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Crystal Structure and Catalytic Mechanism of PglD from Campylobacter jejuni

2008; Elsevier BV; Volume: 283; Issue: 41 Linguagem: Inglês

10.1074/jbc.m801207200

1083-351X

N.B. Olivier, Barbara Imperiali,

Porphyrin Metabolism and Disorders

The carbohydrate 2, 4-diacetamido-2, 4, 6-trideoxy-α-d-glucopyranose (BacAc2) is found in a variety of eubacterial pathogens. In Campylobacter jejuni, PglD acetylates the C4 amino group on UDP-2-acetamido-4-amino-2, 4, 6-trideoxy-α-d-glucopyranose (UDP-4-amino-sugar) to form UDP-BacAc2. Sequence analysis predicts PglD to be a member of the left-handed β helix family of enzymes. However, poor sequence homology between PglD and left-handed β helix enzymes with existing structural data precludes unambiguous identification of the active site. The co-crystal structures of PglD in the presence of citrate, acetyl coenzyme A, or the UDP-4-amino-sugar were solved. The biological assembly is a trimer with one active site formed between two protomers. Residues lining the active site were identified, and results from functional assays on alanine mutants suggest His-125 is critical for catalysis, whereas His-15 and His-134 are involved in substrate binding. These results are discussed in the context of implications for proteins homologous to PglD in other pathogens. The carbohydrate 2, 4-diacetamido-2, 4, 6-trideoxy-α-d-glucopyranose (BacAc2) is found in a variety of eubacterial pathogens. In Campylobacter jejuni, PglD acetylates the C4 amino group on UDP-2-acetamido-4-amino-2, 4, 6-trideoxy-α-d-glucopyranose (UDP-4-amino-sugar) to form UDP-BacAc2. Sequence analysis predicts PglD to be a member of the left-handed β helix family of enzymes. However, poor sequence homology between PglD and left-handed β helix enzymes with existing structural data precludes unambiguous identification of the active site. The co-crystal structures of PglD in the presence of citrate, acetyl coenzyme A, or the UDP-4-amino-sugar were solved. The biological assembly is a trimer with one active site formed between two protomers. Residues lining the active site were identified, and results from functional assays on alanine mutants suggest His-125 is critical for catalysis, whereas His-15 and His-134 are involved in substrate binding. These results are discussed in the context of implications for proteins homologous to PglD in other pathogens. N-Linked glycosylation involves the covalent attachment of a carbohydrate moiety to a protein at the amide nitrogen of an asparagine side chain in the consensus sequence Asn-Xaa-Ser/Thr (1Weerapana E. Imperiali B. Glycobiology. 2006; 16: 91-101Crossref PubMed Scopus (277) Google Scholar). Although the existence of archaeal glycoproteins was described more than 30 years ago (2Mescher M.F. Strominger J.L. J. Biol. Chem. 1976; 251: 2005-2014Abstract Full Text PDF PubMed Google Scholar), N-linked glycosylation was only recently discovered in Campylobacter jejuni (3Szymanski C.M. Yao R. Ewing C.P. Trust T.J. Guerry P. Mol. Microbiol. 1999; 32: 1022-1030Crossref PubMed Scopus (330) Google Scholar). This eubacterium is a Gram-negative pathogen known to be the leading cause of gastroenteritis in developed countries and has been identified as the most frequently occurring infection preceding Guillain-Barre syndrome and its variant Miller-Fisher syndrome (4Jacobs B.C. Rothbarth P.H. van der Meche F.G. Herbrink P. Schmitz P.I. de Klerk M.A. van Doorn P.A. Neurology. 1998; 51: 1110-1115Crossref PubMed Scopus (672) Google Scholar, 5Koga M. Gilbert M. Li J. Koike S. Takahashi M. Furukawa K. Hirata K. Yuki N. Neurology. 