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Originally published In Press as doi:10.1074/jbc.M703044200 on May 22, 2007

J. Biol. Chem., Vol. 282, Issue 30, 22217-22227, July 27, 2007
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Biochemical Characterization of Thepolysialic Acid-specific O-Acetyltransferase NeuO of Escherichia coli K1*Formula

Anne K. Bergfeld{ddagger}, Heike Claus§, Ulrich Vogel§, and Martina Mühlenhoff{ddagger}1

From the {ddagger}Department of Cellular Chemistry, Medical School Hannover, Carl-Neuberg-Strasse 1, Hannover 30625 and the Institute for Hygiene and Microbiology, §University of Würzburg, Josef-Schneider-Str. 2, 97080 Würzburg, Germany

Received for publication, April 11, 2007 , and in revised form, May 14, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Escherichia coli K1 is a leading pathogen in neonatal sepsis and meningitis. The K1 capsule, composed of {alpha}2,8-linked polysialic acid, represents the major virulence factor. In some K1 strains, phase-variable O-acetylation of the capsular polysaccharide is observed, a modification that is catalyzed by the prophage-encoded O-acetyltransferase NeuO. Phase variation is mediated by changes in the number of heptanucleotide repeats within the 5'-coding region of neuO, and full-length translation is restricted to repeat numbers that are a multiple of three. To understand the biochemical basis of K1 capsule O-acetylation, NeuO encoded by alleles containing 0, 12, 24, and 36 repeats was expressed and purified to homogeneity via a C-terminal hexahistidine tag. All NeuO variants assembled into hexamers and were enzymatically active with a high substrate specificity toward polysialic acid with >14 residues. Remarkably, the catalytic efficiency (kcat/Kmdonor) increased linearly with increasing numbers of repeats, revealing a new mechanism for modulating NeuO activity. Using homology modeling, we predicted a three-dimensional structure primarily composed of a left-handed parallel beta-helix with one protruding loop. Two amino acids critical for catalytic activity were identified and corresponding alanine substitutions, H119A and W143A, resulted in a complete loss of activity without affecting the oligomerization state. Our results indicate that in NeuO typical features of an acetyltransferase of the left-handed beta-helix family are combined with a unique regulatory mechanism based on variable N-terminal protein extensions formed by tandem copies of an RLKTQDS heptad.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Escherichia coli K1 is one of the main organisms causing bacterial sepsis and meningitis during the neonatal period (1-3). The pathogenesis of E. coli meningitis is a complex process involving colonization of the gastrointestinal tract, intestinal translocation, bacteremia, passage of the blood-brain barrier, and invasion of the arachnoidal space (4). Cell culture experiments indicated that transfer across the blood-brain barrier is mediated by transcytosis through brain endothelial cells (5, 6) and that, in contrast to other bacterial agents causing meningitis, E. coli K1 invades meningioma cells directly, leading to rapid cell death before an inflammatory response can be induced (7). Despite advances in diagnostics and therapeutics of neonatal infections, E. coli neonatal meningitis is still characterized by high rates of mortality and a high incidence of permanent neurological sequelae in those that survive (8, 9).

E. coli K1 is protected by a thick layer of capsular polysaccharide, which is the major virulence factor important for serum resistance and vital passage of the blood-brain barrier (5, 10, 11). The K1 capsule consists of polysialic acid (polySia)2 formed by the most prevalent sialic acid of humans, 5-N-acetyl neuraminic acid (Neu5Ac). Up to 200 residues are joined in {alpha}2,8-glycosidic linkages, resulting in a linear homopolymer that is structurally identical to polySia of the host organism (12, 13). Due to this antigenic mimicry, the K1 capsule is poorly immunogenic, and no effective polysaccharide-based vaccines are available.

In several K1 strains, modification of the capsule by O-acetylation of the Neu5Ac residues in positions O-7 and O-9 was observed (14-16). This modification is phase-variable, and individual strains can switch between O-acetylated (OAc+) and non-O-acetylated (OAc-) capsule variants (14). Although associated with increased immunogenicity, modification of the K1 capsule by O-acetylation correlates with increased virulence in patients with bacteremia (17). Because O-acetylated polySia resists hydrolysis by neuraminidases, capsule O-acetylation may favor colonization of the intestinal tract (14).

O-Acetylation of the K1 capsule is catalyzed by an acetyl-CoA-dependent O-acetyltransferase with preferential acceptor specificity toward polySia with >14 residues (15). The corresponding gene (neuO) is part of a 40-kb prophage of the P22 family, and phase variation is mediated by changes in the overall length of variable number of tandem repeats (VNTRs) within the 5'-coding region (16, 18). VNTR loci are among the most variable regions of many bacterial genomes and arise through slippage and mispairing during DNA replication due to occasional DNA polymerase dissociation (19, 20). In the neuO-positive K1 strains reported so far, 14-39 copies of the heptanucleotide unit 5'-AAGACTC-3' were inserted two nucleotides downstream of the start codon (16). Only repeat numbers that are a multiple of three allow full-length translation of neuO (phase on), whereas other numbers move the start codon out of frame resulting in truncated and thereby inactive translation products (phase off). In the case of full-length translation, the presence of VNTRs will lead to significant N-terminal protein extensions with every three tandem heptanucleotides encoding an RLKTQDS heptad. Comparison of 10 different OAc+ K1 strains indicated that VNTR-encoded protein extensions may affect NeuO activity (16). However, direct analysis of the enzymatic activity in bacterial lysates is hampered by the high frequency of phase variation, and thus far, isolation of endogenous NeuO failed (15). In the present study, we succeeded in purification of active recombinant enzyme, forming the basis for a detailed biochemical characterization of NeuO. Functional and structural analyses of variants encoded by neuO alleles with 0, 12, 24, and 36 heptanucleotide repeats revealed that NeuO shares the typical features of an acyltransferase of the left-handed-beta-helix (LbetaH) superfamily and assembles into hexamers that might be formed by dimerization of two trimers. In contrast to other members of the LbetaH family, NeuO activity is regulated by changes in the number of N-terminal RLKTQDS heptads and the catalytic efficiency gradually increases with the length of this VNTR-encoded protein extension.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—E. coli K1 strains A160 and C375 were kindly provided by M. Achtman and F. Ørskov, respectively. pET expression vectors and E. coli BL21(DE3) were obtained from Novagen. CMP-Neu5Ac, Neu5Ac, colominic acid, 5,5'-dithiobis(2-nitrobenzoic acid) (Ellman's reagent), acetyl-, propionyl-, and butyryl-CoA were purchased from Sigma. Isolated sialic acid dimers, trimers, tetramers, pentamers, and hexamers were obtained from Nacalai-Tesque (Tokyo, Japan).

Cloning of the Capsule-specific O-Acetyltransferase of E. coli K1—In parallel to Deszo et al. (16), we identified the capsule-specific O-acetyltransferase of E. coli K1 in the unfinished genome of strain RS218 (serotype O18ac:H7:K1, University of Wisconsin) by screening for homologies to the amino acid sequence of the sialic acid O-acetyltransferase of serogroup W-135 and Y meningococci (oatWY, accession number Y13969). A corresponding DNA sequence of the identified gene was amplified by PCR from genomic DNA of the O-acetylation-positive K1 strain C375. The gene was termed oatK1, and the sequence was submitted to the DDBJ/EMBL/GenBankTM data-bases under the accession number AJ783705. DNA sequencing revealed the presence of 18 heptanucleotide repeats and that the coding region is identical to neuO published by Deszo et al. (16).

