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Glycosyltransferases Involved in Biosynthesis of the Outer Core Region of Escherichia coli Lipopolysaccharides Exhibit Broader Substrate Specificities Than Is Predicted from Lipopolysaccharide Structures

2007; Elsevier BV; Volume: 282; Issue: 37 Linguagem: Inglês

10.1074/jbc.m704131200

1083-351X

Michael D. Leipold, Evgeny Vinogradov, Chris Whitfield,

Bacterial Genetics and Biotechnology

The waaJ, waaT, and waaR genes encode α-1,2-glycosyltransferases involved in synthesis of the outer core region of the lipopolysaccharide of Escherichia coli. They belong to the glycosyltransferase CAZy family 8, characterized by the GT-A fold, DXD motifs, and by retention of configuration at the anomeric carbon of the donor sugar. Each enzyme adds a hexose residue at the same stage of core oligosaccharide backbone extension. However, they differ in the epimers for their donor nucleotide sugars, and in their acceptor residues. WaaJ is a UDP-glucose: (galactosyl) LPS α-1,2-glucosyltransferase, whereas WaaR and WaaT have UDP-glucose:(glucosyl) LPS α-1,2-glucosyltransferase and UDP-galactose:(glucosyl) LPS α-1,2-galactosyltransferase activities, respectively. The objective of this work was to examine their ability to utilize alternate donors and acceptors. When expressed in the heterologous host, each enzyme was able to extend the alternate LPS acceptor in vivo but they retained their natural donor specificity. In vitro assays were then performed to test the effect of substituting the epimeric donor sugar on incorporation efficiency with the natural LPS acceptor of the enzyme. Although each enzyme could utilize the alternate donor epimer, activity was compromised because of significant decreases in kcat and corresponding increases in Km(donor). Finally, in vitro assays were performed to probe acceptor preference in the absence of the cellular machinery. The results were enzyme-dependent: while an alternate acceptor had no significant effect on the kinetic behavior of His6-WaaT, His6-WaaJ showed a significantly decreased kcat and increased Km(acceptor). These results illustrate the differences in behavior between closely related glycosyltransferase enzymes involved in the synthesis of similar glycoconjugates and have implications for glycoengineering applications. The waaJ, waaT, and waaR genes encode α-1,2-glycosyltransferases involved in synthesis of the outer core region of the lipopolysaccharide of Escherichia coli. They belong to the glycosyltransferase CAZy family 8, characterized by the GT-A fold, DXD motifs, and by retention of configuration at the anomeric carbon of the donor sugar. Each enzyme adds a hexose residue at the same stage of core oligosaccharide backbone extension. However, they differ in the epimers for their donor nucleotide sugars, and in their acceptor residues. WaaJ is a UDP-glucose: (galactosyl) LPS α-1,2-glucosyltransferase, whereas WaaR and WaaT have UDP-glucose:(glucosyl) LPS α-1,2-glucosyltransferase and UDP-galactose:(glucosyl) LPS α-1,2-galactosyltransferase activities, respectively. The objective of this work was to examine their ability to utilize alternate donors and acceptors. When expressed in the heterologous host, each enzyme was able to extend the alternate LPS acceptor in vivo but they retained their natural donor specificity. In vitro assays were then performed to test the effect of substituting the epimeric donor sugar on incorporation efficiency with the natural LPS acceptor of the enzyme. Although each enzyme could utilize the alternate donor epimer, activity was compromised because of significant decreases in kcat and corresponding increases in Km(donor). Finally, in vitro assays were performed to probe acceptor preference in the absence of the cellular machinery. The results were enzyme-dependent: while an alternate acceptor had no significant effect on the kinetic behavior of His6-WaaT, His6-WaaJ showed a significantly decreased kcat and increased Km(acceptor). These results illustrate the differences in behavior between closely related glycosyltransferase enzymes involved in the synthesis of similar glycoconjugates and have implications for glycoengineering applications. Bacteria produce a variety of glycoconjugates. The diversity in their