2005; 64: 1605-1611Crossref PubMed Scopus (177) Google Scholar, 6Rees J.H. Soudain S.E. Gregson N.A. Hughes R.A. N. Engl. J. Med. 1995; 333: 1374-1379Crossref PubMed Scopus (662) Google Scholar). The phenotypes for impaired N-linked glycosylation in C. jejuni are a reduction in natural transformability (7Larsen J.C. Szymanski C. Guerry P. J. Bacteriol. 2004; 186: 6508-6514Crossref PubMed Scopus (98) Google Scholar), reduced interaction with epithelial cells in vitro (3Szymanski C.M. Yao R. Ewing C.P. Trust T.J. Guerry P. Mol. Microbiol. 1999; 32: 1022-1030Crossref PubMed Scopus (330) Google Scholar), and reduced colonization in animals (3Szymanski C.M. Yao R. Ewing C.P. Trust T.J. Guerry P. Mol. Microbiol. 1999; 32: 1022-1030Crossref PubMed Scopus (330) Google Scholar, 8Hendrixson D.R. DiRita V.J. Mol. Microbiol. 2004; 52: 471-484Crossref PubMed Scopus (308) Google Scholar). In C. jejuni the first sugar of the heptasaccharide that is N-linked to proteins is BacAc 2The abbreviations used are: BacAc2, 2, 4-diacetamido-2, 4, 6-trideoxy-α-d-glucopyranose; AUC, analytical ultracentrifugation; Bicine, N, N-bis(2-hydroxyethyl)glycine; LβH, left-handed β helix; MES, 2-(N-morpholino)ethanesulfonic acid; pgl, protein glycosylation gene; UDP-4-amino-sugar, UDP-2-acetamido-4-amino-2, 4, 6-trideoxy-α-d-glucopyranose; MALDI, matrix-assisted laser desorption ionization time-of-flight; r.m.s.d., root mean square deviation; SeMet, selenomethionine. 2 (9Young N.M. Brisson J.R. Kelly J. Watson D.C. Tessier L. Lanthier P.H. Jarrell H.C. Cadotte N. St. Michael F. Aberg E. Szymanski C.M. J. Biol. Chem. 2002; 277: 42530-42539Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar), a diacetylated form of N-acetylbacillosamine (10Zehavi U. Sharon N. J. Biol. Chem. 1973; 248: 433-438Abstract Full Text PDF PubMed Google Scholar). BacAc2 is initially synthesized as a uridine diphosphate (UDP) derivative. Gene products of the C. jejuni protein glycosylation (pgl) locus form the N-linked heptasaccharide biosynthetic pathway (3Szymanski C.M. Yao R. Ewing C.P. Trust T.J. Guerry P. Mol. Microbiol. 1999; 32: 1022-1030Crossref PubMed Scopus (330) Google Scholar), and knockouts of any enzyme involved in the biosynthesis of UDP-BacAc2 disrupts formation of the heptasaccharide (11Kelly J. Jarrell H. Millar L. Tessier L. Fiori L.M. Lau P.C. Allan B. Szymanski C.M. J. Bacteriol. 2006; 188: 2427-2434Crossref PubMed Scopus (111) Google Scholar). The biosynthesis of UDP-BacAc2 occurs by the sequential modification of UDP-N-acetylglucosamine (GlcNAc) at the C6 and C4 positions (Fig. 1). Initially, PglF, a membrane-bound protein and member of the short-chain dehydrogenase family of enzymes, conducts an NAD+-dependent dehydration at C6 to form UDP-2-acetamido-2, 6-dideoxy-α-d-xylo-hexulose (12Schoenhofen I.C. McNally D.J. Vinogradov E. Whitfield D. Young N.M. Dick S. Wakarchuk W.W. Brisson J.R. Logan S.M. J. Biol. Chem. 2006; 281: 723-732Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). The dehydration step results in formation of a ketone at C4, where PglE conducts a pyridoxal-5′-phosphate-dependent transamination using l-glutamate as the source of the transferred amine to form the UDP-4-amino-sugar (12Schoenhofen I.C. McNally D.J. Vinogradov E. Whitfield D. Young N.M. Dick S. Wakarchuk W.W. Brisson J.R. Logan S.M. J. Biol. Chem. 2006; 281: 723-732Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). PglD, the focus of this study, then transfers an acetyl group from acetyl coenzyme A (AcCoA) to the C4 amine to form UDP-BacAc2 (13Olivier N.B. Chen M.M. Behr J.R. Imperiali B. Biochemistry. 2006; 45: 13659-13669Crossref PubMed Scopus (81) Google Scholar). Bioinformatic analysis shows that PglD contains a series of imperfect tandem repeats collectively known as a hexapeptide repeat motif (14Dicker I.B. Seetharam S. Mol. Microbiol. 