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Structure of UDP Complex of UDP-galactose:β-Galactoside-α-1,3-galactosyltransferase at 1.53-Å Resolution Reveals a Conformational Change in the Catalytically Important C Terminus

2001; Elsevier BV; Volume: 276; Issue: 51 Linguagem: Inglês

10.1074/jbc.m108828200

1083-351X

Ester Boix, G. Jawahar Swaminathan, Yingnan Zhang, R. Natesh, Keith Brew, K. Ravi Acharya,

Metabolism and Genetic Disorders

UDP-galactose:β-galactosyl α-1,3-galactosyltransferase (α3GT) catalyzes the transfer of galactose from UDP-α-d-galactose into an α-1,3 linkage with β-galactosyl groups in glycoconjugates. The enzyme is expressed in many mammalian species but is absent from humans, apes, and old world monkeys as a result of the mutational inactivation of the gene; in humans, a large fraction of natural antibodies are directed against its product, the α-galactose epitope. α3GT is a member of a family of metal-dependent retaining glycosyltransferases including the histo-blood group A and B synthases. A crystal structure of the catalytic domain of α3GT was recently reported (Gastinel, L. N., Bignon, C., Misra, A. K., Hindsgaul, O., Shaper, J. H., and Joziasse, D. H. (2001) EMBO J. 20, 638–649). However, because of the limited resolution (2.3 Å) and high mobility of the atoms (as indicated by high B-factors) this structure (form I) does not provide a clear depiction of the catalytic site of the enzyme. Here we report a new, highly ordered structure for the catalytic domain of α3GT at 1.53-Å resolution (form II). This provides a more accurate picture of the details of the catalytic site that includes a bound UDP molecule and a Mn2+ cofactor. Significantly, in the new structure, the C-terminal segment (residues 358–368) adopts a very different, highly structured conformation and appears to form part of the active site. The properties of an Arg-365 to Lys mutant indicate that this region is important for catalysis, possibly reflecting its role in a donor substrate-induced conformational change. UDP-galactose:β-galactosyl α-1,3-galactosyltransferase (α3GT) catalyzes the transfer of galactose from UDP-α-d-galactose into an α-1,3 linkage with β-galactosyl groups in glycoconjugates. The enzyme is expressed in many mammalian species but is absent from humans, apes, and old world monkeys as a result of the mutational inactivation of the gene; in humans, a large fraction of natural antibodies are directed against its product, the α-galactose epitope. α3GT is a member of a family of metal-dependent retaining glycosyltransferases including the histo-blood group A and B synthases. A crystal structure of the catalytic domain of α3GT was recently reported (Gastinel, L. N., Bignon, C., Misra, A. K., Hindsgaul, O., Shaper, J. H., and Joziasse, D. H. (2001) EMBO J. 20, 638–649). However, because of the limited resolution (2.3 Å) and high mobility of the atoms (as indicated by high B-factors) this structure (form I) does not provide a clear depiction of the catalytic site of the enzyme. Here we report a new, highly ordered structure for the catalytic domain of α3GT at 1.53-Å resolution (form II). This provides a more accurate picture of the details of the catalytic site that includes a bound UDP molecule and a Mn2+ cofactor. Significantly, in the new structure, the C-terminal segment (residues 358–368) adopts a very different, highly structured conformation and appears to form part of the active site. The properties of an Arg-365 to Lys mutant indicate that this region is important for catalysis, possibly reflecting its role in a donor substrate-induced conformational change. β-galactosyl α-1,3-galactosyltransferase 4-morpholineethanesulfonic acid Specific hetero-oligosaccharides on glycoproteins and glycolipids play important roles in cell-cell and cell-matrix interactions, affect the stability and structure of proteins, and modulate cellular interactions with viruses, toxins, and other proteins; they are also epitopes that are recognized by the immune system (1Varki A. Glycobiology. 1993; 3: 97-130Crossref PubMed Scopus (4984) Google Scholar). The range and types of carbohydrate structures present on a cell vary in different tissues and species as a reflection of the specificity of glycosyltransferases, enzymes that catalyze the transfer of a specific monosaccharide from an activated derivative (such as UDP-galactose) into a defined linkage with a specific acceptor (2Natsuka S. Lowe J.B. Curr. Opin. Struct. Biol. 1994; 4: 683-686Crossref Scopus (63) Google Scholar, 3Paulson J.C. Colley K.H. J. Biol. Chem. 1989; 264: 17615-17618Abstract Full Text PDF PubMed Google Scholar). The carbohydrate chains of glycoconjugates have an enormous potential for variation because of the range of different sugars and the large number of alternative glycosidic linkages. Consequently, they are carriers of large amounts of biological information that originates from the specificity of glycosyltransferases. In addition to their biosynthetic roles, some glycosyltransferases also function directly in cellular interactions and regulation (4Gijsen H.J.M. Qiao L. Fitz W. Wong C-H. Chem. Rev. 1996; 96: 443-473Crossref PubMed Scopus (426) Google Scholar, 5Shi X. Amindari S. Paruchuri K. Skalla D. Burkin H. Shur B.D. Miller D.J. Development. 