structures is afforded by an unparalleled range of glycosyltransferase enzymes that transfer sugars from activated donor substrates to acceptor substrates. Bacterial enzymes have provided some influential models to assess glycosyltransferase structure and function because of the relative ease of their manipulation. One source of glycosyltransferase diversity is lipopolysaccharide (LPS) 2The abbreviations used are:LPSlipopolysaccharideCAZycarbohydrateactive enzymescore OScore oligosaccharideKdo3-deoxy-d-manno-octulosonic acidUDP-Glcuridine-5′-diphosphoglucoseGalgalactoseGlcglucoseGlcNAcN-acetylglucosamineGlcNglucosamineEtNethanolamineAmpampicillinGmgentamycinCEcapillary electrophoresisMSmass spectrometryNMRnuclear magnetic resonanceKDN2-keto-3-deoxy-d-glycero-d-galacto-nononic acid assembly, and the focus of this study are the enzymes involved in biosynthesis of the core oligosaccharide region (core OS). The outer leaflet of the Gram-negative outer membrane contains LPS as a major component. LPS is comprised of three structural domains: lipid A, core OS, and O antigen (1Raetz C.R.H. Whitfield C. Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3422) Google Scholar). Escherichia coli isolates produce one of five core OS types: K-12, R1, R2, R3, and R4 (reviewed in Ref. 2Amor K. Heinrichs D.E. Frirdich E. Ziebell K. Johnson R.P. Whitfield C. Infect. Immun. 2000; 68: 1116-1124Crossref PubMed Scopus (137) Google Scholar), and there are at least two core OS types in Salmonella isolates (3Kaniuk N.A. Monteiro M.A. Parker C.T. Whitfield C. Mol. Microbiol. 2002; 46: 1305-1318Crossref PubMed Scopus (18) Google Scholar). The backbone of the inner (lipid A proximal) core OS is typically conserved, and the various core types primarily arise from differences in inner core substitution and the structure of the part of the outer core, which provides the attachment site for O antigen. The genetic basis for these differences has been described (3Kaniuk N.A. Monteiro M.A. Parker C.T. Whitfield C. Mol. Microbiol. 2002; 46: 1305-1318Crossref PubMed Scopus (18) Google Scholar, 4Heinrichs D.E. Yethon J.A. Whitfield C. Mol. Microbiol. 1998; 30: 221-232Crossref PubMed Scopus (286) Google Scholar). lipopolysaccharide carbohydrateactive enzymes core oligosaccharide 3-deoxy-d-manno-octulosonic acid uridine-5′-diphosphoglucose galactose glucose N-acetylglucosamine glucosamine ethanolamine ampicillin gentamycin capillary electrophoresis mass spectrometry nuclear magnetic resonance 2-keto-3-deoxy-d-glycero-d-galacto-nononic acid However, moving from the experimentally derived polysaccharide structure to the assignment of a specific glycosyltransferase involved in a particular linkage is not straightforward. Even when candidate genes have been identified, their DNA sequences alone cannot predict which donor or acceptor substrates will be used by the glycosyltransferase. Experiments involving in vivo complementation of chromosomal insertion mutants and subsequent PAGE analysis of LPS patterns do not necessarily directly address which gene product is producing the LPS alteration, nor do they identify the donor substrate used (5Kadam S.K. Rehemtulla A. Sanderson K.E. J. Bacteriol. 1985; 161: 277-284Crossref PubMed Google Scholar). Of the known and predicted outer core OS glycosyltransferases from E. coli, only WaaJ has undergone in vitro characterization with both native substrates and purified enzyme (6Leipold M.D. Kaniuk N.A. Whitfield C. J. Biol. Chem. 2007; 282: 1257-1264Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Bacterial core OS biosynthesis requires the concerted action of specific glycosyltransferase enzymes. The outer core OS biosynthesis glycosyltransferases in E. coli provide an interesting collection of related (predicted) enzymes to examine principles of substrate (UDP-sugar) and linkage specificity (Fig. 1). WaaJ catalyzes the addition of an α-1,2-linked Glc to the outer core OS in R3 E. coli (7Kaniuk N.A. Vinogradov E. Li J. Monteiro M.A. Whitfield C. J. Biol. Chem. 2004; 279: 31237-31250Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar) and Salmonella enterica serovar Typhimurium (5Kadam S.K. Rehemtulla A. Sanderson K.E. J. Bacteriol. 