3 (157)13.7 (239)11.6 (228)CC1/2cThe resolution cut-off was set to CC1/2 > 0.3, where CC1/2 is the Pearson correlation coefficient of two “half” data sets, each derived by averaging half of the observations for a given reflection. The data set for the equilibrium structure features some mild anisotropy. Following the resolution estimation given by the program Aimless (25) and based on the visual inspection of the electron density maps, only for this data set we included data with a somewhat lower CC1/2 value (26). (%)99.9 (92.6)99.7 (88.1)99.7 (78.0)99.5 (18.8)99.6 (68.0)99.7 (46.6)Completeness (%)98 (92.6)93 (91)93.7 (92)97.5 (97.5)92.4 (90.8)92.6 (83.6)Unique reflections72,64658,36849,095190,28657,074113,316Redundancy4.2 (3.2)1.7 (1.7)1.6 (1.7)3.8 (3.9)9.1 (9.5)6.9 (6.4)I/σ16.9 (4.6)10.2 (1.2)9.3 (2.5)7.0 (1.0)8.4 (1.5)8.7 (1.1)No. of nonhydrogen atoms Protein971449324934986949309988 Ligands176138160470160320 Waters243251298695170242Average B value for protein/ligand atoms (Å2)34.2/25.425.59/25.822.4/18.723.6/22.829.3/26.437.14/ 38.12Rcryst (%)17.8 (21.5)17.0 (28.6)16.8 (24.8)17.2 (32.2)17.2 (29.7)18.7 (32.2)Rfree (%)22.7 (26.5)20.0 (30.7)20.7 (27.1)20.3 (30.9)21.6 (30.7)22.2 (34.0)Root mean square deviations from standard values Bond lengths (Å)0.0870.0100.0090.0110.0090.008 Bond angles (degrees)1.581.681.571.731.601.58Ramachandran plot Preferred (%)97.598.098.097.798.097.3 Allowed (%)2.31.71.72.01.72.2 Outliers (%)0.20.30.30.30.30.4a Values in parentheses are for reflections in the highest-resolution shell.b Rsym = ∑|Ii − 〈I〉|/∑Ii, where Ii is the intensity of the ith observation and is the mean intensity of the reflection.c The resolution cut-off was set to CC1/2 > 0.3, where CC1/2 is the Pearson correlation coefficient of two “half” data sets, each derived by averaging half of the observations for a given reflection. The data set for the equilibrium structure features some mild anisotropy. Following the resolution estimation given by the program Aimless (25Collaborative Computational Project, Number 4The CCP4 suite: programs for protein crystallography.Acta Crystallogr. D Biol. Crystallogr. 1994; 50 (15299374): 760-76310.1107/s0907444994003112Crossref PubMed Scopus (17708) Google Scholar) and based on the visual inspection of the electron density maps, only for this data set we included data with a somewhat lower CC1/2 value (26Karplus P.A. Diederichs K. Linking crystallographic model and data quality.Science. 2012; 336 (22628654): 1030-103310.1126/science.1218231Crossref PubMed Scopus (1169) Google Scholar). Open table in a new tab NAD+ remains tightly associated to the enzyme during the entire purification process (Fig. 2A) (11Kavanagh K.L. Jörnvall H. Persson B. Oppermann U. 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.Cell Mol. Life Sci. 2008; 65 (19011750): 3895-390610.1007/s00018-008-8588-yCrossref PubMed Scopus (515) Google Scholar, 13Eixelsberger T. Sykora S. Egger S. Brunsteiner M. Kavanagh K.L. Oppermann U. Brecker L. Nidetzky B. Structure and mechanism of human UDP-xylose synthase: evidence for a promoting role of sugar ring distortion in a three-step catalytic conversion of UDP-glucuronic acid.J. Biol. Chem. 2012; 287 (22810237): 31349-3135810.1074/jbc.M112.386706Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 35Liu Y. Vanhooke J.L. Frey P.A. UDP-galactose 4-epimerase: NAD+ content and a charge-transfer band associated with the substrate-induced conformational transition.Biochemistry. 