Homology Modeling—The structural fold of NeuO was predicted by the program 3D-PSSM (21), a profile-based method that relies on multiple sequence and multiple structural alignments. Highest scores were obtained for the structural model based on homology to the E. coli maltose O-acetyltransferase (PDB accession number 1ocx), and all structural data were visualized by the PyMOL Molecular Graphics System.

Generation of NeuO Expression Plasmids—Constructs for the expression of wild-type NeuO were constructed in pET22b-Strep. In this modified pET22b vector, the original pelB leader sequence was exchanged by the sequence encoding an N-terminal Strep tag II (WSHPQFEK). NeuO without heptanucleotide repeats was amplified by PCR using genomic E. coli K1 DNA as a template and the primer pair MM275/MM282 (5'-CGGGATCCATGTCGTTTTCCGTTGATG-3',5'-GTCCGCTCGAGTTGCGTGAGCTTCGCATG-3'). BamHI and XhoI sites (underlined) in forward and reverse primers, respectively, were used for subcloning of the PCR products into the BamHI/XhoI sites of pET22b-Strep, resulting in constructs with an N-terminal Strep tag II and a C-terminal hexahistidine tag. NeuO variants with heptanucleotide repeats were amplified by PCR using the primer pair MM301 (5'-CGGGATCCATGTTAAGACTCAAGACTC-3' and MM282 and genomic DNA of an E. coli K1 strain harboring an neuO gene with 36 heptanucleotide repeats as a template. The forward primer MM301 covers two heptanucleotide repeats and, therefore, can bind randomly within the repeat stretch resulting in a wide variety of PCR products containing 2-36 heptanucleotide repeats. PCR products of different length were subcloned by BamHI/XhoI sites into pET22b-Strep as described above. Constructs containing 12, 24, and 36 repeats were selected, and the number of heptanucleotide repeats was confirmed by sequencing.

Site-directed Mutagenesis—Site-directed mutagenesis was performed by PCR using the QuikChange site-directed mutagenesis kit (Stratagene) following the manufacturer's guidelines and the following primer pairs: MM295/MM296 to introduce the amino acid exchange H119A (5'-GCGTGCATCAGATGGCGCGCCTATATTTGATATTC-3' and 5'-GAATATCAAATATAGGCGCGCCATCTGATGCACGC-3') and MM278/MM279 for the mutation W143A (5'-CATTATATCTAGTTACGTAGCGGTAGGGAGAAATGTCTC-3' and 5'-GAGACATTTCTCCCTACCGCTACGTAACTAGATATAATG-3'). BamHI/XhoI fragments of the corresponding PCR products were subcloned in pET22b-Strep for recombinant expression in E. coli BL21(DE3). The identity of all constructs was confirmed by sequencing.

Expression and Purification of Recombinant NeuO—Freshly transformed E. coli BL21(DE3) were cultivated at 37 °C in Luria-Bertani (LB)-medium containing 200 µg/ml carbenicillin. At an optical density (A600) of 0.6 expression was induced by adding 0.1 mM isopropyl 1-thio-beta-D-galactopyranoside and bacteria were harvested 2 h after induction. Purification was performed by immobilized Ni2+-affinity chromatography using HisTrap-chelating HP columns (GE Healthcare). Bacteria were resuspended in binding buffer (20 mM Tris-HCl, pH 8.0/500 mM NaCl/40 mM imidazole) and lysed by sonication, and the soluble fraction was applied to a 1-ml column according to the manufacturer's instructions. After washing with 25 ml of binding buffer, proteins were eluted with a linear imidazole gradient of 40-500 mM imidazole in binding buffer. Imidazole was removed by gel filtration using a HiPrep 26/10 desalting column (GE Healthcare) equilibrated with 100 mM Tris-HCl, pH 8.0/150 mM NaCl.

In Vitro Assay for the Determination of NeuO Activity—NeuO activity was determined in a spectrophotometric assay according to Alpers et al. (22). In a total volume of 100 µl, the standard reaction mixture contained 20 mM Tris-HCl, pH 7.5, 25 mM EDTA, 4 mM 5,5'-dithiobis(2-nitrobenzoic acid), 2 mg/ml colominic acid, and 1 mM acetyl-CoA, and the reaction was initiated by adding 24 pmol of purified enzyme. The reaction was performed at 25 °C and monitored continuously at 405 nm in half-area 96-well plates (Greiner) using a PowerWave 340 microplate spectrophotometer (BioTek). For comparison of NeuO activity toward colominic acid, CMP-Neu5Ac, Neu5Ac, and Neu5Ac oligomers of different length, the concentrations of all acceptor substrates were normalized to equal sialic acid content (6.85 mM) and thereby to an equal number of acceptor sites. For quantification of the sialic acid content, oligomers were hydrolyzed to free sialic acid by treatment with 100 mM trifluoroacetic acid for 4 h at 80 °C followed by the thiobarbituric acid assay according to Skoza and Mohos (23) using Neu5Ac as standard.

Kinetic Analysis—The acceptor and donor kinetics were measured on purified NeuO variants using the spectrophotometric assay described above. The steady-state parameters, Km and kcat, were determined by holding one substrate at saturating concentration while the concentration of the second substrate was varied. Twelve different concentrations of donor (0.025-2 mM acetyl-CoA) and acceptor substrate (2-215 µM colominic acid) were used. Kinetic parameters were obtained by fitting the initial rate data to the Michaelis-Menten equation using nonlinear regression analysis with Prism 4.0 software (GraphPad Software, Inc.).

Analysis of NeuO Activity in Vivo—NeuO lacking the VNTR region was amplified by PCR using the primers HC412 (5'-GCGCGCGGATCCCGTTTTCCGTTGATGATAATGGG-3') and HC406 (5'-GCGCGCCTGCAGTATTTATTGCGTGAGCTTCGC-3') containing BamHI and PstI sites (underlined), respectively. The above described pET-based plasmids containing neuO encoding either wild-type or mutated variants with the amino acid exchanges H119A and W143A were used as a template. PCR products were ligated into BamHI/PstI sites of pQE32 (Qiagen), and the identity of all constructs was confirmed by DNA sequencing. The resulting plasmids were transformed into E. coli K1 strain A160, which lacks endogenous neuO. Bacteria were grown on LB-agar containing 100 µg/ml ampicillin and 20 µl of a bacterial suspension (optical density at 600 nm = 0.1) were added to each well of a microtiter plate (Greiner) coated with poly-D-lysine (Sigma). After drying, bacteria were fixed with 0.05% glutaraldehyde in phosphate-buffer saline. Capsule expression and modification by O-acetylation was monitored in an enzyme-linked immunosorbent assay as described previously (24) using the following monoclonal antibodies (mAbs): mAb 735 (25), which is specific for {alpha}2,8-linked polySia (OAc+ and OAc- forms), and mAb 58-5 (Monosan), which binds exclusively to the O-acetylated form of the K1 antigen.