1985; 161: 277-284Crossref PubMed Google Scholar), and the kinetic properties of the E. coli enzyme have been investigated (6Leipold M.D. Kaniuk N.A. Whitfield C. J. Biol. Chem. 2007; 282: 1257-1264Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). The related WaaT enzyme adds an α-1,2-linked Gal to the outer core OS in R1 and R4 E. coli (8Heinrichs D.E. Yethon J.A. Amor P.A. Whitfield C. J. Biol. Chem. 1998; 273: 29497-29505Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar), whereas WaaR is required for the addition of an α-1,2-glucose to the outer core OS in K-12 and R2 E. coli (8Heinrichs D.E. Yethon J.A. Amor P.A. Whitfield C. J. Biol. Chem. 1998; 273: 29497-29505Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). While WaaJ, WaaR, and WaaT all belong to glycosyltransferase CAZy family 8 and catalyze the same α-1,2 linkage of the donor sugar to their lipid-linked acceptors, they utilize different UDP-sugar donor substrates and have different terminal sugars on their acceptor LPS. However the predicted activities for WaaR and WaaT have not been examined directly. Here we examine the donor and acceptor specificity of these glycosyltransferases enzymes in a combination of in vivo and in vitro approaches to generate a better understanding of their substrate specificities and the potential for their manipulation in glycoengineering. Bacterial Strains and Plasmids—The prototypes for the R1, R2, and R3 core OSs are E. coli F470, F632, and F653 respectively; all are rough mutants, i.e. lacking O antigen (9Schmidt G. Fromme I. Mayer H. Eur. J. Biochem. 1970; 14: 357-366Crossref PubMed Scopus (53) Google Scholar). CWG350 (waaJ:aacC1) is a derivative of F653 (7Kaniuk N.A. Vinogradov E. Li J. Monteiro M.A. Whitfield C. J. Biol. Chem. 2004; 279: 31237-31250Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). CWG309 (waaT: aacC1) and CWG308 (waaO:aacC1) are derivatives of F470 (8Heinrichs D.E. Yethon J.A. Amor P.A. Whitfield C. J. Biol. Chem. 1998; 273: 29497-29505Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). These mutants have been described previously and are marked by the gentamycin-resistance cassette (aacC1). E. coli TOP10 cells F– mcrA Δ(mrr-hsdRMS-mcrBC) f80ΔlacZM15 ΔlacX74 deoR recA1 araD139 Δ(ara-leu)7697 galU galK rpsL (Strr) endA1 nupG were purchased from Invitrogen and used for expression of the glycosyltransferases. Plasmid pWQ272 is a pBAD18-derivative (10Guzman L-M. Belin D. Carson M.J. Beckwith J. J. Bacteriol. 1995; 177: 4121-4130Crossref PubMed Scopus (3976) Google Scholar) containing the coding sequence for an N-terminally hexahistidine-tagged His6-WaaJ fusion protein and was previously reported (6Leipold M.D. Kaniuk N.A. Whitfield C. J. Biol. Chem. 2007; 282: 1257-1264Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Plasmid pWQ269 is a derivative of pBAD18 engineered to contain a plasmid-encoded ribosome binding site and N-terminal His6-tag 5′ of the multicloning site in an organization identical to pWQ272 (supplemental Table S1). The waaR and waaT genes were amplified by PCR and cloned into pWQ269. Plasmid pWQ270 contains the coding sequence for His6-WaaR amplified from E. coli F632 genomic DNA (purified using the InstaGene kit, Bio-Rad), while pWQ271 contains the coding sequence for His6-WaaT from pWQ905 (8Heinrichs D.E. Yethon J.A. Amor P.A. Whitfield C. J. Biol. Chem. 1998; 273: 29497-29505Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). All oligonucleotides were synthesized by Sigma Genosys and are listed in supplemental Table S1. The sequences of all of the constructs were confirmed to be error-free by sequencing at the Guelph Molecular Supercenter (University of Guelph). Complementation Experiments to Assess in Vivo Activity of His6-Waa* Derivatives—Function of the various glycosyltransferases was established by electrotransformation of E. coli CWG350 (waaJ:aacC1), CWG309 (waaT:aacC1), and CWG308 (waaO:aacC1) with plasmids encoding the appropriate Waa* glycosyltransferase. Cultures of transformed bacteria were grown overnight at 37 °C in LB containing 100 μg/ml ampicillin, and 0.1-ml aliquots were then used to inoculate 5-ml cultures of the same medium supplemented with 0.02% l-arabinose to induce expression from the pBAD promoter in pBAD18 (10Guzman L-M. Belin D. Carson M.J. Beckwith J. J. Bacteriol. 