2001; 128: 645-654PubMed Google Scholar). The limited information currently available regarding the structural basis of molecular recognition and catalysis by these important enzymes precludes a clear understanding of the molecular basis of their various biological functions. High resolution structural data are also needed to inform the design of inhibitors and to facilitate the engineering of new catalysts for the enzymatic synthesis of natural and novel glycoconjugates for therapeutic uses (4Gijsen H.J.M. Qiao L. Fitz W. Wong C-H. Chem. Rev. 1996; 96: 443-473Crossref PubMed Scopus (426) Google Scholar, 6Nixon B. Lu Q. Wassler M.J. Foote C.I. Ensslin M.A. Shur B.D. Cells Tissues Organs. 2001; 168: 46-57Crossref PubMed Scopus (64) Google Scholar, 7Watt G.M. Lowden P.A.S. Flitsch S.L. Curr. Opin. Struct. Biol. 1997; 7: 652-656Crossref PubMed Scopus (63) Google Scholar). The majority of glycosyltransferases are type-II membrane proteins with short N-terminal cytosolic domains, a membrane-spanning region, a stem, and a C-terminal catalytic region (3Paulson J.C. Colley K.H. J. Biol. Chem. 1989; 264: 17615-17618Abstract Full Text PDF PubMed Google Scholar). Classification schemes have been proposed for them based on sequence similarity and specificity; however, localized similarities between the sequences of different galactosyltransferases (8Breton C. Bettler E. Joziasse D.H. Geremia R.A. Imberty A. J. Biochem. (Tokyo). 1998; 123: 1000-1009Crossref PubMed Scopus (136) Google Scholar), mannosyl, and other glycosyltransferases (9Wiggins C.A. Munro S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7945-7950Crossref PubMed Scopus (320) Google Scholar, 10Zhou D. Dinter A. Gallego R.G. Kamerling J.P. Vliegenthart J.F.G. Berger E.G. Hennet T. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 406-411Crossref PubMed Scopus (87) Google Scholar) suggest that such schemes may eventually be simplified into larger groups with similar folds and conserved binding sites (see also Ref. 11Ünligil U.M. Zhou S. Yuwaraj S. Sarkar M. Schachter H. Rini J.M. EMBO J. 2000; 19: 5269-5280Crossref PubMed Scopus (238) Google Scholar). A priori mechanistic considerations divide glycosyltransferases into two large groups, those that catalyze a reaction in which the anomeric configuration of the transferred sugar is inverted and those that catalyze a retaining reaction (12Ünligil U.M. Rini J.M. Curr. Opin. Struct. Biol. 2000; 10: 510-517Crossref PubMed Scopus (344) Google Scholar). While the former are likely to act through a displacement (SN2) mechanism that necessarily produces an inversion at C-1, a retaining reaction is expected to involve a double displacement mechanism with formation of an intermediate such as a β-glycosyl-enzyme covalent complex. UDP-galactose β-galactoside α-1,3 galactosyltransferase (α3GT1; EC 2.4.1.151) is an enzyme found in many mammalian species but not in humans and their closest relatives because of the mutational inactivation of its gene (13Galili U. Swanson K. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7401-7404Crossref PubMed Scopus (268) Google Scholar); in species lacking the enzyme, about 1% of circulating endogenous antibodies are directed against the product of its action, the α-galactose epitope (14Galili U. Springer Semin. Immunopathol. 1993; 15: 155-171Crossref PubMed Scopus (182) Google Scholar). These protect against pathogens but are a barrier to the xenotransplantation of organs from species with active α3GT to humans. α3GT is a model for several paralogous retaining glycosyltransferases of varying substrate specificity including the histo-blood group A and B glycosyltransferases (15Yamamoto F-I. Clausen H. White T. Marken J. Hakomori S-I. Nature. 1990; 345: 229-233Crossref PubMed Scopus (893) Google Scholar), Forssman glycolipid synthase (16Haslam D.B. Baenziger J.U. