Characterization of WbpB, WbpE, and WbpD and Reconstitution of a Pathway for the Biosynthesis of UDP-2,3-diacetamido-2,3-dideoxy-d-mannuronic Acid in Pseudomonas aeruginosa

The lipopolysaccharide of Pseudomonas aeruginosa PAO1 contains an unusual sugar, 2,3-diacetamido-2,3-dideoxy-d-mannuronic acid (d-ManNAc3NAcA). wbpB, wbpE, and wbpD are thought to encode oxidase, transaminase, and N-acetyltransferase enzymes. To characterize their functions, recombinant proteins were overexpressed and purified from heterologous hosts. Activities of His6-WbpB and His6-WbpE were detected only when both proteins were combined in the same reaction. Using a direct MALDI-TOF mass spectrometry approach, we identified ions that corresponded to the predicted products of WbpB (UDP-3-keto-d-GlcNAcA) and WbpE (UDP-d-GlcNAc3NA) in the coupled enzyme-substrate reaction. Additionally, in reactions involving WbpB, WbpE, and WbpD, an ion consistent with the expected product of WbpD (UDP-d-GlcNAc3NAcA) was identified. Preparative quantities of UDP-d-GlcNAc3NA and UDP-d-GlcNAc3NAcA were enzymatically synthesized. These compounds were purified by high-performance liquid chromatography, and their structures were elucidated by NMR spectroscopy. This is the first report of the functional characterization of these proteins, and the enzymatic synthesis of UDP-d-GlcNAc3NA and UDP-d-GlcNAc3NAcA. The lipopolysaccharide of Pseudomonas aeruginosa PAO1 contains an unusual sugar, 2,3-diacetamido-2,3-dideoxy-d-mannuronic acid (d-ManNAc3NAcA). wbpB, wbpE, and wbpD are thought to encode oxidase, transaminase, and N-acetyltransferase enzymes. To characterize their functions, recombinant proteins were overexpressed and purified from heterologous hosts. Activities of His6-WbpB and His6-WbpE were detected only when both proteins were combined in the same reaction. Using a direct MALDI-TOF mass spectrometry approach, we identified ions that corresponded to the predicted products of WbpB (UDP-3-keto-d-GlcNAcA) and WbpE (UDP-d-GlcNAc3NA) in the coupled enzyme-substrate reaction. Additionally, in reactions involving WbpB, WbpE, and WbpD, an ion consistent with the expected product of WbpD (UDP-d-GlcNAc3NAcA) was identified. Preparative quantities of UDP-d-GlcNAc3NA and UDP-d-GlcNAc3NAcA were enzymatically synthesized. These compounds were purified by high-performance liquid chromatography, and their structures were elucidated by NMR spectroscopy. This is the first report of the functional characterization of these proteins, and the enzymatic synthesis of UDP-d-GlcNAc3NA and UDP-d-GlcNAc3NAcA. Gram-negative organisms such as Pseudomonas aeruginosa produce lipopolysaccharide (LPS) 4The abbreviations used are: LPS, lipopolysaccharide; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; FPLC, fast-protein liquid chromatography; MS, mass spectrometry; TOCSY, total correlation spectroscopy; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser effect spectroscopy; CE, capillary electrophoresis. as an essential component of the outer leaflet of the outer membrane. LPS can be conceptually divided into three parts: lipid A, which anchors LPS into the membrane; core oligosaccharide, which contributes to membrane stability; and the O-antigen, which is a polysaccharide that extends away from the cell surface. In P. aeruginosa, two types of O-antigen are observed: A-band O-antigen, which is common to most strains, and B-band O-antigen, which is variable and therefore used as the basis of the International Antigenic Typing Scheme (1Liu P.V. Wang S. J. Clin. Microbiol. 