Determination of the Minimal Acceptor Substrate Length—In a final volume of 50 µl, 200 µg of colominic acid was incubated with 1.3 µg of purified NeuO in the presence of [14C]acetyl-CoA (GE Healthcare) for 1 h at 37 °C. The reaction mixture was separated by high percentage PAGE, and the degree of polymerization of the smallest radioactively labeled acceptor molecule was determined by autoradiography. For generation of a radioactively labeled oligosialic acid marker ladder, the solid-phase fixed polysialyltransferase ST8SiaIV/PST-1 was autopolysialylated in vitro in the presence of CMP-[14C]Neu5Ac (GE Healthcare) as described (26). After removal of unbound substrate, radioactively labeled sialic acid oligomers were released by partial endosialidase cleavage.

Analysis of Polysialic Acid by High Percentage PAGE—Sialic acid oligomers and polymers were separated on 25% polyacrylamide gels as described (27). Electrophoresis was performed for 4 h at 4 °C and 400 V, using 14-cm gels (0.8-mm thick). Immediately after electrophoresis, acrylamide gels were vacuumdried and exposed to Hyperfilm-MP (GE Healthcare).

SDS-PAGE, Silver Staining, and Immunoblotting—SDS-PAGE was performed under reducing conditions using 2.5% (v/v) beta-mercaptoethanol. Silver staining and Western blot analysis were performed as described previously (28). For detection of the hexahistidine tag, penta-His antibody (Qiagen) was used at a concentration of 1 µg/ml. Strep-tagged proteins were detected with StrepTactin-AP conjugate (IBA) at a 1:4000 dilution.

Size-exclusion Chromatography—The quaternary structure of wild-type and mutant NeuO was determined on a Superdex 200 HR 10/30 column (GE Healthcare). The column was equilibrated with 100 mM Tris-HCl, pH 8.0/150 mM NaCl and calibrated with molecular mass standards (Sigma) thyroglobulin (669 kDa), beta-amylase (200 kDa), alcohol dehydrogenase (150 kDa), bovine serum albumin (66 kDa), carbonic anhydrase (29 kDa), and cytochrome C (12.4 kDa). 1 mg of affinity-purified wild-type or mutant NeuO was applied to the column, and the eluted protein was monitored by absorbance at 280 nm.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Purification of NeuO Variants with Variable N-terminal Protein Extensions—In all OAc+ E. coli K1 strains investigated so far, neuO contains VNTRs located two nucleotides downstream of the start codon (16). As shown in Fig. 1A, full-length translation and thereby expression of active NeuO depend on repeat numbers that are divisible by three. In the case of full-length translation, every three tandem heptanucleotides encode an RLKTQDS heptad.

To investigate the role of N-terminal heptads on protein expression and activity, a set of neuO alleles with 0, 12, 24, and 36 heptanucleotide repeats was generated, and the corresponding translation products were termed NeuO+0, NeuO+12, NeuO+24, and NeuO+36, respectively. Each variant was expressed in E. coli BL21(DE3) with an N-terminal Strep- and a C-terminal hexahistidine tag (see Fig. 1B for schematic representation). Recombinant proteins were isolated by immobilized metal affinity chromatography (IMAC) using the C-terminal hexahistidine tag. Notably, high imidazole concentrations (≥450 mM) were required for the elution of all variants, indicating tight adsorption to the IMAC matrix. Because NeuO was unstable under these conditions, fractions eluted from the IMAC column were loaded immediately onto a Sephadex G-25 column to remove imidazole. Using this purification protocol, all NeuO variants were purified to homogeneity (Fig. 1, C and D). Silver staining revealed single bands with an apparent molecular mass consistent with the calculated mass of the respective variant (Fig. 1, B and D). Western blot analysis using StrepTactin and anti-penta-His antibody (Fig. 1D, middle and right panel) revealed that neuO alleles with 12, 24, and 36 heptanucleotide units were translated in full-length proteins with the expected N-terminal extensions of 28, 56, and 84 amino acids, respectively, and no proteolytic cleavage or degradation of the heptad region was observed. From 1-liter cultures, 2.5 mg of purified NeuO+0 was obtained (Fig. 1B). Expression levels and protein yields increased with increasing numbers of VNTRs, suggesting that heptads contribute to protein stability. Because the insertion of 12, 24, and 36 nucleotide repeats increases the molecular mass of the translation products by 13, 25, and 37%, respectively, protein yields were compared on the molar level revealing a 2-fold higher protein yield for NeuO+36 than for NeuO+0 (Fig. 1B).


Figure 1
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  		FIGURE 1.
Purification of NeuO variants containing VNTR-encoded heptad repeats. A, schematic representation of the VNTR region of neuO showing alleles with 13 (top) and 12 (bottom) 5'-AAGACTC-3' repeats. RLKTQDS heptads encoded by 3 heptanucleotide repeats are highlighted by gray boxes. B, schematic representation of NeuO variants encoded by alleles containing 0, 12, 24, and 36 repeats. The VNTR-encoded heptad region is shown in black, and N-terminal Strep- and C-terminal hexahistidine tag are shown in light and dark gray, respectively. All variants were expressed in E. coli BL21(DE3), and proteins were purified by IMAC on an Ni2+-chelating column. The amount of purified protein obtained from a 1-liter culture is given as mean ± S.D. from three independent experiments. C, purification of NeuO lacking the VNTR region. After expression in E. coli BL21(DE3), NeuO+0 was isolated by IMAC. Purification steps were monitored by 14% SDS-PAGE and Coomassie staining, including aliquots of bacterial lysate (lane 1), flow through of the IMAC column (lane 2), wash fractions (lanes 3 and 4), NeuO eluted from IMAC column (lane 5), and purified NeuO after subsequent imidazole removal by gel filtration (lane 6). D, analysis of affinity-purified NeuO variants encoded by alleles containing 0, 12, 24, and 36 heptanucleotide repeats. Equal protein amounts of each variant were separated by 14% SDS-PAGE and analyzed by silver staining (left panel) and Western blotting using StrepTactin-AP for detection of the N-terminal Strep tag (middle panel) or anti-penta-His antibody for detection of the C-terminal hexahistidine tag (right panel). E, enzymatic activities of NeuO variants encoded by alleles containing the indicated number of heptanucleotide repeats. NeuO activity was determined spectrophotometrically in a modified Alpers assay using 24 pmol of purified protein.

 
It is worth noting that none of the NeuO variants bound significantly to a StrepTactin column, although the N-terminal Strep tag was detectable by Western blotting (Fig. 1D, middle panel). This observation indicates that in native NeuO the N terminus is not accessible. Remarkably, this finding was irrespective of the presence or absence of heptad repeats (data not shown). For the unequivocal detection of full-length proteins, we kept the Strep tag in all used constructs. Direct comparison of NeuO with and without the Strep tag revealed that this tag did not affect NeuO activity (data not shown).

N-terminal Heptads Are Dispensable for Enzymatic Activity of Isolated NeuO—After demonstrating that heptanucleotide repeats are translated in stable protein extensions, we asked whether RLKTQDS heptads are essential for enzymatic activity of NeuO. To investigate this point, the O-acetyltransferase activity of NeuO variants with and without heptad repeats was determined in a modified Alpers assay (22). In this spectrophotometric assay, the transfer of acetyl groups from acetyl-CoA to polySia is followed by measuring the appearance of the free sulfhydryl group of CoA-SH with Ellman's reagent. Reactions were initiated by adding equimolar amounts of purified NeuO+0, NeuO+12, NeuO+24, or NeuO+36, and the optical density was monitored at 405 nm. Under the applied conditions of high donor and acceptor substrate concentrations, NeuO+0 and NeuO+12 showed comparable enzymatic activities (Fig. 1E), demonstrating that N-terminal RLKTQDS heptads are dispensable for enzymatic activity. Interestingly, increased activities were observed for NeuO+24 and NeuO+36, indicating that VNTR-encoded protein extensions influence the enzymatic properties of NeuO.