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10697-10702Crossref PubMed Scopus (80) Google Scholar), and isogloboside 3 synthase (17Keusch J.J. Manzella S.M. Nyame K.A. Cummings R.D. Baenziger J.U. J. Biol. Chem. 2000; 275: 25308-25314Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). A crystal structure of the catalytic domain of α3GT (in tetragonal form, P41212 space group, one molecule/asymmetric unit, form I) was recently reported at 2.3- and 2.5-Å resolution in UMP- and Hg-UDP-galactose-bound forms, respectively (18Gastinel L.N. Bignon C. Misra A.K. Hindsgaul O. Shaper J.H. Joziasse D.H. EMBO J. 2001; 20: 638-649Crossref PubMed Scopus (179) Google Scholar). These structures identified binding sites for UDP and a Mn2+ cofactor. A region of electron density in the Hg-UDP-galactose-bound structure was interpreted as a β-galactosyl moiety covalently attached to Glu-317, suggesting a covalent catalytic mechanism. However, no direct evidence was obtained for a glycosyl-enzyme covalent bond, and the limited resolution of the structure, reflected also in the high B-factors for all atoms and the disordered C-terminal region of the polypeptide chain, raises questions about this interpretation. To clarify unresolved issues regarding the structure of α3GT and the specific roles of different amino acid residues in the reaction mechanism, we sought to obtain crystals with improved resolution using different crystallization conditions. Here we report the structure of a new crystal form of α3GT obtained with polyethylene glycol as the precipitating agent instead of a high salt concentration buffer (pH 6.0, monoclinic form, P21 space group, dimer/asymmetric unit, form II) at 1.53 Å. The entire structure is highly ordered, with the C terminus (residues 358–368) adopting what appears to be an "active" conformation in the structure. A possibly analogous conformational change in a region of other glycosyltransferases that are unrelated in amino acid sequence to α3GT (12Ünligil U.M. Rini J.M. Curr. Opin. Struct. Biol. 2000; 10: 510-517Crossref PubMed Scopus (344) Google Scholar), including β-4-galactosyltransferase-I (19Ramakrishnan B. Qasba P.K. J. Mol. Biol. 2001; 310: 205-218Crossref PubMed Scopus (174) Google Scholar), has been noted that may have an important role in the catalytic mechanism (12Ünligil U.M. Rini J.M. Curr. Opin. Struct. Biol. 2000; 10: 510-517Crossref PubMed Scopus (344) Google Scholar). Mutagenesis of α3GT indicates that this region is important for catalysis. We discuss the implications of our results for the mechanism of α3GT and its homologues. The catalytic domain of bovine α3GT (residues 80–368) was expressed in Escherichia coli, purified as previously described (20Zhang Y. Wang P.G. Brew K. J. Biol. Chem. 2001; 276: 11567-11574Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar), and stored at −20 °C in 20 mm MES-NaOH buffer (pH 6) in 50% glycerol. Crystals were grown at 16 °C by the vapor diffusion hanging drop method by mixing 2 μl of the protein at 5 mg/ml in 20 mm MES-NaOH buffer, pH 6.0, 10% glycerol, containing 10 mm UDP and 0.1 mm MnCl2, with an equal volume of a reservoir solution containing 5% polyethylene glycol 6000 and 0.1 mTris-HCl, pH 8.0. Single crystals appeared after 2–3 days. Before data collection, crystals were flash-cooled at 100 K in a cryoprotectant containing 10% polyethylene glycol 6000, 0.1 m Tris-HCl, pH 8.0, and 25% glycerol. A high resolution data set to 1.53 Å was collected using a 30-cm MAR research image plate at DESY, EMBL outstation (Hamburg, Germany). The crystals belong to the P21 space group, with two molecules (a noncrystallographic dimer) in the asymmetric unit and some 58% of the crystal volume occupied by the solvent. Raw data images were indexed and scaled using the DENZO and SCALEPACK modules of the HKL Suite (21Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38570) Google Scholar) (Table I).Table IStatistics for data collection and refinementParametersValuesWavelength used during data collection0.909 ÅCell dimensionsa = 45.02, b = 94.14, c = 94.38 Å, β = 98.9°Resolution range40.0–1.53 ÅSpace groupP 21 (2 mol/asymmetric unit)No. of observations386,939No. of unique reflections114,924R symm(%)aRsymm = ΣhΣi[‖I i(h) − 〈I (h)〉‖/ΣhΣi I i(h)], where Ii is the i th measurement and 〈I (h)〉 is the weighted mean of all measurements of I (h). (outermost shell)bOutermost shell is 1.58–1.53 Å.10.6 (49.7)Completeness (%) (outermost shell)bOutermost shell is 1.58–1.53 Å.98.2 (93.8)I/ςI (outermost shell)bOutermost shell is 1.58–1.53 Å.11.73 (1.98)Crystallographic R-factor (%) (R cryst)cRcryst = Σh‖F o −F c‖/ΣhF o, whereF o and F c are the observed and calculated structure factor amplitudes of reflection h.14.05Free R-factor (%) (R free)dRfree is equal toR cryst for a randomly selected 5% subset of reflections, not used in refinement.19.06Deviations from ideality Bond lengths (Å)0.014 Bond angles (degrees)2.17 Dihedral angles (degrees)25.23 Improper dihedrals (degrees)1.43Number of protein atoms2426 (molecule A), 2418 (molecule