Biochemical Characterization of WbpA, a UDP-N-acetyl-d-glucosamine 6-Dehydrogenase Involved in O-antigen Biosynthesis in Pseudomonas aeruginosa PAO1

WbpA (PA3159) is an enzyme involved in the biosynthesis of unusual di-N-acetyl-d-mannosaminuronic acid-derived sugar nucleotides found in the O antigen of Pseudomonas aeruginosa PAO1 (serotype O5). The wbpA gene that encodes this enzyme was cloned into pET-28a, overexpressed as a histidine-tagged fusion protein, and purified by nickel chelation chromatography. Capillary electrophoresis was used to examine substrate conversion by WbpA, and the data revealed that WbpA is a UDP-N-acetyl-d-glucosamine 6-dehydrogenase (EC 1.1.1.136), which uses NAD+ as a coenzyme. The enzyme reaction product was purified by HPLC and analyzed using NMR spectroscopy. Our results showed unequivocally that the product of the WbpA reaction is UDP-N-acetyl-d-glucosaminuronic acid. WbpA requires either NH4+ or K+ for activity and the accompanying anions exert secondary effects on activity consistent with their ranking in the Hofmeister series. Kinetic analysis showed positive cooperativity with respect to UDP-N-acetyl-d-glucosamine binding with a K0.5 of 94 μm, a kcat of 86 min–1, and a Hill coefficient of 1.8. In addition, WbpA has a K0.5 for NAD+ of 220 μm, a kcat of 86 min–1, and a Hill coefficient of 1.1. The oligomerization state of WbpA was analyzed by gel filtration, dynamic light scattering, and analytical ultracentrifugation, with all three techniques indicating that WbpA exists as a trimer in solution. However, tertiary structure predictions suggested a tetramer, which was supported by data from transmission electron microscopy. The electron micrograph of negatively stained WbpA samples revealed structures with 4-fold symmetry. WbpA (PA3159) is an enzyme involved in the biosynthesis of unusual di-N-acetyl-d-mannosaminuronic acid-derived sugar nucleotides found in the O antigen of Pseudomonas aeruginosa PAO1 (serotype O5). The wbpA gene that encodes this enzyme was cloned into pET-28a, overexpressed as a histidine-tagged fusion protein, and purified by nickel chelation chromatography. Capillary electrophoresis was used to examine substrate conversion by WbpA, and the data revealed that WbpA is a UDP-N-acetyl-d-glucosamine 6-dehydrogenase (EC 1.1.1.136), which uses NAD+ as a coenzyme. The enzyme reaction product was purified by HPLC and analyzed using NMR spectroscopy. Our results showed unequivocally that the product of the WbpA reaction is UDP-N-acetyl-d-glucosaminuronic acid. WbpA requires either NH4+ or K+ for activity and the accompanying anions exert secondary effects on activity consistent with their ranking in the Hofmeister series. Kinetic analysis showed positive cooperativity with respect to UDP-N-acetyl-d-glucosamine binding with a K0.5 of 94 μm, a kcat of 86 min–1, and a Hill coefficient of 1.8. In addition, WbpA has a K0.5 for NAD+ of 220 μm, a kcat of 86 min–1, and a Hill coefficient of 1.1. The oligomerization state of WbpA was analyzed by gel filtration, dynamic light scattering, and analytical ultracentrifugation, with all three techniques indicating that WbpA exists as a trimer in solution. However, tertiary structure predictions suggested a tetramer, which was supported by data from transmission electron microscopy. The electron micrograph of negatively stained WbpA samples revealed structures with 4-fold symmetry. Pseudomonas aeruginosa produces two chemically and anti-genically distinct forms of lipopolysaccharide (LPS) 1The abbreviations used are: LPS, lipopolysaccharide; CE, capillary electrophoresis; CE-IEF, capillary electrophoresis isoelectric focusing; COSY, correlation spectroscopy; HMBC, heteronuclear multiple bond correlation; HMQC, heteronuclear multiple quantum coherence; HSQC, heteronuclear single quantum coherence; IPTG, isopropyl thio-β-d-galactopyranoside; MIP, 1-l-myo-inositol-1-phosphate; TEM, transmission electron microscopy; TOCSY, total correlation spectroscopy; UDP-d-GalNAc, UDP-N-acetyl-d-galactosamine; UDP-d-GalNAcA, UDP-N-acetyl-d-galactosaminuronic acid; UDP-d-GlcNAc, UDP-N-acetyl-d-glucosamine; UDP-d-GlcNAcA, UDP-N-acetyl-d-glucosaminuronic acid; UDP-d-ManNAc, UDP-N-acetyl-d-mannosamine; UDP-d-ManNAcA, UDP-N-acetyl-d-mannosaminuronic acid; MES, 4-morpholineethanesulfonic acid. molecules, known as A-band and B-band O antigen. The B-band O antigen is an important virulence factor for evasion of host defenses and confers resistance to phagocytosis and serum-mediated killing (1Hancock R.E. Mutharia L.M. Chan L. Darveau R.P. Speert D.P. Pier G.B. Infect. Immun. 