1995; 177: 4121-4130Crossref PubMed Scopus (3976) Google Scholar). After growth at 37 °C for 5 h, SDS-proteinase K whole cell lysate samples were made following the procedure of Hitchcock and Brown (11Hitchcock P.J. Brown T.M. J. Bacteriol. 1983; 154: 269-277Crossref PubMed Google Scholar). LPS molecular species in these samples were then separated by electrophoresis using 4–12% gradient NuPage gels (Invitrogen). Electrophoresis was carried out at 150 V for 75 min. The gels were silver-stained using standard methods (12Tsai C.M. Frasch C.E. Anal. Biochem. 1982; 119: 115-119Crossref PubMed Scopus (2317) Google Scholar). The extent of complementation was determined by scanning the gels using a Bio-Rad GS-800 Calibrated Densitometer and determining the relative amounts of the two major bands with QuantityOne software. Production and Purification of LPS and Core Oligosaccharides—The LPS (3453 g/mol calculated molecular weight CWG350 LPS; 3368 g/mol calculated molecular weight CWG309 LPS) was purified from F653, CWG350, CWG309, CWG350 (pWQ271), CWG350 (pWQ270), and CWG309 (pWQ272). The LPS was extracted from cells harvested from 6-liter cultures and isolated by phenol/chloroform/petroleum ether method (13Galanos C. Luderitz O. Westphal O. Eur. J. Biochem. 1969; 9: 245-249Crossref PubMed Scopus (1369) Google Scholar), as previously described (7Kaniuk N.A. Vinogradov E. Li J. Monteiro M.A. Whitfield C. J. Biol. Chem. 2004; 279: 31237-31250Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). The isolated LPS was frozen and lyophilized. Working stocks for in vitro assays were stored as 2 mg/ml or 5 mg/ml aqueous solutions at –20 °C. Lipid A was removed by treating LPS (100 mg) in 5 ml of 2% AcOH at 100 °C for 3 h. Lipid A was removed as a precipitate by centrifugation, and soluble products were separated on a Sephadex G-50 column (2.5 × 95 cm) eluted in pyridinium/acetate buffer, pH 4.5 (4 ml of pyridine and 10 ml of AcOH in 1 liter of water). The eluate was monitored using a refractive index detector. The samples were then filtered through a SepPak C18 column (Waters) in water. Anion-exchange chromatography was performed on a 5-ml Hitrap Q column (Amersham Biosciences) in water for 10 min, then in a linear gradient of 0 to 1 M NaCl over 60 min with UV detection at 220 nm. To obtain a cleaner NMR spectrum, core oligosaccharide from CWG309 (pWQ272) (5 mg) was dephosphorylated by 48% aqueous hydrofluoric acid (0.1 ml) for 20 h at 4 °C. The HF was removed under a stream of nitrogen, and the product was then desalted by gel chromatography on a Sephadex G-15 column (1.6 × 80 cm) column using the pyridinium acetate buffer, pH 4.5 (4 ml of pyridine and 10 ml of AcOH in 1 liter of water) as eluant. The eluant was monitored by a refractive index detector and collected fractions were then lyophilized before use. Compositional and Methylation Analysis—For compositional analysis, oligosaccharides were hydrolyzed in 4 m CF3CO2H (120 °C, 3 h), and monosaccharides were converted into the alditol acetate derivatives. The products were analyzed by gas-liquid chromatography (GC) on an Agilent 6850 chromatograph equipped with DB-17 (30 m × 0.25 mm) fused-silica column using a temperature gradient of 180 °C (2 min) to 240 °C at 2 °C/min. Methylation analysis was performed using Ciucanu-Kerek procedure (14Ciucanu I. Kerek F. Carbohydr. Res. 1984; 131: 209-217Crossref Scopus (3215) Google Scholar). Methylated products were hydrolyzed, and the monosaccharides were converted to 1d-alditol acetates by conventional methods and analyzed by GC-MS. GC-MS was performed on Varian Saturn 2000 system equipped with an ion-trap mass spectral detector using the same column. NMR Spectroscopy—NMR spectra were recorded at 25 °C in D2O on a Varian UNITY INOVA 600 instrument using acetone as reference (1H, 2.225 ppm and 13C, 31.45 ppm). Varian standard programs COSY, NOESY (mixing time of 300 ms), TOCSY (spinlock time 120 ms), HSQC, and gHMBC (evolution delay of 100 ms) were used with digital resolution in F2 dimension <2 Hz/point for proton-proton correlations. Spectra were assigned using the computer program Pronto. The chemical shift data is presented in supplemental Table S2 according to the labeling scheme in supplemental Fig. S1. Mass Spectrometry—CE-MS spectra were acquired using a 4000 QTrap mass spectrometer (Applied Biosystems/Sciex, Concord, ON, Canada) with