B)B-factor statistics (Å2) Overall B-factor17.05 Main chain14.39 Side chain19.47 Solvent atoms (757 in total)31.62 UDP atoms (2 in total, 1/monomer)10.61 Mn2+ ion (2 in total, 1/monomer)10.07, 9.99 Glycerol atoms (2 in total, 1/monomer)40.41Mean anisotropyeMean anisotropy calculated using the program PARVATI (37). of the structure0.41a Rsymm = ΣhΣi[‖I i(h) − 〈I (h)〉‖/ΣhΣi I i(h)], where Ii is the i th measurement and 〈I (h)〉 is the weighted mean of all measurements of I (h).b Outermost shell is 1.58–1.53 Å.c Rcryst = Σh‖F o −F c‖/ΣhF o, whereF o and F c are the observed and calculated structure factor amplitudes of reflection h.d Rfree is equal toR cryst for a randomly selected 5% subset of reflections, not used in refinement.e Mean anisotropy calculated using the program PARVATI (37Merritt E.A. Acta Crystallogr. Sec. D. 1999; 55: 1109-1117Crossref PubMed Scopus (165) Google Scholar). Open table in a new tab The structure of α3GT dimer was determined by the molecular replacement method using the 2.5-Å tetragonal (form I, monomer) structure (18Gastinel L.N. Bignon C. Misra A.K. Hindsgaul O. Shaper J.H. Joziasse D.H. EMBO J. 2001; 20: 638-649Crossref PubMed Scopus (179) Google Scholar) with the program AMoRe (22Navaza J. Acta Crystallogr. Sec. A. 1994; 50: 157-163Crossref Scopus (5029) Google Scholar). The structure was initially refined using the program CNS (23Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sec. D. 1998; 54: 905-921Crossref PubMed Scopus (16966) Google Scholar), with temperature factors for all atoms kept isotropic. The behavior of the cross-validation R-factor (R free) was monitored throughout the refinement (24Brünger A.T. Nature. 1992; 355: 472-474Crossref PubMed Scopus (3864) Google Scholar). Several rounds of energy minimization, individualB-factor refinement, simulated annealing using CNS, and model building using the program O (25Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sec. A. 1991; 47: 110-119Crossref PubMed Scopus (13010) Google Scholar) were performed until convergence of the R free value. The water molecules were picked using the program ARP/wARP (26Lamzin V.S. Wilson K.S. Acta Crystallogr. Sec. D. 1993; 49: 129-149Crossref PubMed Google Scholar). These water molecules were manually inspected carefully with the aid ofF o − F c and 2F o − F c difference electron density maps and accepted only if peaks existed in both the maps at the 3ς and 1ς level, respectively, and were at hydrogen bonding distance from the appropriate atoms. The model during CNS refinement converged to a crystallographic R-factor (R cryst) of 18.4 and R free of 20.0%. Further refinement was carried out using SHELXL-97 (27Sheldrick G.M. Schneider T.R. Methods Enzymol. 1997; 277: 319-343Crossref PubMed Scopus (1885) Google Scholar). CGLS refinement in SHELXL-97 was carried out restraining all of the 1,2 and 1,3 distances with the Engh and Huber (28Engh R.A. Huber R. Acta Crystallogr. Sec. A. 1991; 47: 392-400Crossref Scopus (2545) Google Scholar) restraints. Initially, all of the atomic displacement parameters were kept isotropic. The data to parameter ratio >2 enabled us to carry out anisotropic refinement on atomic displacement parameters, which was subsequently justified by a 1.3% drop inR free and an improved Fourier map. All of the alternate conformations were modeled after the initial anisotropic refinement. Any new atoms added to the molecule were refined isotropically for at least two cycles before they were refined anisotropically. The multiple conformation site occupation factors were refined constraining their sum to be unity. The model converged to anR cryst/R free of 14.82/20.09%. The final refinement was carried out with hydrogens included in the calculated positions (for protein atoms alone, except for multiple conformations). The addition of hydrogen atoms as riding model was justified by a drop in R free of 1.03%, leading to a final model withR cryst/R free of 14.05/19.06%. All nonhydrogen atoms were refined anisotropically including water molecules (excluding protein atoms with multiple conformations). The final model of α3GT comprises residues 82–368 for both molecules and contains one UDP molecule and one Mn2+ ion per monomer at the active site. The structure contains 757 water molecules and two glycerol molecules (from the crystallization medium or cryoprotectant). All of the residues of the dimer lie in allowed regions of the Ramachandran (ϕ-ψ) map. The expression vector for wild type α3GT, pET15b-α3GT, was constructed as described previously (20Zhang Y. Wang P.G. Brew K. J. Biol. Chem. 2001; 276: 11567-11574Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). The mutant coding sequence was generated by amplification of