Figure 2
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  		FIGURE 2.
Effect of heptad repeats on the catalytic efficiency of NeuO. Catalytic efficiencies for the donor (A) and the acceptor substrate (B) were determined for NeuO encoded by alleles containing 0, 12, 24, and 36 heptanucleotide repeats (see Table 1), and the obtained kcat/Km values were plotted versus the number of heptanucleotide repeats.

 
RLKTQDS Heptad Repeats Increase the Catalytic Efficiency of NeuO—To further investigate the effect of heptad repeats on the activity of NeuO, substrate kinetics of purified NeuO variants with and without RLKTQDS repeats were compared. In a first step, steady-state kinetic constants of NeuO+0, NeuO+12, NeuO+24, and NeuO+36 were determined for the donor substrate using 12 different concentrations of acetyl-CoA. All kinetic analyses were performed at 25 °C and yielded linear Lineweaver-Burk plots (see supplemental Figs. S1 and S2). The donor Km value of NeuO+0 was determined to be 0.4 mM (Table 1), whereas all Km values of heptad-containing variants were decreased by almost 50%. Vmax and thereby kcat values slightly increased with the number of heptads leading to a 2.3-fold higher kcat for NeuO+36 compared with NeuO+0.


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  		TABLE 1
Kinetic parameters of NeuO variants with different numbers of heptad repeats

NeuO activity was measured in the spectrophotometric assay at pH 7.5 and 25 °C. Donor kinetics were determined using 0.025-2 mM acetyl-CoA with colominic acid at 2 mg/ml. Acceptor kinetics were determined using 0.015-2 mg/ml colominic acid (corresponding to 0.05-6.85 mM sialic acid residues) with acetyl-CoA at 2 mM. The steady-state parameters, Km and kcat, were determined from initial velocity measurements from at least three independent experiments measured in triplicate and values are given as mean ± S.D.

 
A lower Km value in combination with an increased kcat resulted in a 4-fold higher catalytic efficiency (kcat/Kmdonor) of NeuO+36 compared with NeuO+0 (Table 1). Remarkably, kcat/Kmdonor values increased linearly with the number of heptad repeats (Fig. 2A). These results suggest that the presence of heptad repeats result in gradual changes in enzyme conformation allowing an increase in enzyme-donor substrate affinity and facilitation of the reaction rate.

In a second step, kinetic parameters for the acceptor were determined using 12 different polySia concentrations. As an acceptor substrate, we used colominic acid, a commercially available mixture of oligomeric {alpha}2,8-linked sialic acid. Analysis of the batch used in the present study revealed oligomer lengths ranging from 2 to over 60 residues and an average degree of polymerization (DP) of 32. To acknowledge the fact that colominic acid is heterogeneous in polymer length, and that longer polymers contain more acceptor sites than shorter chains, acceptor concentrations were expressed as concentration of total sialic acids. Because acceptor affinities may vary with polySia chain length and also between internal and terminal sialic acids residues, acceptor Km values will represent average values. For NeuO+0, the acceptor Km value was determined to be 1.17 mM (Table 1). In contrast to the donor Km values, no significant differences in Kmacceptor were observed for NeuO variants with and without heptad repeats. Therefore, the kcat/Kmacceptor value was only 2-fold higher for NeuO+36 compared with NeuO+0 (Table 1), and the catalytic efficiency also increased with the number of repeats (Fig. 2B).


Figure 3
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  		FIGURE 3.
Determination of the minimal acceptor length. Purified NeuO+36 was incubated with colominic acid and [14C]acetyl-CoA. The reaction mixture was separated by 25% PAGE, and radioactively labeled products were visualized by autoradiography (lane 1). The shortest detectable sialic acid oligomer is marked by an arrow. For size determination, radioactively labeled sialic acid oligomers with DP 3-18 were used as a marker (lane 2).

 
Donor Specificity of NeuO—In addition to acetyl-CoA, the alternative acyl donors propionyl-CoA and butyryl-CoA were assayed as substrates for NeuO. Butyryl-CoA turned out to be a very poor substrate, and even at a concentration of 1 mM, no transfer activity was observed. Propionyl-CoA, however, was used as a donor substrate. Similar to the results obtained with acetyl-CoA, increased enzymatic activities were monitored for NeuO+36 compared with NeuO+0 (data not shown). Kinetic parameters were evaluated only for NeuO+36, and results are summarized in Table 1 (bottom row).

Interestingly, identical Km values were obtained for acetyl- and propionyl-CoA, indicating that the binding pocket for the donor substrate can accommodate the larger acyl-chain of propionyl-CoA. However, a 13-fold decrease in kcat was observed for propionyl-CoA, indicating that the free energy for the transition state of the rate-limiting step is significantly higher for propionyl-CoA compared with acetyl-CoA.

Acceptor Specificity of NeuO—To investigate the acceptor substrate specificity of NeuO, the enzymatic activity of purified enzyme was monitored in the presence of sialic acid monomers (Neu5Ac), activated sialic acid (CMP-Neu5Ac), and a set of short {alpha}2,8-linked sialic acid oligomers with defined DPs in the range of dimer to hexamer. Because different kinetic properties were observed for NeuO variants with and without heptad repeats, acceptor substrate specificities were analyzed in parallel for NeuO+0 and NeuO+36. In contrast to colominic acid, none of the assayed compounds was used as an acceptor (Table 2). This result demonstrates that NeuO is highly specific for polySia and that even an {alpha}2,8-linked sialic acid hexamer is too short for NeuO-catalyzed O-acetylation.


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  		TABLE 2
Acceptor specificity of NeuO

Relative enzymatic activities of purified NeuO+0 and NeuO+36 were determined in the presence of different acceptor substrates. All acceptor substrates were used at a concentration of 6.85 mM of total sialic acids, and the activity obtained for colominic acid was set to 100%. Values are mean ± S.D. from two independent experiments measured in triplicate.

 
To determine the minimal acceptor length of NeuO, we performed a radioactive incorporation assay. Purified NeuO+0 and NeuO+36 were incubated in the presence of 14C-labeled acetyl-CoA and colominic acid as acceptor substrate. After separation by high percentage PAGE, reaction products were visualized by autoradiography. Because only those oligomers modified by O-acetylation will become radioactively labeled, the length of the shortest visible oligomer will represent the minimal acceptor length required. As a size marker, 14C-labeled polySia was partially digested with endosialidase, resulting in an oligomer ladder starting with DP 3 (26). For the two enzyme variants, NeuO+0 and NeuO+36, reaction products of similar length were obtained and results are shown for NeuO+36 as an example (Fig. 3). Faint radioactive signals became visible for oligomers with DP 14-16, and strong signals were obtained for oligomers >16 residues. Together, these results demonstrate high acceptor substrate specificity of NeuO for sialic acid oligomers >14 residues.