1983; 42: 170-177Crossref PubMed Google Scholar, 2Cryz Jr., S.J. Pitt T.L. Furer E. Germanier R. Infect. Immun. 1984; 44: 508-513Crossref PubMed Google Scholar, 3Engels W. Endert J. Kamps M.A. van Boven C.P. Infect. Immun. 1985; 49: 182-189Crossref PubMed Google Scholar, 4Dasgupta T. de Kievit T.R. Masoud H. Altman E. Richards J.C. Sadovskaya I. Speert D.P. Lam J.S. Infect. Immun. 1994; 62: 809-817Crossref PubMed Google Scholar, 5Goldberg J.B. Pier G.B. Trends Microbiol. 1996; 4: 490-494Abstract Full Text PDF PubMed Scopus (44) Google Scholar, 6Schiller N.L. Hatch R.A. Diagn. Microbiol. Infect. Dis. 1983; 1: 145-157Crossref PubMed Scopus (22) Google Scholar). It is also required for virulence, as demonstrated in mouse challenge infection models, and it contributes to initial tissue damage and inflammatory responses in the lungs of cystic fibrosis patients (2Cryz Jr., S.J. Pitt T.L. Furer E. Germanier R. Infect. Immun. 1984; 44: 508-513Crossref PubMed Google Scholar, 7Tang H.B. DiMango E. Bryan R. Gambello M. Iglewski B.H. Goldberg J.B. Prince A. Infect. Immun. 1996; 64: 37-43Crossref PubMed Google Scholar). The B-band O antigen of P. aeruginosa PAO1 (serotype O5) contains a trisaccharide repeating unit composed of 2-acetamido-3-acetamidino-2,3-dideoxy-β-d-mannuronic acid, 2,3-diacetamido-2,3-dideoxy-β-d-mannuronic acid, and N-acetyl-α-d-fucosamine (8Knirel Y.A. Kochetkov N.K. Biochemistry (Moscow). 1994; 59: 1325-1383Google Scholar). These three sugar residues are postulated to be synthesized from the common precursor UDP-N-acetyl-d-glucosamine (UDP-d-GlcNAc) (9Burrows L.L. Charter D.F. Lam J.S. Mol. Microbiol. 1996; 22: 481-495Crossref PubMed Scopus (115) Google Scholar), and the initial steps in the biosynthesis of the fucosamine moiety have previously been described (10Creuzenet C. Schur M.J. Li J. Wakarchuk W.W. Lam J.S. J. Biol. Chem. 2000; 275: 34873-34880Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 11Creuzenet C. Lam J.S. Mol. Microbiol. 2001; 41: 1295-1310Crossref PubMed Scopus (49) Google Scholar). N-Acetyl-d-mannosaminuronic acid residues are present in both the enterobacterial common antigen (ECA) of Escherichia coli and the polysaccharide capsule of Staphylococcus aureus, and the biosynthesis of the nucleotide-activated precursor to these molecules, UDP-N-acetyl-d-mannosaminuronic acid (UDP-d-ManNAcA), is synthesized from UDP-d-GlcNAc via a two-step pathway. In the first step, WecB (E. coli) or Cap5P (S. aureus) catalyze a C-2 epimerization to form UDP-N-acetyl-d-mannosamine (UDP-d-ManNAc), and in the second step, WecC (E. coli) or Cap5O (S. aureus) catalyze a C-6 dehydrogenation to form UDP-d-ManNAcA (12Meier-Dieter U. Starman R. Barr K. Mayer H. Rick P.D. J. Biol. Chem. 1990; 265: 13490-13497Abstract Full Text PDF PubMed Google Scholar, 13Kiser K.B. Bhasin N. Deng L. Lee J.C. J. Bacteriol. 1999; 181: 4818-4824Crossref PubMed Google Scholar, 14Portoles M. Kiser K.B. Bhasin N. Chan K.H. Lee J.C. Infect. Immun. 2001; 69: 917-923Crossref PubMed Scopus (49) Google Scholar). WbpA and WbpI of P. aeruginosa are essential for B-band O-antigen biosynthesis, since wbpA and wbpI knockout mutants are devoid of B-band polysaccharide and produce rough LPS (15Burrows L.L. Pigeon K.E. Lam J.S. FEMS Microbiol. Lett. 2000; 189: 135-141Crossref PubMed Google Scholar). We previously proposed that biosynthesis of the mannosaminuronic acid-derived residues of P. aeruginosa would follow a similar pathway to UDP-d-ManNAcA biosynthesis based on ∼50% amino acid similarity between WecB and WbpI, and between WecC and WbpA (9Burrows L.L. Charter D.F. Lam J.S. Mol. Microbiol. 