pET15b-α3GT using a T7 promoter and the mutagenic primer, 5′­CGCGGATCC¯TCA GAC ATT ATTTTT¯AAC CAC ATT ATA CTC­3′BamHIR365KSequence 1 The amplification product was cleaved with Xba I and Bam HI and cloned into a pET42b vector that had been previously treated with the same enzymes. The mutant was characterized by automated DNA sequence analysis of the entire coding sequence. The enzyme was expressed as described for wild-type α3GT (20Zhang Y. Wang P.G. Brew K. J. Biol. Chem. 2001; 276: 11567-11574Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Steady state kinetic studies were carried out as described previously (20Zhang Y. Wang P.G. Brew K. J. Biol. Chem. 2001; 276: 11567-11574Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) using a radiochemical assay; enzyme activity was measured at varying concentrations of lactose (acceptor substrate) and a series of fixed concentrations of UDP-galactose, and the data were analyzed by fitting to the equation, v=Vm[A][B]/(KiaKb+Ka[B]+Kb[A]+[A][B])Eq. 1 using the Curvefitter program of SigmaPlot™. [A] and [B] represent the concentrations of UDP-galactose and lactose, respectively. The Arg-365 to Lys mutant was much less active than wild type enzyme and was assayed at a concentration of 92 μg/ml, as compared with 4.6 μg/ml for α3GT. Near and far UV CD spectra of the mutant enzyme were determined with a JASCO J-710/720 spectropolarimeter as described (20Zhang Y. Wang P.G. Brew K. J. Biol. Chem. 2001; 276: 11567-11574Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) using protein (0.5 mg/ml) dissolved in 20 mm Tris-HCl buffer, pH 7.4, containing 50% glycerol. The structures of the two α3GT monomers are virtually identical to one another with a root mean square deviation of 0.14 Å over all Cα atoms. Alternate conformations are found for Glu-145, Phe-184, Val-229, Glu-241, and Trp-249 in molecule A and Glu-145, Phe-184, Glu-241, and Glu-360 in molecule B. The overall structure of α3GT-form II (dimer) is similar to the previously reported structure of α3GT (form I, monomer) except at the catalytic site (Fig. 1,a and c). Briefly, the molecule exhibits a α/β fold and encompasses a central region with a "Rossmann fold" similar to those found in nucleotide binding domains (29Rossmann M.G. Moras D. Olsen K.W. Nature. 1974; 250: 194-199Crossref PubMed Scopus (1167) Google Scholar). The active site is identified as a deep tunnel inside the molecule based on the presence of a bound UDP molecule and a Mn2+ ion (Fig.2, a and b). It contains several regions of sequence that are conserved between different homologues of α3GT. If the C-terminal 10 residues are excluded, the form II structure (residues 82–358) superimposes closely with that of form I, exhibiting a root mean square deviation of 0.31 Å for backbone atoms and 0.92 Å for all atoms. However, the C-terminal region comprising residues 358–368, which is highly disordered in form I (18Gastinel L.N. Bignon C. Misra A.K. Hindsgaul O. Shaper J.H. Joziasse D.H. EMBO J. 2001; 20: 638-649Crossref PubMed Scopus (179) Google Scholar), has undergone a large positional change in form II. The mean root mean square deviation for the Cα atoms for the two structures in this segment is 15.2 Å with a maximum of 21 Å for Arg-365 and Asn-366. This stretch of the molecule is also highly ordered in form II as evidenced by the B-factors; in general, both molecules in form II are well ordered, reflecting the high resolution and much lower mobility for all residues (TableI). In form II, the C terminus forms a lid to the active site of the molecule (Fig. 1 c) so that the large change in structure between the two forms is associated with reduced active site accessibility.Figure 2a, schematic figure showing the main hydrogen bond interactions between UDP and α3GT residues at the catalytic site of the enzyme. The Mn2+ ion and water molecules are also shown. This image was created using the program MOLSCRIPT (38Kraulis P.J. J. Appl. Crystallogr. 1991; 24: 946-950Crossref Google Scholar) and rendered using Raster3D (41Merrit E.A. Bacon D.J. Methods Enzymol. 1997; 277: 505-524Crossref PubMed Scopus (3875) Google Scholar). b, the location of UDP molecule in the active site tunnel. Thisimage was created using the program DINO (A. Philippsen; available on the World Wide Web at www.dino3d.org).