Homology Modeling of the Catalytic Part of NeuO—Primary sequence analysis of NeuO revealed similarity to the hexapeptide repeat family of acyltransferases (16, 29). Proteins of this family are characterized by tandem repeats of a hexapeptide with the consensus motif [LIV]-[GAED]-X2-[STAV]-X, which forms alternating short beta-strands and tight turns resulting in a triangular left-handed beta-helix (LbetaH) (30). All members of the hexapeptide acyltransferase superfamily assemble into catalytic trimers with three symmetrically arranged catalytic sites at the interface between the monomers.


Figure 4
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  		FIGURE 4.
Homology model of NeuO in comparison with the crystal structure of MAT. A, ribbon representation of MAT (PDB accession number 1ocx; side view of the monomer). B, trimeric organization of MAT (top view), with the three subunits shown in green, red, and blue. The location of His-113 and Trp-137, which are part of the active site, are indicated by arrows. C, three-dimensional model of NeuO lacking VNTR-encoded heptads (side view of the monomer). The model was obtained by using the program 3D-PSSM (21) with the structure of MAT as a template. D, the predicted structure of the NeuO monomer (purple) was modeled into the trimer of MAT (gray). The locations of His-119 and Trp-143 of NeuO are indicated by arrows. All structural data were visualized by PyMOL.

 
To gain first insight into the structure of a polySia modifying O-acetyltransferase, homology models of NeuO lacking heptad repeats were generated using the program 3D-PSSM (21). The best structural match with an E-value of 2.2 x 10-4 (≥95% certainty) was obtained with the crystal structure of the maltose O-acetyltransferase (MAT) of E. coli (Protein Data Bank number 1ocx) (31) (see supplemental Fig. S3). The obtained model consists primarily of an LbetaH fold with five and one-third triangular coils (Fig. 4). The LbetaH domain covers the central and C-terminal part of NeuO (amino acids 61-216) and is interrupted by a single loop formed by residues 116-133 (Fig. 4C). For the first 60 amino acids of NeuO, however, only poor predictions were obtained, indicating structural differences between the N-terminal parts of NeuO and MAT. The LbetaH domain of MAT is N-terminally capped by three {alpha}-helices (residues 1-55), whereas only one {alpha}-helix was predicted for NeuO.

Notably, two amino acids that are part of the active site of MAT were highly conserved in NeuO. His-113, which corresponds to His-119 in NeuO, is located in the loop that protrudes between beta-sheets four and five. This residue is proposed to abstract the proton from the hydroxyl group of the acceptor prior to acetyl transfer (31, 32). Trp-137, which corresponds to Trp-143 in NeuO, is located in a beta-sheet of the LbetaH domain and is involved in binding of acetyl-CoA (31, 32). In the homo-trimer, the tryptophan of one subunit is located opposite to the catalytic histidine of the next subunit (Fig. 4B). In the predicted NeuO model, His-119 and Trp-143 are located at equivalent positions, suggesting similar functions in enzymatic catalysis.

Identification of Catalytic Residues—To prove whether His-119 and Trp-143 are crucial for enzymatic activity of NeuO, single alanine substitutions were performed. For both mutations, H119A and W143A, variants with and without 36 heptanucleotide repeats were generated, and the resulting proteins were expressed in E. coli BL21(DE3) with N-terminal Strep- and C-terminal hexahistidine tags. For either variant, similar expression levels than for the corresponding wild-type proteins were observed (data not shown), indicating that the introduced amino acid exchanges did not affect the stability of NeuO. The four mutant variants were purified to homogeneity (Fig. 5A) and assayed for enzymatic activity. However, in contrast to the wild-type forms of NeuO+0 and NeuO+36, no enzymatic activity was detected for the corresponding mutant forms (Fig. 5B). In addition to the in vitro analysis, the activity of NeuO variants was studied in vivo. To circumvent phase variation, the in vivo approach was restricted to variants lacking the VNTR region. Corresponding wild-type and mutant forms were cloned into the expression vector pQE32, and the resulting plasmids were transformed into E. coli K1 strain A160. As shown by PCR analysis, this strain lacks endogenous neuO (data not shown). Consequently, the expressed K1 capsule is OAc-negative as confirmed by an enzyme-linked immunosorbent assay using two capsule-specific antibodies: (i) mAb 735 (25), which binds to {alpha}2,8-linked polySia irrespective of the presence or absence of O-acetylation, and (ii) mAb 58-5, which recognizes exclusively the O-acetylated form of the K1 antigen (Fig. 5C). O-Acetylation of the K1 capsule of strain A160 was only observed after expression of wild-type NeuO. By contrast, no capsule O-acetylation was detected after expression of NeuO-H119A and NeuO-W143A, although K1 expression per se was not affected (Fig. 5C). Together, these results demonstrate that His-119 and Trp-143 are essential for NeuO activity in vitro and in vivo.

NeuO Assembles into Hexamers—The quaternary structure of all known hexapeptide acyltransferases is trimeric with the exception of the serine acetyltransferase (SAT) of E. coli and Haemophilus influenzae, which adopts a hexameric structure that is formed by dimers of trimers (33-35). Because trimerization is prerequisite for activity of hexapeptide acyltransferases, we asked whether the introduced mutations H119A and W143A induced conformational changes that prevent oligomerization and thereby abolished enzymatic activity of NeuO. Therefore, the quaternary structure of wild-type and mutant NeuO was determined by size-exclusion chromatography, and results are shown for variants based on neuO alleles with 36 heptanucleotide repeats (Fig. 6). For wild-type NeuO+36, a molecular mass of 217 kDa was determined corresponding to an oligomeric state of 5.9. Although the shape of NeuO is not yet known, this result indicates that NeuO is another example of a hexapeptide acyltransferase that assembles into hexamers. Similar to the structure of SAT, NeuO hexamers might be formed by dimers of trimers. Notably, both mutants, H119A and W143A, showed an identical oligomeric state, demonstrating that the introduced amino acid exchanges did not interfere with complex assembly. In summary, our data indicate that alanine substitution of His-119 and Trp-143 did not result in substantial alterations in the overall structure of NeuO. However, exchange of either residue completely abolished activity, suggesting that both amino acids are part of the active site.


Figure 5
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  		FIGURE 5.
Identification of amino acid residues critical for NeuO activity. A, the single amino acid exchanges H119A and W143A were introduced into NeuO+0 (left panel) and NeuO+36 (right panel). After expression in E. coli BL21(DE3) and affinity purification on Ni2+-chelating columns, wild-type (wt) and mutant forms were analyzed by 14% SDS-PAGE and silver staining (upper panel) or Western blotting using anti-penta-His antibody (lower panel). B, the enzymatic activities of NeuO+0 (wt), NeuO+36 (wt+36), and the corresponding mutants H119A and W143A were determined spectrophotometrically in the modified Alpers assay using equimolar concentrations of each purified protein. Data represent mean values ± S.D. of three independent experiments measured in triplicates. C, in vivo analysis of NeuO mutants. E. coli K1 strain A160 (Ø), which lacks endogenous neuO, was transformed with the empty vector pQE32 (mock), pQE32-based plasmids containing wild-type neuO lacking the VNTR region (wt), and the corresponding mutant variants H119A and W143A. Bacteria were analyzed for O-acetylated capsular polysaccharide by an enzyme-linked immunosorbent assay using mAb 58-5 (black bars). K1 capsule expression was controlled using mAb 735 (gray bars), which recognizes both OAc+ and OAc- forms of the K1 antigen. Negative controls were performed using bovine serum albumin instead of primary antibody.