1996; 22: 481-495Crossref PubMed Scopus (115) Google Scholar, 15Burrows L.L. Pigeon K.E. Lam J.S. FEMS Microbiol. Lett. 2000; 189: 135-141Crossref PubMed Google Scholar). The current investigation of the activity of WbpA was prompted by the inability of wbpI and wbpA to complement wecB and wecC knockout mutants (15Burrows L.L. Pigeon K.E. Lam J.S. FEMS Microbiol. Lett. 2000; 189: 135-141Crossref PubMed Google Scholar). Interestingly, di-N-acetylated sugars have also been found to be constituents of LPS in other bacterial pathogens, including Bordetella pertussis and Bordetella parapertusis, causative agents of whooping cough, and Bordetella bronchiseptica, which causes upper respiratory tract infections in animals (16Preston A. Allen A.G. Cadisch J. Thomas R. Stevens K. Churcher C.M. Badcock K.L. Parkhill J. Barrell B. Maskell D.J. Infect. Immun. 1999; 67: 3763-3767Crossref PubMed Google Scholar). Here we show that WbpA (PA3159, Pseudomonas genome data base www.pseudomonas.com) is a 50.3 kDa protein that catalyzes a C-6 dehydrogenation reaction, but its activity differs from that of Cap5O and WecC in that it uses UDP-d-GlcNAc as a substrate instead of UDP-d-ManNAc. Furthermore, we propose that this represents the first step in a common pathway for the biosynthesis of di-N-acetylated hexosaminuronic acid sugar nucleotides. Materials—UDP-N-acetyl-d-glucosamine, UDP-N-acetyl-d-galactosamine (UDP-d-GalNAc), UDP-d-glucose, NAD+, NADH, NADP+, NADPH, kanamycin, and gel filtration molecular weight standards were obtained from Sigma-Aldrich (Oakville, ON, Canada). Ribonuclease A, carbonic anhydrase II, and β-lactoglobulin standards were obtained from were from and were from ON, Canada). The and E. coli were from and were from were with purified by a was to the of PubMed Scopus Google Scholar). were stained with The of purified WbpA was as described by PubMed Scopus Google Scholar). and of was cloned into the with a wbpA was from aeruginosa by using the which an which a The reaction of of of of and was the of of by of 94 and step of was the product and the were with and purified using and using The was by and and into the E. coli using for the of of was with of an and was to a of and was to for were by for and were of from of was in of binding The were by on and and were by for using the was as by the with nickel as the and binding as the the of and in purified WbpA was to and using a to the by the The of WbpA was analyzed by and purified WbpA was the of to a of of by Capillary were using a with and the The and acid and were as by the and the capillary was a capillary with a of and carbonic anhydrase and β-lactoglobulin as and was to the that an step of for was to of was using the and were in from a of the three of and UDP-d-GlcNAc and of the of enzyme and in a reaction of and were in and and in and were and the of the was in a the of was with the of a by of the of and in a reaction of and were or substrate conversion for the was by of of of enzyme in a reaction of UDP-d-GlcNAc, NAD+, and of of the in II, and of were and the of was in the as the described of and anions of the on the initial of WbpA using UDP-d-GlcNAc and NAD+ as in a of Kinetic and of enzyme in a of of for UDP-d-GlcNAc, NAD+ and of UDP-d-GlcNAc that from to of for NAD+, and of NAD+ that from to were and the of the was by in a the of was from and The data were to the Hill and were by from The Hill was from Hill The results are the of three of the by Capillary were using a with The was and the capillary was with a The capillary was by with for for and for were by for and the was and by was using the of the WbpA reaction of UDP-d-GlcNAc, NAD+, and of purified WbpA was for in a and was from the reaction by a The WbpA reaction product was purified by using an and a of the WbpA reaction product were and the was by of the by filtration to the the to by of the were and analyzed by NMR of the WbpA were using a with a The sugar was in of The were with of the The of was used as an and acid was used for the from COSY, HSQC, HMBC, and and were used for R. C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, B. K. Li J. Lee J.C. Lam J.S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. T. NMR of Google Scholar). HPLC molecular weight of