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The UDP binding domain is located between a twisted central β-sheet (β2–β5), two long α-helices (α3 and α4), and the C-terminal region of the molecule (Figs. 1 (a and b) and 2 (a and b)). The new structure shows highly ordered, clear binding of UDP molecule through the conserved DVD motif (9Wiggins C.A. Munro S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7945-7950Crossref PubMed Scopus (320) Google Scholar) and a Mn2+ ion. Some interactions of the UDP moiety with α3GT are similar to those in form I (Fig. 2, Table II) and involve residues that are located at the end of strands β2 and β5, helix α4, and the D225VD227 motif (Fig. 1 B). In addition, in the present structure (form II), the C-terminal region (β10 and α7) also constitutes part of the active site and makes direct interaction with the UDP phosphates (Fig.2, Table II) (see below). The C terminus (residues 358–368) that is shown by the present structure to be important for UDP binding is also highly conserved in all currently known α3GT amino acid sequences (30Shetterly S. Tom I. Yen T-Y. Joshi R. Lee L. Wang P.G. Macher B.A. Glycobiology. 2001; 11: 645-653Crossref PubMed Scopus (11) Google Scholar).Table IIHydrogen bonds and Van der Waals interactions between UDP, Mn2+, bound water molecules, and α3GT protein atomsInteracting atoms (hydrogen bond interactions)DistanceÅUracil O2Val-136 N2.82Uracil N3Val-136 O3.04Uracil O4Wat 1312.84Ribose O2*Phe-134 O2.63Ribose O2*Wat 12.75Ribose O3*Val-226 N3.07Ribose O3*Asp-227 OD12.96O1 P-αAsp-225 OD23.26O1 P-αAsp-227 OD22.95O1 P-αAsp-227 OD12.96O1 P-αMn2+2.24O2 P-αArg-365 NH22.83O2 P-αTyr-139 OH2.55O2 P-αWat 272.99O1 P-βMn2+2.11O1 P-βWat 7502.81O1 P-βLys-359 NZ3.13O1 P-βWat 1572.60O1 P-βWat 613.20O1 P-βAsp-225 OD23.29O2 P-βWat 2602.94O2 P-βWat 872.64O3 P-βTyr-361 OH2.61O3 P-βLys-359 NZ2.79O3 P-βWat 273.23Mn2+O1 P-α2.24Mn2+O1 P-β2.11Mn2+Asp-225 OD22.13Mn2+Wat 7502.16Mn2+Asp-227 OD12.18Mn2+Asp-227 OD22.39Interacting atoms (hydrophic interactions)aThese residues have several interatomic distances between 3 and 4.1 Å with the UDP atoms.Number of contactsVal-136>10Tyr-139>10Ile-198>10Arg-20210Asp-22510Val-2265Asp-2274Lys-3593Tyr-3613Arg-3653a These residues have several interatomic distances between 3 and 4.1 Å with the UDP atoms. Open table in a new tab Mn2+ is required for catalysis by many UDP-sugar-utilizing glycosyltransferases, and activity has been shown to be dependent on two metal ions in both β-4-galactosyltransferase-I (31Powell J.T. Brew K. J. Biol. Chem. 1976; 251: 3645-3652Abstract Full Text PDF PubMed Google Scholar) and α3GT (20Zhang Y. Wang P.G. Brew K. J. Biol. Chem. 2001; 276: 11567-11574Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). In the α3GT (both forms I and II) structure, one Mn2+ ion was identified. This Mn2+ forms an octahedral coordination through interactions with two oxygen atoms from the α- and β-phosphates of the UDP molecule, a single interaction with the OD2 atom of Asp-225, bidentate coordination with OD1 and OD2 atoms of Asp-227, and an interaction with a water molecule (Table II). The Mn2+ ion appears to stabilize the DVD sequence motif and binds the diphosphate moiety of the UDP molecule. Previous studies have shown that mutants of α3GT in which either aspartate of the motif is changed to asparagine have undetectable catalytic activity, in keeping with its key role in Mn2+ and donor substrate binding and catalysis (20Zhang Y. Wang P.G. Brew K. J. Biol. Chem. 2001; 276: 11567-11574Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). The location of the binding site for the second Mn2+ cofactor in our structure was not identified. The last 11 residues at the C terminus (residues 358–368) adopt distinct conformations in the form I and form II structures (Fig. 1 c). In the form I structure, this loop is disordered, with high B-factors of about 90 Å2, suggesting more than one conformation. In the present form II structure, residues 354–356 form a short β-strand, and residues 361–364 adopt a definite α-helical structure and are well defined in the electron density map. The conformational change observed in the present structure is associated with the formation of hydrogen bonds and van der Waals contacts between the α- and β-phosphates of UDP and Lys-359, Tyr-361, and Arg-365 and increased rigidity of all residues in this region (Fig. 2, Table II). The averageB-factors for the above contact residues are 15.0, 14.9, and 16.2 Å2, respectively, significantly less than the averageB-factor for the overall structure of 17.05 Å2. The conformational change in the C terminus between the two structures, the sequence conservation among other α3GT, and analogous transconformations in other glycosyltransferases (12Ünligil U.M. Rini J.M. Curr. Opin. Struct. Biol. 2000; 10: 510-517Crossref PubMed Scopus (344) Google Scholar) all suggest that this region could have a key role in the catalytic action. To test this, we constructed and characterized a mutant enzyme with a structurally conservative substitution, Lys for Arg-365, a residue in this region that is conserved in all homologues of α3GT. R365K was