1990; 28: 922-925Crossref PubMed Google Scholar). P. aeruginosa serotypes O2, O5, O16, O18, and O20 collectively belong to serogroup O2, because they all share common backbone sugar structures in their O-antigen repeat units consisting of two di-N-acetylated uronic acids and one 2-acetamido-2,6-dideoxy-d-galactose (N-acetyl-d-fucosamine). The minor structural variations in the O-antigen repeat units that differentiate this serogroup into five serotypes are: the type of glycosidic linkage between O-units (alpha versus beta) that is formed by the O-antigen polymerase (Wzy), isomers present (d-mannuronic or l-guluronic acid), and acetyl group substituents (2Knirel Y.A. Vinogradov E.V. Kocharova N.A. Paramonov N.A. Kochetkov N.K. Dmitriev B.A. Stanislavsky E.S. Lanyi B. Acta Microbiol. Hung. 1988; 35: 3-24PubMed Google Scholar, 3Knirel Y.A. Kochetkov N.K. Biochemistry. 1994; 59: 1325-1382Google Scholar, 4Newton G.J. Daniels C. Burrows L.L. Kropinski A.M. Clarke A.J. Lam J.S. Mol. Microbiol. 2001; 39: 1237-1247Crossref PubMed Google Scholar). The B-band O-antigen of P. aeruginosa PAO1 (serotype O5) contains a repeating trisaccharide of 2-acetamido-3-acetamidino-2,3-dideoxy-d-mannuronic acid (d-ManNAc3NAmA), 2,3-diacetamido-2,3-dideoxy-d-mannuronic acid (d-ManNAc3NAcA), and 2-acetamido-2,6-dideoxy-d-galactose (3Knirel Y.A. Kochetkov N.K. Biochemistry. 1994; 59: 1325-1382Google Scholar). The biosynthesis of the two mannuronic acid derivatives has yet to be fully understood and has been the subject of investigation by our group. To produce UDP-d-ManNAc3NAcA, a five-step pathway has been proposed (Fig. 1) that requires the products of five genes localized to the B-band O-antigen biosynthesis cluster (5Raymond C.K. Sims E.H. Kas A. Spencer D.H. Kutyavin T.V. Ivey R.G. Zhou Y. Kaul R. Clendenning J.B. Olson M.V. J. Bacteriol. 2002; 184: 3614-3622Crossref PubMed Scopus (117) Google Scholar). The O-antigen biosynthesis cluster was shown to be identical for all serotypes within serogroup O2, which further underscores the high similarity between these serotypes (5Raymond C.K. Sims E.H. Kas A. Spencer D.H. Kutyavin T.V. Ivey R.G. Zhou Y. Kaul R. Clendenning J.B. Olson M.V. J. Bacteriol. 2002; 184: 3614-3622Crossref PubMed Scopus (117) Google Scholar). The five genes, including wbpA, wbpB, wbpE, wbpD, and wbpI, have been shown to be essential for B-band LPS biosynthesis, because knockout mutants of each of these genes are deficient in B-band O-antigen (6Burrows L.L. Pigeon K.E. Lam J.S. FEMS Microbiol. Lett. 2000; 189: 135-141Crossref PubMed Google Scholar, 7Westman E.L. Preston A. Field R.A. Lam J.S. J. Bacteriol. 2008; 190: 6060-6069Crossref PubMed Scopus (26) Google Scholar, 8Wenzel C.Q. Daniels C. Keates R.A. Brewer D. Lam J.S. Mol. Microbiol. 2005; 57: 1288-1303Crossref PubMed Scopus (24) Google Scholar). Homologs of all five of the proteins required for the UDP-d-ManNAc3NAcA biosynthesis pathway are conserved in other bacterial pathogens, including Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica. Cross-complementation of P. aeruginosa knockout mutants lacking wbpA, wbpB, wbpE, wbpD, or wbpI with the homologues from B. pertussis could fully restore LPS production in the P. aeruginosa LPS mutants, suggesting that the genes from B. pertussis are functional homologs of the wbp genes (7Westman E.L. Preston A. Field R.A. Lam J.S. J. Bacteriol. 2008; 190: 6060-6069Crossref PubMed Scopus (26) Google Scholar). Homologs of these genes could be identified in diverse bacterial species, demonstrating the importance of UDP-d-ManNAc3NAcA biosynthesis beyond its role in P. aeruginosa (7Westman E.L. Preston A. Field R.A. Lam J.S. J. Bacteriol. 