WbpA in was with the of a gel filtration with a of and The was with carbonic anhydrase and as was as the correlation between and and of the with of WbpA was of the standards and WbpA the was from the is the and is the total The molecular of WbpA was from the of WbpA was by dynamic light in a with WbpA was a of in and of were as described previously J.S. Lam R.E. J. Bacteriol. PubMed Google with the The were on a of WbpA, the and on for were with a transmission electron an of for structure for WbpA were using both the M.J. J. Mol. Biol. 2000; PubMed Scopus Google and the were with known structures using the P. M.J. 2000; PubMed Scopus Google and protein structures were using R.A. Trends Full Text PDF PubMed Scopus Google acid were using the 1994; 22: PubMed Scopus Google Scholar). and of was to using the and E. coli host and of WbpA was of purified WbpA as from the be obtained from of The molecular of WbpA as from with the molecular of 50.3 analysis of WbpA the step showed with from to the step was WbpA as with an of which is the of analysis of were in from a of three carbonic anhydrase and β-lactoglobulin A, WbpA and standards with an WbpA and standards of WbpA analysis of UDP-d-GlcNAc and NAD+ revealed two reaction that and The product was to be by to an the product was as UDP-N-acetyl-d-glucosaminuronic acid by NMR analysis analysis showed that of NAD+ were for of UDP-d-GlcNAc and of were for of revealed conversion of UDP-d-GlcNAc revealed with UDP-d-glucose, or as to show the for UDP-d-GlcNAc as its of by is in were using and the on the sugar and the be obtained of and and of and an the the were the the C-6 and the were The and are in I. The the to the with and the was found to be and was The for the moiety were similar to previously R. C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). These results the that the structure of the WbpA reaction product is data for and in a of by is to catalyze the conversion of UDP-d-GlcNAc a with an between and WbpA is from to with activity between and The activity of WbpA be by in for of of is to catalyze the conversion of UDP-d-GlcNAc in the of and but conversion is in the of or The initial are the accompanying is and the accompanying is with a correlation between initial and the of the the Hofmeister 1985; PubMed Scopus Google Scholar). The of has a on initial on substrate in with an in the initial substrate conversion as to a of this as the of the substrate conversion of Kinetic of the data from with UDP-d-GlcNAc as the substrate and NAD+ as the substrate to the analyzed by of the data showed a of positive cooperativity a the data were to the Hill and the are in Hill of the UDP-d-GlcNAc data revealed a Hill coefficient of Kinetic analysis of with NAD+ as the substrate UDP-d-GlcNAc showed with a Hill coefficient of 1.1. In all were by from The data were also analyzed from and with similar for WbpA from Hill were in by from by from Hill from Hill were in by from in a of of results from analytical gel filtration showed that WbpA has an of of this to the molecular weight of for WbpA that WbpA to form a trimer in solution. results were obtained WbpA was analyzed by dynamic light scattering, which showed WbpA to have a in of and a molecular weight of However, WbpA was analyzed by and the revealed that have two of with and with 4-fold for of WbpA, including were by and Interestingly, the of 6-dehydrogenase from P. aeruginosa as the The of WbpA by contains based on of this of total amino acid residues of WbpA are in and of these are WbpA amino acid similarity and amino acid to It is that this WbpA has residues in of in that form bond with NADH, of that form to the of that form to and of that form to the WbpA is essential for B-band O-antigen biosynthesis in P. aeruginosa PAO1 (15Burrows L.L. Pigeon K.E. Lam J.S. FEMS Microbiol. Lett. 2000; 189: 135-141Crossref PubMed Google Scholar), and in this we show that it is a UDP-d-GlcNAc UDP-d-GlcNAc 6-dehydrogenase activity was previously in both and D.F. J. PubMed Scopus Google Scholar, T. N. H. N. E. J. 1985; PubMed Scopus Google Scholar), the enzyme was this is the first of and of this including analysis of