1992; 6: 817-823Crossref PubMed Scopus (38) Google Scholar, 15Vaara M. FEMS Microbiol. Lett. 1992; 76: 249-254Crossref PubMed Google Scholar). The pattern of the repeated unit generally conforms to the sequence (LIV)1, (GAED)2, X3, X4, (STAV)5, and X6. The first crystal structure solved of a protein containing this signature sequence was that of UDP-N-acetylglucosamine acetyltransferase (16Raetz C.R. Roderick S.L. Science. 1995; 270: 997-1000Crossref PubMed Scopus (299) Google Scholar). The tertiary structure formed by residues in the hexapeptide repeat was shown to be a left-handed β helix. LβH enzymes that have been characterized biochemically are known to acylate substrates such as UDP-GlcNAc (17Anderson M.S. Bull H.G. Galloway S.M. Kelly T.M. Mohan S. Radika K. Raetz C.R. J. Biol. Chem. 1993; 268: 19858-19865Abstract Full Text PDF PubMed Google Scholar), galactosides (18Lewendon A. Ellis J. Shaw W.V. J. Biol. Chem. 1995; 270: 26326-26331Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar), and antibiotics (19Rende-Fournier R. Leclercq R. Galimand M. Duval J. Courvalin P. Antimicrob. Agents Chemother. 1993; 37: 2119-2125Crossref PubMed Scopus (116) Google Scholar, 20Tian Y. Beaman T.W. Roderick S.L. Proteins. 1997; 28: 298-300Crossref PubMed Scopus (3) Google Scholar). The proposed mechanism of catalysis for LβH enzymes has been summarized in a review by Field and Naismith (21Field R.A. Naismith J.H. Biochemistry. 2003; 42: 7637-7647Crossref PubMed Scopus (29) Google Scholar). Briefly, the substrate is activated by abstraction of a proton from either a hydroxyl group or a protonated primary amine by a side-chain functional group, usually the imidazole of histidine. The activated substrate then conducts a nucleophilic attack on the acetyl group of AcCoA, forming a tetrahedral intermediate which is followed by the subsequent release of the deacetylated coenzyme and product. Sequence alignments of PglD and other LβH proteins with existing crystal structures share ∼25% sequence similarity that is primarily localized to the LβH domain (data not shown). Sequences that align more favorably with the full-length PglD are proteins from a variety of pathogenic and non-pathogenic organisms (supplemental Fig. S1). Noteworthy among these are homologs found in the Neisseria bacterial species. PglB, a bifunctional transmembrane protein, has been implicated in the biosynthesis of a diacetyl-trideoxy hexose found O-linked to pilin from Neisseria gonorrhoeae and Neisseria meningitides (22Aas F.E. Vik A. Vedde J. Koomey M. Egge-Jacobsen W. Mol. Microbiol. 2007; 65: 607-624Crossref PubMed Scopus (101) Google Scholar, 23Hegge F.T. Hitchen P.G. Aas F.E. Kristiansen H. Lovold C. Egge-Jacobsen W. Panico M. Leong W.Y. Bull V. Virji M. Morris H.R. Dell A. Koomey M. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 10798-10803Crossref PubMed Scopus (110) Google Scholar). Another PglD homolog is NeuD from Mannheimia hemolytica and Streptococcus agalactiae. In S. agalactiae this protein is known to O-acetylate sialic acid and is required for capsular polysaccharide sialylation (24Lewis A.L. Hensler M.E. Varki A. Nizet V. J. Biol. Chem. 2006; 281: 11186-11192Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). There have been several recent advances toward determining the three-dimensional structure of PglD. A crystal structure of PglD in the apo state has been solved by the Protein Structure Initiative. This structure has been made publicly available from the Protein Data Bank (www.rcsb.org) under the identifier 2NPO (Fig. 2). Although analysis of the structure by the authors is pending publication, it can be seen that the crystal structure is unique and non-redundant, thus providing a valuable representation of other sequences. More recently, two other crystal structures of PglD were solved, one in the presence of citrate (3BFP) and the other in the presence of coenzyme A (2VHE) (25Rangarajan E.S. Ruane K.M. Sulea T. Watson D.C. Proteau A. Leclerc S. Cygler M. Matte A. Young N.M. Biochemistry. 