2008; 190: 6060-6069Crossref PubMed Scopus (26) Google Scholar). The first enzyme of the UDP-d-ManNAc3NAcA biosynthesis pathway, WbpA, is a 6-dehydrogenase that converts UDP-2-acetamido-2-deoxy-d-glucose (N-acetyl-d-glucosamine; UDP-d-GlcNAc) to UDP-2-acetamido-2-deoxy-d-glucuronic acid (N-acetyl-d-glucosaminuronic acid, UDP-d-GlcNAcA) using NAD+ as a coenzyme (9Miller W.L. Wenzel C.Q. Daniels C. Larocque S. Brisson J.R. Lam J.S. J. Biol. Chem. 2004; 279: 37551-37558Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar) (Fig. 1). Following this, the second step in UDP-d-ManNAc3NAcA biosynthesis is proposed to be an oxidation reaction catalyzed by WbpB, forming UDP-2-acetamido-2-deoxy-d-ribo-hex-3-uluronic acid (3-keto-d-GlcNAcA), which in turn is used as the substrate for transamination by WbpE, creating UDP-2-acetamido-3-amino-2,3-dideoxy-d-glucuronic acid (d-GlcNAc3NA). This residue is thought to be the substrate for WbpD, a putative N-acetyltransferase of the hexapeptide acyltransferase superfamily (10Vuorio R. Hirvas L. Vaara M. FEBS Lett. 1991; 292: 90-94Crossref PubMed Scopus (24) Google Scholar) that requires acetyl-CoA as a co-substrate (8Wenzel C.Q. Daniels C. Keates R.A. Brewer D. Lam J.S. Mol. Microbiol. 2005; 57: 1288-1303Crossref PubMed Scopus (24) Google Scholar). WbpD has been proposed to synthesize UDP-2,3-diacetamido-2,3-dideoxy-d-glucuronic acid (UDP-d-GlcNAc-3NAcA), which is utilized in the B-band O-antigen of P. aeruginosa serotype O1. In P. aeruginosa serogroup O2, the UDP-d-GlcNAc3NAcA is then epimerized by WbpI to create the UDP-d-ManNAc3NAcA required for incorporation into B-band LPS (11Westman E.L. McNally D.J. Rejzek M. Miller W.L. Kannathasan V.S. Preston A. Maskell D.J. Field R.A. Brisson J.R. Lam J.S. Biochem. J. 2007; 405: 123-130Crossref PubMed Scopus (17) Google Scholar). A derivative of UDP-d-ManNAc3NAcA is also used in the synthesis of B-band O-antigen of P. aeruginosa serogroup O2. UDP-d-ManNAc3NAmA is thought to be produced through additional modification of UDP-d-ManNAc3NAcA via the action of WbpG, an amidotransferase, which has also been demonstrated to be essential for the production of B-band O-antigen (12Burrows L.L. Charter D.F. Lam J.S. Mol. Microbiol. 1996; 22: 481-495Crossref PubMed Scopus (115) Google Scholar, 13Rocchetta H.L. Burrows L.L. Lam J.S. Microbiol. Mol. Biol. Rev. 1999; 63: 523-553Crossref PubMed Google Scholar). In the current study, our aim was to define the of WbpB, WbpE, and WbpD, because only has been for the of and (7Westman E.L. Preston A. Field R.A. Lam J.S. J. Bacteriol. 2008; 190: 6060-6069Crossref PubMed Scopus (26) Google and the reaction catalyzed by WbpD could be demonstrated to the of its UDP-d-GlcNAc3NA (8Wenzel C.Q. Daniels C. Keates R.A. Brewer D. Lam J.S. Mol. Microbiol. 2005; 57: 1288-1303Crossref PubMed Scopus (24) Google Scholar). The functional characterization of these proteins is also for LPS biosynthesis in B. pertussis, because the genes in the LPS of this species, and could of wbpB, wbpE, and wbpD, when in P. aeruginosa PAO1 (7Westman E.L. Preston A. Field R.A. Lam J.S. J. Bacteriol. 2008; 190: 6060-6069Crossref PubMed Scopus (26) Google Scholar). these three proteins a for the of and be for the biosynthesis of a of other and MALDI-TOF mass spectrometry were used to reaction of WbpB and WbpE and that the expected products were produced only when both were present the enzymatic synthesis of the product of both which was demonstrated to be UDP-d-GlcNAc3NA by NMR was a because this sugar has been produced by UDP-d-GlcNAc3NA was also essential for as the substrate of WbpD, which only to the enzymatic of this also the enzymatic synthesis of UDP-d-GlcNAc3NAcA