analysis of WbpA demonstrated that it is to UDP-d-GlcNAc as a and based on the inability of wbpA to complement a wecC (15Burrows L.L. Pigeon K.E. Lam J.S. FEMS Microbiol. Lett. 2000; 189: 135-141Crossref PubMed Google Scholar), WbpA is to as a Our results that biosynthesis of the di-N-acetylated mannosaminuronic acid-derived sugar nucleotides of P. aeruginosa differs from that of the sugars of E. coli and of the with a C-6 dehydrogenation reaction biosynthesis of the first via an epimerization C-2 C-6 is also the with biosynthesis of UDP-N-acetyl-d-galactosaminuronic acid which with an epimerization C-6 dehydrogenation C. M. Wakarchuk W.W. Lam J.S. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar, C. M. E. Li J. Lam J.S. J. Biol. Chem. 2000; 275: Full Text Full Text PDF Google Scholar). Furthermore, to the of WbpA in P. aeruginosa that di-N-acetylated acid and di-N-acetylated acid, we that the WbpA reaction represents a common first step in the biosynthesis of di-N-acetylated hexosaminuronic acid from analysis also showed that WbpA requires of NAD+ for of UDP-d-GlcNAc and produces of for of which was by NMR analysis unequivocally to be on these and the known of van de I. J. PubMed Scopus Google Scholar), we that WbpA catalyzes the in WbpA has a of which with the of UDP-d-GlcNAc 6-dehydrogenase from A. and from M. and of from E. coli D.F. J. PubMed Scopus Google Scholar, T. N. H. N. E. J. 1985; PubMed Scopus Google Scholar, R. PubMed Scopus (22) Google Scholar). also with the results of that was previously by C. M. E. Li J. Lam J.S. J. Biol. Chem. 2000; 275: Full Text Full Text PDF Google Scholar). The for be to the of a since the for both 42: PubMed Scopus Google and R.E. van de I. J. Chem. Scopus Google by a to form a and a would to the that would be required for a is The based on amino acid with is which with from the of In initial of WbpA we that was essential for revealed that WbpA requires either NH4+ or K+ in for is in to that was required to of UDP-d-GlcNAc 6-dehydrogenase activity in from M. T. N. H. N. E. J. 1985; PubMed Scopus Google Scholar). Furthermore, results are consistent with of other including a from E. coli and that requires or Jr., J. PubMed Scopus (44) Google Scholar, E. A. K. H. J. 2001; PubMed Google Scholar). activity was in or the effects with NH4+ or K+ from of WbpA of the of these but that NH4+ and K+ are of NH4+ and K+ have both been as of from E. and the structure of that enzyme revealed a common binding in which either of these be R. J. Biol. 1996; PubMed Scopus Google Scholar). The for these in the binding of was to the of these to in with which is the bond of all other and R. J. Biol. 1996; PubMed Scopus Google Scholar). WbpA have a binding since the of a similar binding would a for the of NH4+ and K+ for It is to that which similar on NH4+ or K+ for this the that NH4+ are K+ are involved in the of of this is from with from both of which catalyze the conversion of to L. C. H. 2000; PubMed Scopus Google Scholar). the is by and the structure of this enzyme the of in the reaction of this enzyme J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). We also that the of the or to a in the enzyme activity of The effects of these anions with their the Hofmeister 1985; PubMed Scopus Google Scholar), the initial reaction is by anions of and by anions of as in These results are similar to that the of in enzyme activity J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Scholar), since WbpA is negatively These results are also consistent with the of by anions to their in the Hofmeister PubMed Scopus Google Scholar). of the data both a a The is the since an in the of with an in initial of the conversion of substrate to product with an in an initial is consistent with the with The also have initial the with as the their ranking in the Hofmeister series. WbpA has a K0.5 of 94 for its UDP-d-GlcNAc, which is for the M. or A. D.F. J. PubMed Scopus Google Scholar, T. N. H. N. E. J. 1985; PubMed Scopus Google indicating a binding between WbpA and its be since the two were purified The K0.5 for NAD+ for WbpA also showed binding the obtained for the M. and A. but these are to the of the enzyme In addition, the K0.5 of WbpA for NAD+ is also that for but this be to the in the substrate also in be by the that to be analysis be C. M. E. Li J. Lam J.S. J. Biol. Chem. 2000; 275: Full Text Full Text PDF Google Scholar). both UDP-d-GlcNAc and NAD+, the is Interestingly, the kcat of WbpA for both UDP-d-GlcNAc and are the for for and for indicating for a in activity or be a of the of It is that the kcat of WbpA for UDP-d-GlcNAc is for enzyme from P. aeruginosa PAO1 O-antigen biosynthesis, which is known to be in its activity C. Lam J.S. Mol. Microbiol. 