2008; 47: 1827-1836Crossref PubMed Scopus (29) Google Scholar). Although lacking the critical acetyl group on coenzyme A, the authors combined structural analysis with functional assays on site-directed mutants to identify several residues lining the active site as important for catalysis. Moreover, computational and molecular modeling efforts were incorporated into the study resulting in a proposal for the mechanism of catalysis and a mode for binding of the sugar substrate to protein. Although previously modeled by computational methods, a crystal structure of the native sugar substrate would provide physical evidence for describing the mode of substrate binding. Furthermore, structural data that present the active site in dissimilar chemical environments may aid in understanding the function of specific residues during catalysis. In this report we describe the results of biophysical and biochemical studies designed to further elucidate the catalytic mechanism of PglD. The co-crystal structures of PglD in the presence of citrate, AcCoA, or the UDP-4-amino-sugar have been solved. Each structure shows a chemical environment in the active site that is distinct from that in the previously published crystal structures. Using sedimentation velocity AUC, we also show that PglD self-associates as a homotrimer in solution. Comparison of the structures reveals that the extreme C-terminal portion of the protein undergoes a coenzyme-dependent cis-trans amide bond isomerization between Val-190 and Pro-191, resulting in an interchange of coils between protomers in the biological assembly. Combining the results from structure and function experiments, we propose a detailed mechanism of catalysis for PglD in the formation of UDP-BacAc2 and discuss some of the implications for homologs in other pathogenic organisms. Molecular Biology—The pglD gene was amplified from genomic DNA (ATCC 700819, designation NCTC 11168) as described elsewhere (13Olivier N.B. Chen M.M. Behr J.R. Imperiali B. Biochemistry. 2006; 45: 13659-13669Crossref PubMed Scopus (81) Google Scholar). The amplicon encoding the fulllength protein was engineered with the restriction sites NcoI and XhoI, then subcloned into the pETGQ vector (26Chen G.Q. Gouaux E. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13431-13436Crossref PubMed Scopus (146) Google Scholar). This vector was used to express constructs with a thrombin-cleavable octahistidine tag at the N terminus. Site-directed mutagenesis was accomplished using the QuikChange site-directed mutagenesis protocol from Stratagene. Protein Expression and Purification—Heterologous expression was accomplished using the Escherichia coli BL-21 (DE3) strain (Stratagene). Cells were transformed with pETGQ-construct plasmids and grown to an A600 of ∼0.6 absorbance units at 37 °C in Luria-Bertani broth; the cultures were cooled to ∼16 °C and then induced by the addition of 0.5 mm isopropyl-β-d-thiogalactopyranoside. Incorporation of selenomethionine was accomplished following a protocol described elsewhere (27Guerrero S.A. Hecht H.J. Hofmann B. Biebl H. Singh M. Appl. Microbiol. Biotechnol. 