1996; 35 (8652544): 7615-762010.1021/bi960102vCrossref PubMed Scopus (30) Google Scholar). It is bound in an elongated conformation as typically observed in other SDR enzymes (21Thoden J.B. Wohlers T.M. Fridovich-Keil J.L. Holden H.M. Human UDP-galactose 4-epimerase: accommodation of UDP-N-acetylglucosamine within the active site.J. Biol. Chem. 2001; 276 (11279032): 15131-1513610.1074/jbc.M100220200Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 28Polizzi S.J. Walsh Jr., R.M. Peeples W.B. Lim J.M. Wells L. Wood Z.A. Human UDP-α-d-xylose synthase and Escherichia coli ArnA conserve a conformational shunt that controls whether xylose or 4-keto-xylose is produced.Biochemistry. 2012; 51 (23072385): 8844-885510.1021/bi301135bCrossref PubMed Scopus (15) Google Scholar, 36Thoden J.B. Holden H.M. Dramatic differences in the binding of UDP-galactose and UDP-glucose to UDP-galactose 4-epimerase from Escherichia coli.Biochemistry. 1998; 37 (9708982): 11469-1147710.1021/bi9808969Crossref PubMed Scopus (80) Google Scholar). Its binding is extensively stabilized through several interactions with residues of the Rossmann fold domain. The amine group of the adenine ring hydrogen-bonds to Asn-101 and Asp-62, the adenine-ribose hydroxyl groups interact with Asp-32 and Lys-43, the β-phosphate is ionically bound to Arg-185, and the nicotinamide ribose is hydrogen-bonded to Tyr-149 and Lys-153. These last two residues belong to the Tyr-X-X-X-Lys motif of the Ser/Thr-Tyr-Lys triad, the typical hallmark of SDR enzymes (32Jörnvall H. Persson B. Krook M. Atrian S. Gonzàlez-Duarte R. Jeffery J. Ghosh D. Short-chain dehydrogenases/reductases (SDR).Biochemistry. 1995; 34 (7742302): 6003-601310.1021/bi00018a001Crossref PubMed Scopus (1121) Google Scholar, 37Jörnvall H. Multiplicity and complexity of SDR and MDR enzymes.Adv. Exp. Med. Biol. 1999; 463 (10352706): 359-36410.1007/978-1-4615-4735-8_44Crossref PubMed Scopus (15) Google Scholar, 38Jörnvall H. Höög J.-O. Persson B. SDR and MDR: completed genome sequences show these protein families to be large, of old origin, and of complex nature.FEBS Lett. 1999; 445 (10094468): 261-26410.1016/S0014-5793(99)00130-1Crossref PubMed Scopus (167) Google Scholar, 39Rossman M.G. Liljas A. Brändén C.-I. Banaszak L.J. 2 Evolutionary and structural relationships among dehydrogenases.in: Boyer P.D. The Enzymes. Academic Press, New York1975: 61-102Google Scholar). This binding mode orients the si-face on the nicotinamide ring toward the substrate-binding site to cope with the sugar moiety and mediate hydride transfer. In the NAD+ complex, two loops around the catalytic site (residues 86–91 and 269–276) are disordered as gathered from the absence of well-defined electron density around them (Fig. 2B). The crystal structure of the enzyme bound to UDP (1.7 Å resolution; Table 1) reveals that these two loops become ordered in the presence of the nucleotide diphosphate. In particular, Glu-276 anchors the hydroxyl groups of the nucleotide ribose, whereas loop 86–91 lends more rigidity to the part of the cavity hosting the pyrophosphate (Fig. 2, B and C). UDP binding affects the conformation of a third loop, formed by residues 204–214. This loop moves closer to the UDP-binding site so that Gln-211 and Arg-213 can hydrogen-bond to the UDP's ribose hydroxy groups and the β-phosphate, respectively. Moreover, Phe-206 is implicated in π-stacking with the uracil ring, whereas Thr-204 is hydrogen-bonded to the NH group of the base. This com
Referência(s)