constructed as described and expressed as soluble protein in good yield (11 mg/liter of cell culture). Both far and near UV CD spectra of this mutant are closely similar to those of the wild type enzyme (data not shown), indicating that the mutation does not introduce any global conformational change. The kinetic parameters of this mutant determined at 10 mm Mn2+ are summarized in TableIII. Compared with wild type,k cat of the mutant is reduced 38-fold, while parameters associated with the binding of donor and acceptor substrate showed insignificant or minor changes. The catalytic efficiency (k cat/K ia ×K b) is also reduced 44-fold, reflecting the reduction in k cat; thus, this highly conservative substitution specifically reduces the stabilization of the transition state by the enzyme with essentially no effect on substrate binding in the α3GT·Mn2+·UDP-galactose·lactose complex.Table IIIEffects of the Arg-365 to Lys mutation on the catalytic properties of α3GTEnzymeα3GTaData for wild-type α3GT are taken from Zhang et al. (20).α3GTR365Kk cat(s−1)6.4 ± 0.70.17 ± 0.007K a(mm)bSubscript a denotes parameters for the donor substrate UDP-galactose, and subscriptb denotes parameters for the acceptor substrate, lactose.0.43 ± 0.070.72 ± 0.06K ia(mm)0.14 ± 0.030.14 ± 0.03K b (mm)19.9 ± 3.423.8 ± 1.9K ib (mm)6.5 ± 1.84.6 ± 1.1k cat/K ia*K b× 106 (s−1m−2)cThis parameter is the catalytic efficiency for a bisubstrate reaction.2.3 ± 0.70.052 ± 0.012a Data for wild-type α3GT are taken from Zhang et al. (20Zhang Y. Wang P.G. Brew K. J. Biol. Chem. 2001; 276: 11567-11574Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar).b Subscript a denotes parameters for the donor substrate UDP-galactose, and subscriptb denotes parameters for the acceptor substrate, lactose.c This parameter is the catalytic efficiency for a bisubstrate reaction. Open table in a new tab In the present structure, Arg-365 directly interacts with the α-phosphate of UDP and the OH atom of Tyr-139, which in turn interacts with the α-phosphate of UDP and makes stacking interactions against the uracil ring (Table II, Fig. 2). Furthermore, Arg-365 is involved in van der Waals interactions with UDP, Tyr-361, and Trp-195. Based on modeling studies, we predict that Arg-365 Lys substitution would not provide all of the interactions observed in the present structure. Among the 10 or so currently known glycosyltransferase structures, five, including bovine α3GT, are from eukaryotes: bovine β-4 galactosyltransferase (32Gastinel L.N. Cambillau C. Bourne Y. EMBO J. 1999; 18: 3546-3557Crossref PubMed Scopus (250) Google Scholar), the complex of mouse β-4-galactosyltransferase with mouse α-lactalbumin (19Ramakrishnan B. Qasba P.K. J. Mol. Biol. 2001; 310: 205-218Crossref PubMed Scopus (174) Google Scholar), human glucuronyltransferase (33Pederson L.C. Tsuchida K. Kitagawa H. Sugahara K. Darden T.A. Negishi M. J. Biol. Chem. 2000; 275: 34580-34585Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar), and rabbitN-acetylglucosaminyltransferase (11Ünligil U.M. Zhou S. Yuwaraj S. Sarkar M. Schachter H. Rini J.M. EMBO J. 2000; 19: 5269-5280Crossref PubMed Scopus (238) Google Scholar). Fig.3 shows a comparison of these and two relevant prokaryotic transferases, the retaining galactosyltransferase LgtC from Neisseria meningitidis (34Persson K. Ly H.D. Dieckelmann M. Wakarchuk W.W. Withers S.G. Strynadka N.C. Nat. Struct. Biol. 2001; 8: 166-175Crossref PubMed Scopus (313) Google Scholar) and nucleotide diphosphosugar transferase, SpsA, from Bacillus subtilis (35Charnock S.J. Davies G.J. Biochemistry. 1999; 38: 6380-6385Crossref PubMed Scopus (310) Google Scholar). These enzymes share a "Rossmann fold" topology in their UDP-binding region together with a metal-binding cluster of aspartates. Similarities have been noted in the mode of interaction with UDP (or UDP-sugar), particularly the interaction of the uridine moiety with the β-sheet and the phosphates with Mn2+ and the aspartate cluster (12Ünligil U.M. Rini J.M. Curr. Opin. Struct. Biol. 2000; 10: 510-517Crossref PubMed Scopus (344) Google Scholar). The presence of a contiguous flexible loop that undergoes a conformational change to a more rigid structure on UDP or donor substrate binding also appears to be a common feature (12Ünligil U.M. Rini J.M. Curr. Opin. Struct. Biol. 2000; 10: 510-517Crossref PubMed Scopus (344) Google Scholar), although the structure and location of this region differs in the various transferases. For example, in β-4 galactosyltransferase-I, a flexible loop interacts with UDP in an analogous fashion to that of α3GT upon binding to α-lactalbumin, the modulator protein of β-4 galactosyltransferase-I, in the presence of glucose (19Ramakrishnan B. Qasba