 


Figure 6
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  		FIGURE 6.
Quaternary structure of NeuO. The oligomeric state of purified NeuO+36 and the corresponding mutants H119A and W143A was analyzed by size exclusion chromatography on a Superdex 200 column. Void volume (Vo) and elution positions of standard proteins are indicated by arrows.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Surface sialic acids are a common component and virulence factor of a variety of pathogenic bacteria (36). In many cases, modification by O-acetylation has been observed and might play an important role in immunogenicity and survival in the host organism (14, 37-42). In the present study, we succeeded for the first time in purification and characterization of a polySia-specific O-acetyltransferase. NeuO, which is essential for O-acetylation of the capsular polysaccharide of E. coli K1, was purified to homogeneity using a C-terminal hexahistidine tag. Previous attempts to purify endogenous NeuO required detergent extraction, and, after partial purification on DEAE-Sepharose, further isolation steps abolished activity (15). By contrast, recombinant NeuO was obtained from the soluble protein fraction, and the isolated enzyme was enzymatically active as demonstrated in a spectrophotometric assay and by incorporation of radioactively labeled acetyl groups into polySia. A possible explanation for the different outcome during purification of endogenous and recombinant NeuO is that, in E. coli K1, efficient O-acetylation of the capsular polysaccharide might depend on tight association of NeuO with the polySia biosynthesis and transport machinery. In this case, detergent might be required to dissociate NeuO from the membrane-bound protein complex, a step that can be omitted if NeuO is expressed in an E. coli strain lacking the K1 capsular polysaccharide gene complex.

The idea that in vivo NeuO-dependent O-acetylation occurs exclusively during or after polymerization of the polysaccharide but not on polySia building blocks is supported by our finding that in vitro neither Neu5Ac nor CMP-Neu5Ac served as acceptor substrates for NeuO. In line with the initial observation made by Higa and Varki (15), purified NeuO was highly specific for sialic acid oligomers with more than 14 residues, and efficient O-acetylation seemed to depend on oligomers with DP >16. However, to understand the structural basis of the acceptor specificity, further studies, including detailed product analyses and characterization of the protein-carbohydrate interactions are required.

Using homology modeling followed by mutational analysis, we characterized NeuO as a typical member of the superfamily of hexapeptide acyltransferases. Enzymes of this family use phosphopantothenyl-based cofactors to transfer acetyl, succinyl, or long chain fatty acyl groups to free hydroxyl or amino groups of a variety of acceptor substrates. All family members contain tandem-repeated imperfect copies of a hexapeptide repeat sequence with the consensus motif [LIV]-[GAED]-X2-[STAV]-X (29, 30). Several members of the family have been crystallized, and the solved structures revealed that this characteristic hexapeptide repeat sequence encodes folding of a left-handed parallel beta-helix domain (31, 43-50). In all known cases, hexapeptide acyltransferases assemble into catalytic trimers with three symmetrical active sites, which are located at the interfaces of two subunits formed by a loop protruding out of one LbetaH domain embracing the LbetaH domain of the neighboring monomer. Based on the structural characteristics, the hexapeptide repeat family is also called LbetaH family and includes not only acyltransferases but also a zinc-dependent {gamma}-carbonic anhydrase (51).

The homology modeling performed in the present study indicated close structural similarity of NeuO to MAT (31), which is closely related to the lacA-encoded galactoside O-acetyltransferase (GAT) of E. coli (45). Similar to MAT and GAT, the predicted model of NeuO contains a short LbetaH domain with five and one-third triangular coils and one protruding loop. Two amino acid residues, which are critical for activity of MAT and GAT, were found in equivalent positions in the NeuO model: His-119 located in the protruding loop and Trp-143 located within a beta-sheet of the LbetaH domain. Subsequent single alanine substitutions completely abolished NeuO activity in vitro and in vivo, indicating important roles in catalysis. All acyltransferases of the LbetaH family crystallized so far posses a catalytic histidine located in a loop embracing the adjacent subunit. The predicted role of this residue is to abstract a proton from the acceptor hydroxyl group, facilitating the attack of the resulting carbonyl by the acyl-donor (32, 52). The second critical amino acid identified in the model of NeuO, Trp-143, is conserved only in a subset of acetyltransferases, including MAT, GAT, xenobiotic acetyltransferases, and the Rhizobium leguminosarum nodulation factor NodL (31, 32, 44, 53). In GAT, replacement of the corresponding Trp-139 by phenylalanine abolished the intrinsic fluorescence quench observed on acetyl-CoA binding (32), and the crystal structure of the binary complex of GAT with acetyl-CoA revealed a direct contact between the indole side chain of Trp-139 and the phosphopantothenyl arm of the cofactor (45). In addition to donor binding, Trp-139 may serve to position the catalytic histidine relative to the hydroxyl group of the acceptor and alter its pKa (45). In line with this dual role in donor binding and positioning of the catalytic histidine, a complete loss of NeuO activity was observed when Trp-143 was substituted by alanine. Together, these results provided convincing evidence that NeuO consists primarily of an LbetaH fold with His-119 and Trp-143 as part of the active site.

Using size-exclusion chromatography, we demonstrated that NeuO assembles into hexamers. So far, the only example of an LbetaH-acyltransferase with a quaternary structure other than trimeric is SAT (54). Similar to NeuO, SAT of E. coli and H. influenzae adopts a hexameric structure, and the solved crystal structures revealed that two SAT trimers assemble into hexamers with the dimer of trimers interface formed by the N-terminal {alpha}-helical domain (34, 35). The observation that a Strep tag placed at the N terminus of NeuO was not accessible for purification might be an indication that, also in NeuO, the N-terminal domain is involved in dimerization of NeuO trimers.

Recently, several genes encoding sialic acid specific O-acetyltransferases have been cloned by us and others: (i) oatC responsible for O-acetylation of the {alpha}2,9-linked polySia capsule of N. meningitidis of serogroup C (55); (ii) oatWY, involved in O-acetylation of sialic acids within the galactose and glucose containing heteropolymeric polySia capsules of serogroup W-135 and Y meningococci, respectively (55); (iii) neuO of E. coli K1 (16); (iv) neuD of group B streptococci required for O-acetylation of terminal {alpha}2,3-linked sialic acids capping the group B streptococci capsule (56); and (v) neuD of Campylobacter jejuni, which was shown to O-acetylate terminal {alpha}2,8-linked sialic acids of the bacterial lipo-oligosaccharide (42). With the notable exception of OatC, which shares no sequence similarity to any known protein in the data base, all other sialic acid-specific O-acetyltransferases were found to contain hexapeptide repeat sequences, indicating that these proteins are all members of the LbetaH family. However, the NeuD proteins might form a separate branch within the family, because amino acid sequence alignments revealed the lack of a potential catalytic histidine, which might be substituted by a lysine residue (56).