2001; 56: 718-723Crossref PubMed Scopus (85) Google Scholar). Twenty hours after induction the cells were harvested by centrifugation and resuspended in ice-cold buffer composed of 50 mm HEPES, 10 mm imidazole, 150 mm NaCl, pH 7.1, at 1/20 the original culture volume. Maintaining a working temperature of 4 °C, the cells were lysed by sonication, and the lysate was cleared by centrifugation in a Type 45 Ti rotor (Beckman/Coulter) at 35,000 rpm. The construct was bound to nickel-nitrilotriacetic acid (Qiagen) in batch using 1 ml of resin per liter of culture, overnight with gentle tumbling. The protein-bound resin was washed with 25 column volumes of lysis buffer containing 60 mm imidazole, and the protein was eluted in lysis buffer containing 250 mm imidazole. The octahistidine tag was removed by thrombinolysis, and after the digest reached completion, the reaction was diluted 10-fold with a buffer composed of 20 mm HEPES at pH 7.1. This solution was loaded onto an SP-Sepharose cation exchange column (GE Healthcare), and the protein eluted with a linear NaCl gradient. Fractions containing the isolated protein were pooled and concentrated to 5 mg/ml for further purification by size exclusion chromatography using a Superdex 200 XK16-60 column (GE Healthcare) in a running buffer of 20 mm HEPES, 150 mm NaCl, pH 7.1. Fractions containing monodispersed material were pooled, and this sample was used for AUC, crystallization, and function assays. MALDI-mass spectrometry was used to verify the molecular mass of purified material and detect incorporation of selenomethionine (data not shown). Sedimentation Velocity AUC—Experiments were conducted in an Optima XL-I ultracentrifuge (Beckman/Coulter) using an An60 Ti four-hole rotor at the Boston Biomedical Research Institute (Watertown, MA). Each experiment was conducted with the temperature in the centrifugation chamber at 37 °C and a rotor speed of 50,000 rpm. The centrifuge was retrofitted with a turbo diffusion pump, circumventing contamination of the optics with oil from the conventional diffusion pump when conducting experiments at temperatures above 25 °C. Data were acquired with the interference optics system using sapphire windows. Each cell assembly was composed of 12-mm double-sector Epon centerpieces with interference slit window holders (Biomolecular Interaction Technologies Center). Samples were dialyzed for 24 h in the gel-filtration running buffer before the experiment. Three sample cells were loaded, with each having a different concentration of PglD (70 ± 1, 25 ± 1, and 6 ± 1 μm) and analyzed in the centrifuge simultaneously. Data were analyzed with the software package SEDANAL (28Stafford W.F. Sherwood P.J. Biophys. Chem. 2004; 108: 231-243Crossref PubMed Scopus (226) Google Scholar), and the program SEDNTERP was used to estimate the partial specific volume of the protein and density of the solvent. Crystallization and Data Collection—Protein solutions with UDP-4-amino-sugar or AcCoA were made such that the final concentration of the added substrate was 5 mm and were then incubated on ice for 1 h. The UDP-4-amino-sugar was enzymatically synthesized in vitro using the method previously described (13Olivier N.B. Chen M.M. Behr J.R. Imperiali B. Biochemistry. 2006; 45: 13659-13669Crossref PubMed Scopus (81) Google Scholar). The protein solution was concentrated and diluted three times with the filtrate using Amicon 10,000 Mr cut-off concentrators. Before setting up trays with sitting or hanging drops, the protein was concentrated to 10 mg/ml. The crystallization drops were formed by mixing 1.5 μl of protein solution with 1.5 μl of reservoir solution. All crystals except for the SeMet derivative were grown with a reservoir solution of 20% polyethylene glycol 1000, 100 mm phosphate-citrate, 200 mm Li2SO4, pH 4.2. The SeMet derivative crystal grew with a reservoir solution containing 1.0 m sodium citrate and 100 mm