2001; 41: 1295-1310Crossref PubMed Scopus (49) Google Scholar). analysis of WbpA data for UDP-d-GlcNAc showed the effects of positive an that is from of the In addition, these data to the Hill to the and with a Hill coefficient of that WbpA is an These results are since which is involved in biosynthesis, also positive cooperativity R. C. L. P. 41: PubMed Scopus Google Scholar). catalyze steps in and be in the form of or Furthermore, catalyze a step that represents the first step in a of the pathway. WbpA catalyzes the first step in the biosynthesis of di-N-acetylated mannosaminuronic acid residues of the B-band O the effects of positive cooperativity be an that this is a Hill coefficient of also that WbpA is in solution. analysis using gel filtration, dynamic light and analytical the that WbpA is a trimer in solution. These results are since that have been to have been as or structures R. PubMed Scopus (22) Google Scholar, 42: PubMed Scopus Google Scholar, R.E. van de I. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar). However, by and of WbpA with be the of the protein showed an of and an of 4-fold of a is in with a of the structure of from P. in which data suggested a structure in R. C. L. P. 41: PubMed Scopus Google the structure of the enzyme was the protein was found to as a of 42: PubMed Scopus Google Scholar). are between WbpA and amino acid the residues that are in WbpA the of which are for with and the sugar substrate in as as postulated to be involved in and in 42: PubMed Scopus Google Scholar). are also that be by of the structure of WbpA with the structure of but also in the electron of WbpA with the structure of In the electron micrograph of WbpA, are structures with and 4-fold as as structures is of the structures 4-fold is with the structure of similar be Furthermore, the structure of in is the structures similar to the other in the WbpA micrograph be These to that WbpA has a structure similar to gel filtration and dynamic light data that a light also that would be a and the for these results be to the of the contains and and data that WbpA has a similar this would the of the tetramer, it would also predictions by gel filtration and dynamic light scattering, which are based on is to WbpA in to this In we have that WbpA is a UDP-d-GlcNAc 6-dehydrogenase involved in the biosynthesis of di-N-acetylated mannosaminuronic acid-derived residues of the B-band O antigen in P. aeruginosa We have also that WbpA has and positive cooperativity with respect to UDP-d-GlcNAc we propose that the WbpA reaction represents a common first step in biosynthesis of di-N-acetylated hexosaminuronic acid sugar and the analysis of WbpA is an important initial step in the pathway. The results of this in with in the that UDP-d-GlcNAc is a precursor in bacterial polysaccharide has been to that this sugar is the precursor in the B. K. Li J. Lee J.C. Lam J.S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, B. N. Lam J.S. J. PubMed Google Scholar), and K. K. H. and J. for the proposed precursor for in the and (10Creuzenet C. Schur M.J. Li J. Wakarchuk W.W. Lam J.S. J. Biol. Chem. 2000; 275: 34873-34880Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 11Creuzenet C. Lam J.S. Mol. Microbiol. 2001; 41: 1295-1310Crossref PubMed Scopus (49) Google for UDP-d-GlcNAcA, the proposed precursor for in the and We the for in electron of the of the purified WbpA, and the for their with the dynamic light and analytical of The of for the electron and of the of for of the We the and for the of a

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