Although NeuO shares the typical features of the LbetaH family, the present study revealed a unique regulatory mechanism based on changes in VNTR-encoded N-terminal protein extensions. The neuO gene is characterized by the presence of a variable number of heptanucleotide repeats within the coding region (16). Because only repeat numbers that are a multiple of three allow full-length translation and thereby capsule O-acetylation, changes in the overall number provide a reversible switch between an on and off phase. Oscillation between OAc+ and OAc- variants might permit adaptation to changes in environmental conditions. However, our results demonstrate that this is not a simple "all-or-none" mechanism. By expressing neuO alleles containing 0, 12, 24, and 36 heptanucleotide repeats, we found that VNTRs are translated in stable N-terminal protein extensions that affect the actual enzymatic activity of the translation product. Every three heptanucleotide repeats encode an RLKTQDS heptad, and, in the presence of 36 heptanucleotide repeats, the translation product is N-terminally elongated by 12 heptads, which account for 37% of the total molecular mass. The presence of RLKTQDS heptads was not essential for NeuO activity but decreased the Km for acetyl-CoA by ~50% without affecting the Km for the acceptor substrate. Remarkably, a linear correlation between the catalytic efficiency (kcat/Kmdonor) and the number of repeats was observed. Thus, stochastic genetic variation generated by VNTRs during the on phase is translated in protein variation that provides a gradual modification of NeuO activity.

kcat/Km may be considered as a measure of the apparent firstorder rate constant for enzyme-substrate interaction and reflects the affinity between enzyme and substrate to form a complex. Thus, the results are consistent with the interpretation that the presence of heptad repeats leads to a change in enzyme conformation with the resultant increase in enzymedonor substrate affinity and facilitation of the reaction rate.

As suggested by Deszo et al. (16), heptad repeats may assemble into a triple coiled-coil. In the hexameric NeuO complex, variation in the length of such a coil might gradually change the subunit arrangement and, thereby, the conformation of the catalytic sites formed at the subunit interfaces of each trimer. However, the complete arrangement of all six subunits and the structure of the heptad repeats will only unequivocally be solved by analyzing the crystal structure of the NeuO complex.

In vivo, direct analysis of NeuO activity is hampered by frequent oscillation between on and off phases. However, comparison of 10 different OAc+ K1 strains indicated that VNTR-encoded protein extensions affect NeuO activity also in vivo, and the highest activities were found in strains expressing neuO alleles with >21 heptanucleotide repeats (16). The analysis of capsular polysaccharides from four individual OAc+ K1 strains revealed different degrees of O-acetylation ranging from 5 to 85% (14, 15). Remarkably, the lowest O-acetyl content and lowest activity were found in a strain without detectable phase variation (14, 15), indicating that, in vivo, modulation of NeuO activity has an impact on the degree of capsule O-acetylation.


   FOOTNOTES
 
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AJ783705.

* This work was supported by grants of the Deutsche Forschungsgemeinschaft (Grant VO 718/4-1 to U. V. and Grant MU 1774/2-1 to M. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S3. Back

1 To whom correspondence should be addressed: Tel.: 49-511-532-9807; Fax: 49-511-532-3956; E-mail: muehlenhoff.martina{at}mh-hannover.de.

2 The abbreviations used are: polySia, polysialic acid; DP, degree of polymerization; GAT, galactoside acetyltransferase; IMAC, immobilized metal affinity chromatography; LbetaH, left-handed beta-helix; mAb, monoclonal antibody; MAT, maltose acetyltransferase; Neu5Ac, 5-N-acetylneuraminic acid; OAc, O-acetylation; SAT, serine acetyltransferase; VNTR, variable number of tandem repeats; Strep, Strep tag II. Back