imidazole, pH 8.0. Crystals were cryoprotected in a reservoir solution supplemented with 20% glycerol and 5 mm substrate as necessary. Intensity data were collected at 110K on beamline X6A (National Synchrotron Light Source, Brookhaven National Laboratory, Upton, NY) as summarized in Table 1. All data sets were indexed, integrated, and scaled using the HKL2000 software suite of programs, and the scaled intensities were converted to structure factors using the program TRUNCATE (29French S. Wilson K. Acta Crystallogr. Sect. A. 1978; 34: 517-525Crossref Scopus (909) Google Scholar).TABLE 1Data collection and refinement statisticsUDP-4-aminoAcCoACitrateSeMet derivativeData collectionSpace groupP4332P212121P63P63Unit cell dimension, a, b, c (Å)162, 162, 16256, 92, 12586, 86, 6586, 86, 65Resolution (Å)30-2.3050-1.8030-1.7730-2.2Rmerge (%)aThe number in parentheses represents the highest resolution bin; 2.38-2.30 Å, 1.86-1.80 Å, 1.83-1.77 Å, and 2.28-2.20 Å for the data sets of UDP-4amino, AcCoA, citrate, and the SeMet derivative, respectively.,bRmerge = ∑|I - |/∑I, where I is the intensity of a reflection, and is the mean intensity of a group of equivalent reflections.11.0 (60.7)6.3 (36.1)6.2 (57.1)13.6 (61.8)I/σIbRmerge = ∑|I - |/∑I, where I is the intensity of a reflection, and is the mean intensity of a group of equivalent reflections.29.4 (4.5)32.0 (5.0)27.7 (2.9)12.2 (2.6)Completeness (%)bRmerge = ∑|I - |/∑I, where I is the intensity of a reflection, and is the mean intensity of a group of equivalent reflections.100 (100)99.9 (99.9)99.7 (99.6)100 (100)RedundancybRmerge = ∑|I - |/∑I, where I is the intensity of a reflection, and is the mean intensity of a group of equivalent reflections.30.2 (30.8)8.0 (7.5)5.7 (5.7)5.6 (5.6)Riso (%)bRmerge = ∑|I - |/∑I, where I is the intensity of a reflection, and is the mean intensity of a group of equivalent reflections.12.0RefinementResolution (Å)30-2.3030-1.8030-1.77Unique reflections31,26556,91325,746Rwork/Rfree (%)dRwork = ∑h||F(h)obs| - |F(h)calc| /∑h|F(h)obs|.,eRfree was calculated for 5% of reflections randomly excluded from the refinement.17.8/19.118.4/22.018.3/19.9No, atoms1,6875,0061,616Protein1,4474,2261,397Organic3815313Water202627206B-factors (Å2)Overall28.619.625.9Protein27.017.022.9Organic23.725.721.4Water41.436.346.4r.m.s.d.Bond lengths (Å)0.0130.0100.010Bond angles (°)1.3831.6201.189Ramachandran plot, %fRamachandran plot statistics are given as core/allowed/generously allowed and are for all chains.85.1/14.9/0.086.2/13.6/0.289.0/11.0/0.0PDB code3BSS3BSY3BSWc Riso = ∑(|FPH - FP|)/∑|FP|, the mean fractional isomorphous change between the native amplitudes (FP) and the amplitudes from the SeMet derivative data set (FPH).a The number in parentheses represents the highest resolution bin; 2.38-2.30 Å, 1.86-1.80 Å, 1.83-1.77 Å, and 2.28-2.20 Å for the data sets of UDP-4amino, AcCoA, citrate, and the SeMet derivative, respectively.b Rmerge = ∑|I - |/∑I, where I is the intensity of a reflection, and is the mean intensity of a group of equivalent reflections.d Rwork = ∑h||F(h)obs| - |F(h)calc| /∑h|F(h)obs|.e Rfree was calculated for 5% of reflections randomly excluded from the refinement.f Ramachandran plot statistics are given as core/allowed/generously allowed and are for all chains. Open table in a new tab c Riso = ∑(|FPH - FP|)/∑|FP|, the mean fractional isomorphous change between the native amplitudes (FP) and the amplitudes from the SeMet derivative data set (FPH). Structure Determination and Refinement—The citrate-bound structure was solved by the method of single isomorphous replacement with anomalous scattering. Heavy atom sites in the substructure were identified using SHELX-D against data collected at the selenium