P.K. J. Mol. Biol. 2001; 310: 205-218Crossref PubMed Scopus (174) Google Scholar). It is interesting to note that in α3GT and the only other structurally characterized retaining glycosyltransferase, LgtC, the UDP (UDP component of UDP- 2-F-galactose in LgtC) is buried in the enzyme complex. It has been suggested that in LgtC, the C-terminal loop would adopt an alternate conformation in the absence of UDP (34Persson K. Ly H.D. Dieckelmann M. Wakarchuk W.W. Withers S.G. Strynadka N.C. Nat. Struct. Biol. 2001; 8: 166-175Crossref PubMed Scopus (313) Google Scholar). This may reflect a need to protect a reactive intermediate in catalysis from solvent in retaining transferases. However, it has also been suggested that the flexible region in both retaining and inverting glycosyltransferases has a role in product release (12Ünligil U.M. Rini J.M. Curr. Opin. Struct. Biol. 2000; 10: 510-517Crossref PubMed Scopus (344) Google Scholar). The new crystal form of α3GT described here provides the structure of the UDP complex at far higher resolution than those reported previously for a UMP complex and a Hg-UDP-galactose complex (18Gastinel L.N. Bignon C. Misra A.K. Hindsgaul O. Shaper J.H. Joziasse D.H. EMBO J. 2001; 20: 638-649Crossref PubMed Scopus (179) Google Scholar); the more ordered structure is also highlighted by the much lower B-factors for all atoms in the structure. A major conformational change relative to the previous structures results in greater order in the C-terminal 10 residues and new enzyme-ligand contacts in this region. This may reflect, in part, the different uridine nucleotides present in the complex. Previous work suggests that the reaction mechanism of α3GT may be ordered with donor substrate binding preceding acceptor binding, but conclusive proof of this is lacking (see Zhang et al. (20Zhang Y. Wang P.G. Brew K. J. Biol. Chem. 2001; 276: 11567-11574Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) for a discussion). In this context, it is possible that the observed conformational change could be linked to the formation of the binding site for the acceptor substrate. Previously, Henion et al. (36Henion T.R. Macher B.A. Anaraki F. Galili U. Glycobiology. 1994; 4: 193-201Crossref PubMed Scopus (93) Google Scholar) found that deletion of as few as 3 amino acid residues from the C terminus of a primate α3GT results in complete loss of catalytic activity. This supports the view that the C terminus is crucial for catalysis, although the physical properties of the truncated enzyme were not characterized. Here we show that the substitution of lysine for the highly conserved Arg-365 does not affect substrate binding but specifically reduces the stability of the transition state in the reaction. This is distinct from the effects of a previously described mutation, Val-226 → Ala, within the D225VD227motif, which perturbs metal cofactor and UDP-galactose binding as well as catalytic efficiency (20Zhang Y. Wang P.G. Brew K. J. Biol. Chem. 2001; 276: 11567-11574Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar), in keeping with the role of this region in interacting with phosphates of UDP and metal ion. Additional work is needed to fully understand the role of the flexible C terminus in the reaction mechanism. The catalytic mechanism of α3GT is currently unknown. Although the formation of a glycosyl-enzyme intermediate is a plausible mechanism for a retaining glycosyltransferase, this type of mechanism is difficult to reconcile with steady state kinetic studies that indicate a sequential mechanism in which all substrates bind prior to catalysis (20Zhang Y. Wang P.G. Brew K. J. Biol. Chem. 2001; 276: 11567-11574Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Interestingly, structural and mutational studies of the LgtC also do not support the formation of a covalent β-galactosyl-enzyme or even a β-galactosyl-substrate intermediate (34Persson K. Ly H.D. Dieckelmann M. Wakarchuk W.W. Withers S.G. Strynadka N.C. Nat. Struct. Biol. 2001; 8: 166-175Crossref PubMed Scopus (313) Google Scholar). The structural evidence for a covalent intermediate in α3GT is not compelling, and an SN i mechanism, as suggested for LgtC, is possible in which nucleophilic attack by the acceptor substrate occurs simultaneously with UDP release and on the same side of the galactose ring (34Persson K. Ly H.D. Dieckelmann M. Wakarchuk W.W. Withers S.G. Strynadka N.C. Nat. Struct. Biol. 2001; 8: 166-175Crossref PubMed Scopus (313) Google Scholar). The present high resolution crystal form and structure provide a platform for further direct structural studies to establish the binding modes of donor and acceptor substrates that may help to unravel the details of the mechanism of action of α3GT.