   ACKNOWLEDGMENTS
 
We thank Rita Gerardy-Schahn for helpful discussions and continuous support and Katharina Stummeyer for her assistance with PyMOL.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Robbins, J. B., McCracken, G. H., Jr., Gotschlich, E. C., Ørskov, F., Ørskov, I., and Hanson, L. A. (1974) N. Engl. J. Med. 290, 1216-1220[Medline] [Order article via Infotrieve]
  2. Sarff, L. D., McCracken, G. H., Schiffer, M. S., Glode, M. P., Robbins, J. B., Ørskov, I., and Ørskov, F. (1975) Lancet 1, 1099-1104[Medline] [Order article via Infotrieve]
  3. McCracken, G. H., Sarff, L. D., Glode, M. P., Mize, S. G., Schiffer, M. S., Robbins, J. B., Gotschlich, E. C., Ørskov, I., and Ørskov, F. (1974) Lancet 2, 246-250[Medline] [Order article via Infotrieve]
  4. Bonacorsi, S., and Bingen, E. (2005) Int. J. Med. Microbiol. 295, 373-381[CrossRef][Medline] [Order article via Infotrieve]
  5. Kim, K. S. (2003) Nat. Rev. Neurosci. 4, 376-385[CrossRef][Medline] [Order article via Infotrieve]
  6. Xie, Y., Kim, K. J., and Kim, K. S. (2004) FEMS Immunol. Med. Microbiol. 42, 271-279[CrossRef][Medline] [Order article via Infotrieve]
  7. Fowler, M. I., Weller, R. O., Heckels, J. E., and Christodoulides, M. (2004) Cell Microbiol. 6, 555-567[CrossRef][Medline] [Order article via Infotrieve]
  8. Harvey, D., Holt, D. E., and Bedford, H. (1999) Semin. Perinatol. 23, 218-225[CrossRef][Medline] [Order article via Infotrieve]
  9. Saez-Llorens, X., and McCracken, G. H., Jr. (2003) Lancet 361, 2139-2148[CrossRef][Medline] [Order article via Infotrieve]
  10. Pluschke, G., Mayden, J., Achtman, M., and Levine, R. P. (1983) Infect. Immun. 42, 907-913[Abstract/Free Full Text]
  11. Leying, H., Suerbaum, S., Kroll, H. P., Stahl, D., and Opferkuch, W. (1990) Infect. Immun. 58, 222-227[Abstract/Free Full Text]
  12. Rohr, T. E., and Troy, F. A. (1980) J. Biol. Chem. 255, 2332-2342[Abstract/Free Full Text]
  13. Mühlenhoff, M., Eckhardt, M., and Gerardy-Schahn, R. (1998) Curr. Opin. Struct. Biol. 8, 558-564[CrossRef][Medline] [Order article via Infotrieve]
  14. Ørskov, F., Ørskov, I., Sutton, A., Schneerson, R., Lin, W., Egan, W., Hoff, G. E., and Robbins, J. B. (1979) J. Exp. Med. 149, 669-685[Abstract/Free Full Text]
  15. Higa, H. H., and Varki, A. (1988) J. Biol. Chem. 263, 8872-8878[Abstract/Free Full Text]
  16. Deszo, E. L., Steenbergen, S. M., Freedberg, D. I., and Vimr, E. R. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 5564-5569[Abstract/Free Full Text]
  17. Frasa, H., Procee, J., Torensma, R., Verbruggen, A., Algra, A., Rozenberg-Arska, M., Kraaijeveld, K., and Verhoef, J. (1993) J. Clin. Microbiol. 31, 3174-3178[Abstract/Free Full Text]
  18. Stummeyer, K., Schwarzer, D., Claus, H., Vogel, U., Gerardy-Schahn, R., and Mühlenhoff, M. (2006) Mol. Microbiol. 60, 1123-1135[CrossRef][Medline] [Order article via Infotrieve]
  19. van Belkum, A., Scherer, S., van Alphen, L., and Verbrugh, H. (1998) Microbiol. Mol. Biol. Rev. 62, 275-293[Abstract/Free Full Text]
  20. Viguera, E., Canceill, D., and Ehrlich, S. D. (2001) EMBO J. 20, 2587-2595[CrossRef][Medline] [Order article via Infotrieve]
  21. Kelley, L. A., MacCallum, R. M., and Sternberg, M. J. (2000) J. Mol. Biol. 299, 499-520[Medline] [Order article via Infotrieve]
  22. Alpers, D. H., Appel, S. H., and Tomkins, G. M. (1965) J. Biol. Chem. 240, 10-13[Free Full Text]
  23. Skoza, L., and Mohos, S. (1976) Biochem. J. 159, 457-462[Medline] [Order article via Infotrieve]
  24. Vogel, U., Morelli, G., Zurth, K., Claus, H., Kriener, E., Achtman, M., and Frosch, M. (1998) J. Clin. Microbiol. 36, 2465-2470[Abstract/Free Full Text]
  25. Frosch, M., Görgen, I., Boulnois, G. J., Timmis, K. N., and Bitter-Suermann, D. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 1194-1198[Abstract/Free Full Text]
  26. Mühlenhoff, M., Eckhardt, M., Bethe, A., Frosch, M., and Gerardy-Schahn, R. (1996) EMBO J. 15, 6943-6950[Medline] [Order article via Infotrieve]
  27. Pelkonen, S., Häyrinen, J., and Finne, J. (1988) J. Bacteriol. 170, 2646-2653[Abstract/Free Full Text]
  28. Gerardy-Schahn, R., Bethe, A., Brennecke, T., Mühlenhoff, M., Eckhardt, M., Ziesing, S., Lottspeich, F., and Frosch, M. (1995) Mol. Microbiol. 16, 441-450[CrossRef][Medline] [Order article via Infotrieve]
  29. Vaara, M. (1992) FEMS Microbiol. Lett. 76, 249-254[Medline] [Order article via Infotrieve]
  30. Jenkins, J., and Pickersgill, R. (2001) Prog. Biophys. Mol. Biol. 77, 111-175[CrossRef][Medline] [Order article via Infotrieve]
  31. Lo Leggio, L., Dal Degan, F., Poulsen, P., Andersen, S. M., and Larsen, S. (2003) Biochemistry 42, 5225-5235[CrossRef][Medline] [Order article via Infotrieve]
  32. Lewendon, A., Ellis, J., and Shaw, W. V. (1995) J. Biol. Chem. 270, 26326-26331[Abstract/Free Full Text]
  33. Hindson, V. J., Moody, P. C., Rowe, A. J., and Shaw, W. V. (2000) J. Biol. Chem. 275, 461-466[Abstract/Free Full Text]
  34. Pye, V. E., Tingey, A. P., Robson, R. L., and Moody, P. C. (2004) J. Biol. Chem. 279, 40729-40736[Abstract/Free Full Text]
  35. Olsen, L. R., Huang, B., Vetting, M. W., and Roderick, S. L. (2004) Biochemistry 43, 6013-6019[CrossRef][Medline] [Order article via Infotrieve]
  36. Vimr, E., and Lichtensteiger, C. (2002) Trends Microbiol. 10, 254-257[CrossRef][Medline] [Order article via Infotrieve]
  37. Lewis, A. L., Nizet, V., and Varki, A. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 11123-11128[Abstract/Free Full Text]
  38. Bhattacharjee, A. K., Jennings, H. J., Kenny, C. P., Martin, A., and Smith, I. C. (1976) Can. J. Biochem. 54, 1-8[CrossRef][Medline] [Order article via Infotrieve]
  39. Lemercinier, X., and Jones, C. (1996) Carbohydr. Res. 296, 83-96[CrossRef][Medline] [Order article via Infotrieve]
  40. Gamian, A., Romanowska, E., Ulrich, J., and Defaye, J. (1992) Carbohydr. Res. 236, 195-208[CrossRef][Medline] [Order article via Infotrieve]
  41. Feng, L., Senchenkova, S. N., Tao, J., Shashkov, A. S., Liu, B., Shevelev, S. D., Reeves, P. R., Xu, J., Knirel, Y. A., and Wang, L. (2005) J. Bacteriol. 187, 758-764[Abstract/Free Full Text]
  42. Houliston, R. S., Endtz, H. P., Yuki, N., Li, J., Jarrell, H. C., Koga, M., van Belkum, A., Karwaski, M. F., Wakarchuk, W. W., and Gilbert, M. (2006) J. Biol. Chem. 281, 11480-11486[Abstract/Free Full Text]
  43. Raetz, C. R., and Roderick, S. L. (1995) Science 270, 997-1000[Abstract/Free Full Text]
  44. Beaman, T. W., Sugantino, M., and Roderick, S. L. (1998) Biochemistry 37, 6689-6696[CrossRef][Medline] [Order article via Infotrieve]
  45. Wang, X. G., Olsen, L. R., and Roderick, S. L. (2002) Structure 10, 581-588[Medline] [Order article via Infotrieve]
  46. Olsen, L. R., and Roderick, S. L. (2001) Biochemistry 40, 1913-1921[CrossRef][Medline] [Order article via Infotrieve]
  47. Beaman, T. W., Binder, D. A., Blanchard, J. S., and Roderick, S. L. (1997) Biochemistry 36, 489-494[CrossRef][Medline] [Order article via Infotrieve]
  48. Brown, K., Pompeo, F., Dixon, S., Mengin-Lecreulx, D., Cambillau, C., and Bourne, Y. (1999) EMBO J. 18, 4096-4107[CrossRef][Medline] [Order article via Infotrieve]
  49. Sugantino, M., and Roderick, S. L. (2002) Biochemistry 41, 2209-2216[CrossRef][Medline] [Order article via Infotrieve]
  50. Sulzenbacher, G., Gal, L., Peneff, C., Fassy, F., and Bourne, Y. (2001) J. Biol. Chem. 276, 11844-11851[Abstract/Free Full Text]
  51. Kisker, C., Schindelin, H., Alber, B. E., Ferry, J. G., and Rees, D. C. (1996) EMBO J. 15, 2323-2330[Medline] [Order article via Infotrieve]
  52. Wyckoff, T. J., and Raetz, C. R. (1999) J. Biol. Chem. 274, 27047-27055[Abstract/Free Full Text]
  53. Dunn, S. M., Moody, P. C., Downie, J. A., and Shaw, W. V. (1996) Protein Sci. 5, 538-541[Medline] [Order article via Infotrieve]
  54. Johnson, C. M., Roderick, S. L., and Cook, P. F. (2005) Arch. Biochem. Biophys. 433, 85-95[CrossRef][Medline] [Order article via Infotrieve]
  55. Claus, H., Borrow, R., Achtman, M., Morelli, G., Kantelberg, C., Long-worth, E., Frosch, M., and Vogel, U. (2004) Mol. Microbiol. 51, 227-239[CrossRef][Medline] [Order article via Infotrieve]
  56. Lewis, A. L., Hensler, M. E., Varki, A., and Nizet, V. (2006) J. Biol. Chem. 281, 11186-11192[Abstract/Free Full Text]

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A. K. Bergfeld, H. Claus, N. K. Lorenzen, F. Spielmann, U. Vogel, and M. Muhlenhoff
The Polysialic Acid-specific O-Acetyltransferase OatC from Neisseria meningitidis Serogroup C Evolved Apart from Other Bacterial Sialate O-Acetyltransferases
J. Biol. Chem., January 2, 2009; 284(1): 6 - 16.
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