peak wavelength and truncated to 2.5 Å. Three of five possible selenium sites for a single molecule of PglD in the asymmetric unit were located. The correlation coefficients for all/weak reflections were 17.2/11.1, and the Patterson figure of merit was 18.3. Structure factors from the native data were merged with initial phases using CAD, phase extension to 1.77 Å, and density modification was carried out using SHELX-E. Values for contrast, connectivity, mean mapCC, and pseudo-free CC were 1.1, 96, 94, and 80%, respectively. The initial model was built with ARP/wARP (30Perrakis A. Harkiolaki M. Wilson K.S. Lamzin V.S. Acta Crystallogr. D Biol. Crystallogr. 2001; 57: 1445-1450Crossref PubMed Scopus (462) Google Scholar), fitting 190 of 198 residues in the construct sequence using the automated tracing function. The structures of UDP-4-amino-sugar-bound and AcCoA-bound protein were solved by molecular replacement using the software programs PHASER and MOLREP, respectively. In each case the search model was that of the citrate-bound structure, omitting the citrate molecule (3BSW). Refinement and model building were performed using Refmac (31Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (14025) Google Scholar), COOT (32Emsley P. Cowtan K. Acta Crystallogr. D Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (24326) Google Scholar), and O (33Jones T.A. Zou J.-Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13055) Google Scholar) with the assistance of 2Fo - Fc and Fo - Fc maps in addition to simulated-annealing Fo - Fc omit maps generated with CNS (34Brunger 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. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (17024) Google Scholar). Five percent of the data were used to calculate the Rfree factor for cross-validation of the refinement process (35Brunger A.T. Acta Crystallogr. D Biol. Crystallogr. 1993; 49: 24-36Crossref PubMed Google Scholar). For each structure, ligand atoms were modeled after the Rfree was below 30%, and water molecules were added gradually using COOT and ARP/wARP. Side chain alternate conformations were modeled during the final stages of refinement. Additional crystallographic calculations, including LSQMAN and PROCHECK, were employed from the CCP4 suite (36Collaborative Computational Project N. Acta Crystallogr. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19879) Google Scholar). The final refinement statistics are presented in Table 1. Capillary Electrophoresis Analysis—Capillary electrophoresis was performed using a Beckman/Coulter Proteome 800 or P/ACE MDQ cE system with detection at 254 nm, and manual integration was performed using the Beckman/Coulter 32 Karat, Version 8.0 software package. The running buffer was composed of 25 mm sodium tetraborate, pH 9.4, using a bare silica capillary (75 μm inner diameter × 80 cm) with a detector distance of 72 cm. The capillary was conditioned before each run with 1.0 m NaOH, water, and then buffer. The conditioning solutions were applied using 25 p.s.i. for 3 min. Samples were prepared by passing through a 10,000 Mr weight cut-off membrane, and the filtrate was diluted with water at a ratio of 1:2. Samples were introduced to the capillary by pressure injection for 15 s at 0.5 p.s.i., and separation was performed at 25 kV in negative mode. Kinetic Data Measurement and Analysis—Histidine mutant reactions were conducted in size exclusion chromatography buffer supplemented with 6 μg of bovine serum albumin to serve as a carrier protein and 2 mm AcCoA while varying the concentration of the UDP-4-amino-sugar in a volume of 30 μl. Protein quantities per reaction per mutant construct are provided in Table 2. pH-dependent reactions