CE injection system (Prince Technologies, Netherlands). CE separation was obtained on a 90-cm length of bare fused-silica capillary (365 μm OD × 50 μm ID) with CE-MS coupling using a liquid sheath-flow interface and isopropyl alcohol:methanol (2:1) as the sheath liquid. An aqueous buffer consisting of 30 mm morpholine was used for all experiments in the negative-ion mode. The MS data are presented in supplemental Table S3. Overexpression, Localization, and Purification of Waa* Proteins—Overexpression and cellular location of the His6-WaaT and His6-WaaR enzymes was monitored by Western immunoblotting, essentially as described previously for His6-WaaJ (6Leipold M.D. Kaniuk N.A. Whitfield C. J. Biol. Chem. 2007; 282: 1257-1264Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Cell-free lysates of E. coli CWG309 containing pWQ271 and E. coli TOP10 containing pWQ270 were separated by ultracentrifugation. The soluble fraction was collected and the membrane pellet was washed twice with 2 ml of 50 mm Tris-HCl, pH 7.5. The fraction volumes were adjusted to facilitate direct comparison of the amount of membrane protein corresponding to a given amount of soluble protein. Protein samples were separated by SDS-PAGE and transferred onto polyvinylidene difluoride membrane. The Western immunoblots were developed using HisProbe H3 mouse anti-His6 primary antibody (Santa Cruz Biotechnology, Santa Cruz, CA), and goat anti-mouse alkaline phosphatase-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, Inc., Montréal, QB). Nitro blue tetrazolium from Sigma and 5-bromo-4-chloro-3-indolylphosphate from Roche were used as substrates to develop the Western blots. The bands were quantified by densitometry using a Bio-Rad GS-800 Calibrated Densitometer with QuantityOne software. The purification of His6-WaaJ from overexpression in E. coli TOP10 cells has been described elsewhere (6Leipold M.D. Kaniuk N.A. Whitfield C. J. Biol. Chem. 2007; 282: 1257-1264Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Purification of His6-WaaT was done in a similar fashion using Ni2+-NTA affinity chromatography, except batch binding was done directly using cell-free lysate, without prior removal of membrane material. In addition, 150 mm NaCl and 7.5 mm imidazole were included in the lysis buffer during sonication and in initial washing of the affinity column. The purified proteins were dispensed in 0.2-ml aliquots for single use, and total protein concentration was determined using the Bio-Rad Protein Assay with bovine serum albumin as the standard. His6-WaaJ and His6-WaaT were stable in this form at 4 °C for periods of up to 2 weeks. Storage at –20 °C offered no additional stability and enzyme samples thawed after storage at –80 °C showed significant loss of activity. It should be noted that the level of overexpression of His6-WaaT was lower than His6-WaaJ. Yields of purified protein were also compromised by the tendency of His6-WaaT to adsorb to filters used in concentration. In Vitro Determination of the Activity of His6-Waa* Enzymes—The activity of the various constructs was determined as previously described (6Leipold M.D. Kaniuk N.A. Whitfield C. J. Biol. Chem. 2007; 282: 1257-1264Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). The reaction is based on the transfer of radioactivity from [14C-Glc]UDP (Perkin Elmer, 200.0 mCi/mmol) or [14C-Gal]UDP (Perkin Elmer, 258.0 mCi/mmol) donor to an acceptor comprising the LPS isolated from E. coli CWG350 (waaJ:aacC1) or CWG309 (waaT:aacC1). Final reaction conditions after the addition of enzyme were: 1–750 μm UDP-sugar donor, 2–750 μm LPS, 100 mm Tris pH 7.5, 0.4 mm EDTA, 5 mm MgCl2, and 200–900 nm His6-WaaJ or His6-WaaT in a final volume of 0.1 ml. The His6-WaaJ or His6-WaaT concentration was adjusted to ensure that it was always at least 5-fold below the lowest substrate concentration. The rates at each substrate concentration were then fit to the Michaelis-Menten equation to determine kcat and Km. These values should be considered as “apparent” because of the stopped nature of the assay. Comparison of WaaJ, WaaR, and WaaT Sequences—Previous work from our laboratory established the in vivo and in vitro properties of WT WaaJ (6Leipold M.D. Kaniuk N.A. Whitfield C. J. Biol. Chem. 2007; 282: 1257-1264Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). The purpose of this work is to expand that analysis to other CAZy family 8 glycosyltransferases involved in E. coli LPS biosynthesis, particularly ones employing alternate donor sugars and terminal acceptor linkages. As might be anticipated for E. coli LPS core oligosaccharide biosynthesis proteins that all belong to glycosyltransferase family 8, WaaJ, WaaR, and WaaT share significant primary sequence identity (∼40%) and similarity (∼60%) and several highly conserved regions (supplemental Fig. S2). The sequences of these proteins predict no transmembrane helices. However, their nascent lipid A-core OS acceptor is membrane-associated, indicating that a membrane association might be beneficial for activity. His6-WaaT was found to be 75% membrane-associated, and His6-WaaR was found to be 64% membrane-associated (data not shown), compared with 55% for His6-WaaJ (6Leipold M.D. Kaniuk N.A. Whitfield C. J. Biol. Chem. 2007; 282: 1257-1264Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). As described for WaaJ (6Leipold M.D. Kaniuk N.A. Whitfield C. J. Biol. Chem. 2007; 282: 1257-1264Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar), the WaaT and WaaR sequences could be threaded onto the LgtC crystal structure with good alignment of secondary structure (data not shown), but the difference in primary sequences limited the usefulness of this information and precluded any meaningful prediction of residues that might dictate specificity. Function of His6-Waa* Derivatives in Vivo—To investigate whether His6-WaaT, His6-WaaJ, and His6-WaaR could function to extend waaJ:aacC1 or waaT:aacC1 LPS in vivo, plasmids encoding the relevant protein were transformed into CWG350 (waaJ:aacC1) and CWG309 (waaT:aacC1) and the resulting LPS profile was investigated by silver-stained PAGE. As shown in Fig. 2, WaaR was able to extend the LPS acceptor from CWG350 (waaJ:aacC1) to generate a product that comigrated with the wild-type LPS, despite the fact that the native acceptor residue for WaaR is a 1,3-linked Glc, rather than the 1,3-linked Gal provided by CWG350 LPS. The CWG350 LPS acceptor lacks three hexose residues (see below) compared with the wild type and migrates significantly faster, thus WaaR is able to form a product that can then be further extended by additional glycosyltransferases. WaaT was also able to extend the 1,3-linked Gal acceptor but the product remained smaller than the wild type; its migration was consistent with the absence of a single residue. Each of the glycosyltransferases was able to extend the 1,3-linked Glc acceptor provided by CWG309 (waaT:aacC1) LPS to restore a wild-type profile, indicating that WaaW (Fig. 1) could utilize their products to add the final core OS residue in the F470 (R1 core OS) background. To further probe the acceptor specificities, each of these enzymes were expressed in CWG308 (waaO:aacC1) cells. Interestingly, no core OS extension occurred in this mutant (Fig. 2C). Despite the observation (above), indicating that WaaJ, WaaR, and WaaT did not discriminate between 1,3-linked Gal or 1,3-linked Glc acceptors, the enzymes are apparently sensitive to the position of the acceptor residue in the context of the core OS structure. While silver-stained PAGE LPS profiling is a facile method to test the ability of a protein to extend a given LPS, it does not give any information about the identity of the sugars added. To identify the sugars added in the above LPS extensions, LPS was isolated from CWG350, CWG350 (pWQ271), CWG350 (pWQ270), CWG350 (pWQ272), and CWG309 (pWQ272). The structures of the LPS molecules were determined by NMR and MS methods (supplemental Tables S2 and S3). The resulting structures are shown graphically in Fig. 3. The CWG350 LPS contains a Gal-(1, 2)-Glc outer core and lacks the 2 terminal residues expected from the waaJ mutation (Fig. 1). Also missing is the side-chain GlcNAc residue. This result was consistent with the PAGE profile with respect to the size of the products but differs from the CWG350 structure obtained previously, where traces of GlcNAc were reported (7Kaniuk N.A. Vinogradov E. Li J. Monteiro M.A. Whitfield C. J. Biol. Chem. 2004; 279: 31237-31250Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). The