contained 50-150 pg of enzyme and 6 μg of bovine serum albumin. Reactions in the range of pH 6.5-9.0 were conducted in duplicate with a three-component buffer system consisting of 20 mm MES (pKa 6.2), 20 mm HEPES (pKa 7.6), 20 mm BICINE (pKa 8.3), and 150 mm NaCl. All reaction mixtures were incubated for 20 min at 37 °C, boiled for 2 min, and then filtered through a 10,000 Mr cut-off membrane and analyzed by capillary electrophoresis as described above. Kinetic parameters were determined by fitting the initial reaction rates to the Michaelis-Menten equation for one substrate using the program SigmaPlot Version 9.0. kcat/Km was obtained from the fitted values of Vmax and Km with propagation of the standard errors. These pH-dependent values were fitted to Equation 1, where Y is the observed kcat/Km,[H+] is the proton concentration of the solution, Ka and pKb are the dissociation constants for ionization of groups that ionize at low and high pH, respectively, and C is the pH-independent value.TABLE 2Kinetic parameters for native and point mutants of pgIDConstructProteinKmkcatkcat/KmVmaxmg/assaymms−1s−1 mm−1nmol/s/mgNative2.5 × 10−81.0 ± 0.13143141.47 × 104H15A5.0 × 10−72.5 ± 0.3138556.46 × 103H125A6.5 × 10−41.0 ± 0.29.1 × 10−29.1 × 10−24.25H134A2.0 × 10−60.8 ± 0.210134.71 × 102 Open table in a new tab Self-association in Solution—Proteins belonging to the LβH superfamily of enzymes are generally expected to form trimers (16Raetz C.R. Roderick S.L. Science. 1995; 270: 997-1000Crossref PubMed Scopus (299) Google Scholar). To investigate the possibility for self-association by PglD, we analyzed a purified protein sample using sedimentation velocity AUC at 37 °C in the absence of exogenous coenzyme or sugar substrate. The sedimentation coefficient of PglD was found to be 5.80 ± 0.02 Svedberg units and showed no concentration dependence to the sedimentation coefficient over a protein concentration range of 6-70 μm (supplemental Fig. S2A). Sample concentrations were calculated using the F-statistics function in SEDANAL (28Stafford W.F. Sherwood P.J. Biophys. Chem. 2004; 108: 231-243Crossref PubMed Scopus (226) Google Scholar); the S.D. of the fit was 0.006 fringes using the Levenberg-Marquardt fitting method. A single species model fit using a 95% confidence interval resulted in a molecular mass of 66 ± 3 kDa (supplemental Fig. S2B). The molecular weight of the construct analyzed was 21.4 kDa, calculated by sequence and verified by MALDI-mass spectrometry (data not shown), which suggests that PglD associates as a homotrimer in solution at 37 °C. Citrate-bound Structure—The co-crystal structure of PglD in the presence of citrate was solved by single isomorphous replacement with anomalous scattering using native data combined with data collected from an isomorphous selenomethionine derivative protein crystal. Statistics for data collection and structure refinement are presented in Table 1. The citrate-bound and 2NPO structures were both solved in the hexagonal space group P63. A single protomer of PglD may be defined as having three sections: the N terminus (Met-1—Asn77), the left-handed β-helix (Leu-78—Gly-185), and the C-terminal coenzyme gate (Val-186—Met-195) (Fig. 2). Superposition of the Cα carbons from 2NPO and the citrate-bound model resulted in a r.m.s.d. of 0.23 Å. The similarity of the citrate-bound model to the apo-state model suggests that the citrate-bound form also represents the apo state. Although the asymmetric unit in both structures contains a single protomer of PglD, the homotrimer is observed centered on a crystallographic 3-fold axis. As seen in the structures of several other proteins that contain a hexapeptide repeat motif (37Beaman T.W. Binder D.A.