NMR analysis of the CWG350 core OS was repeated and no evidence of the GlcNAc residue could be detected; there was no detectable peak corresponding to an N-acetyl group. Moreover, the MS data also reflected a core OS species lacking GlcNAc. The reason for this disparity between the structures of CWG350 is unknown and the results shown below indicate that the currently unidentified transferase required for GlcNAc addition (7Kaniuk N.A. Vinogradov E. Li J. Monteiro M.A. Whitfield C. J. Biol. Chem. 2004; 279: 31237-31250Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar) is still active in the strains used here. In CWG350 cells expressing either WaaR or WaaJ, the authentic LPS structure was restored. WaaR is normally a UDP-glucose:(glucosyl) LPS α-1,2-glucosyltransferase. However, when active in the context of CWG350, it shows UDP-glucose: (galactosyl) LPS α-1,2-glucosyltransferase activity. As expected, the LPS molecule resulting from WaaR or WaaJ activity serves as an acceptor for WaaD and the still unidentified GlcNAc transferase to complete the core OS structure. In contrast, WaaT added only a single residue to CWG350 LPS. While WaaT is a UDP-galactose:(glucosyl) LPS α-1,2-galactosyltransferase in its wildtype background, in CWG350 it exhibits UDP-galactose:(galactosyl) LPS α-1,2-galactosyltransferase activity. It therefore retains its normal donor specificity but can effectively utilize a different acceptor. The resulting product is not further extended by either WaaD or the GlcNAc transferase, indicating that these transferases are either sensitive to perturbations in acceptor structure, or lose critical protein-protein interactions when the precise combination of glycosyltransferase enzymes changes. CWG309 cells expressing WaaJ were able to form a complete core OS. However, Glc was incorporated as the second-to-last hexose in the core OS backbone. WaaJ is normally a UDP-glucose:(galactosyl) LPS α-1,2-glucosyltransferase. Thus, in the CWG309 background, WaaJ shows UDP-glucose:(glucosyl) LPS α-1,2-glucosyltransferase activity. This demonstrates that, like WaaT and WaaR, it also retains its normal donor specificity in a heterologous background. The published structure for CWG309 core OS lacks the β-Glc sidechain, suggesting this residue is added late in the assembly process (8Heinrichs D.E. Yethon J.A. Amor P.A. Whitfield C. J. Biol. Chem. 1998; 273: 29497-29505Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). The full extension of the CWG309 (pWQ272) LPS product implies that WaaV and WaaW are unaffected by the local changes in their acceptor structure. This differs from the situation described above for WaaD acting in CWG350 (Fig. 1). In Vitro Kinetic Behavior of His6-WaaJ and His6-WaaT Proteins—The in vivo results demonstrate two basic principles: (i) His6-WaaT, His6-WaaR, and His6-WaaJ have a marked preference for their specific donor substrates; (ii) these enzymes appear to have a relaxed specificity for alternate LPS acceptor residues, providing the overall size of the acceptor is conserved. His6-WaaJ and His6-WaaT were therefore selected for in vitro analysis to more precisely determine the effect of alternate donor and acceptor substrates on their kinetic properties. Those results are summarized in Table 1.TABLE 1Kinetic parameters for His6-WaaJ and His6-WaaT glycosyltransferasesProteinAcceptor LPSDonorkcataData were calculated as the average and propagated error of the kcat values as determined from the acceptor and donor Michaelis-Menten fits.Km(LPS)bValues were determined at saturating concentrations of UDP-sugar donor (150 μm).Km(donor)cValues were determined at saturating concentrations of LPS acceptor (150 μm).min-1μmμmWaaJCWG350 (waaJ::aacC1)UDP-Glc28 ± 4dValues reproduced from Ref. 6.11 ± 3dValues reproduced from Ref. 6.32 ± 8dValues reproduced from Ref. 6.CWG350 (waaJ::aacC1)UDP-Gal4.1 ± 0.327 ± 7200 ± 30CWG309 (waaT::aacC1)UDP-Glc8.2 ± 0.535 ± 340 ± 5WaaTCWG350 (waaJ::aacC1)UDP-Gal3.7 ± 0.525 ± 836 ± 9CWG309 (waaT::aacC1)UDP-Gal4.6 ± 0.237 ± 342 ± 7CWG309 (waaT::aacC1)UDP-GlcNDeLimitations of protein concentration and reduced catalytic activity prevented full characterization. See “Results” for details.NDeLimitations of protein concentration and reduced catalytic activity prevented full characterization.