to be as this sugar been produced through a synthesis (11Westman E.L. McNally D.J. Rejzek M. Miller W.L. Kannathasan V.S. Preston A. Maskell D.J. Field R.A. Brisson J.R. Lam J.S. Biochem. J. 2007; 405: 123-130Crossref PubMed Scopus (17) Google Scholar, M. Kannathasan V.S. C. Preston A. E.L. Lam J.S. Maskell D.J. Field R.A. Chem. PubMed Scopus Google the enzymatic reaction the of with and a of both and as with our also the in in the pathway for the biosynthesis of UDP-d-ManNAc3NAcA in P. and of and used have been 1). was in with cell were in and the was by for and then to of The was with and then with A was used to the into and and used in this or O5, type A.M. J. Bacteriol. PubMed Google with with for of by to of polymerase to with or of with between and D. Biochem. J. 1999; PubMed Scopus Google from PAO1 into to W.L. Wenzel C.Q. Daniels C. Larocque S. Brisson J.R. Lam J.S. J. Biol. Chem. 2004; 279: 37551-37558Abstract Full Text Full Text PDF PubMed Scopus (36) Google from PAO1 into to His6-WbpB E.L. Preston A. Field R.A. Lam J.S. J. Bacteriol. 2008; 190: 6060-6069Crossref PubMed Scopus (26) Google from PAO1 into to His6-WbpE E.L. Preston A. Field R.A. Lam J.S. J. Bacteriol. 2008; 190: 6060-6069Crossref PubMed Scopus (26) Google from PAO1 into to C.Q. Daniels C. Keates R.A. Brewer D. Lam J.S. Mol. Microbiol. 2005; 57: 1288-1303Crossref PubMed Scopus (24) Google E.L. McNally D.J. Rejzek M. Miller W.L. Kannathasan V.S. Preston A. Maskell D.J. Field R.A. Brisson J.R. Lam J.S. Biochem. J. 2007; 405: 123-130Crossref PubMed Scopus (17) Google Scholar) in a His6-WbpE was in using the for that were with the was and and the used was a with Using FPLC, His6-WbpE was with The was and His6-WbpE was using the Biochem. PubMed Scopus Google Scholar). and were purified as (8Wenzel C.Q. Daniels C. Keates R.A. Brewer D. Lam J.S. Mol. Microbiol. 2005; 57: 1288-1303Crossref PubMed Scopus (24) Google Scholar, W.L. Wenzel C.Q. Daniels C. Larocque S. Brisson J.R. Lam J.S. J. Biol. Chem. 2004; 279: 37551-37558Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, E.L. McNally D.J. Rejzek M. Miller W.L. Kannathasan V.S. Preston A. Maskell D.J. Field R.A. Brisson J.R. Lam J.S. Biochem. J. 2007; 405: 123-130Crossref PubMed Scopus (17) Google that an was used for in with purified were and using using from C. A. S. A. The Google Scholar). was from using purified as W.L. McNally D.J. Brewer D. Brisson J.R. A.M. Lam J.S. J. Biol. Chem. 2008; Full Text Full Text PDF PubMed Scopus (36) Google Scholar). His6-WbpB reactions and with His6-WbpE also and with additional reactions used of each enzyme were to and were capillary WbpI reactions were as (11Westman E.L. McNally D.J. Rejzek M. Miller W.L. Kannathasan V.S. Preston A. Maskell D.J. Field R.A. Brisson J.R. Lam J.S. Biochem. J. 2007; 405: 123-130Crossref PubMed Scopus (17) Google Scholar). was using a with The capillary was with a and was each by with for and then for were by for and was were from the reactions using other was were with of acid in of in an and was the and to In were by an additional was using a MALDI-TOF with a were in and ion from to and using ion to all the ion and were and and The laser was to was using the and of acid in of acid in of The mass with using acid is to be of and a reaction and each of and the reaction was using The was to using an and a of from were and by putative UDP-d-GlcNAc3NA were and and then NMR the same was used that acetyl-CoA and of were in the reaction The for sugar was by using The synthesis of UDP-d-GlcNAc3NAcA be NMR sugar were in of and using a with a and NMR TOCSY, and and from were used for with a were used for residue and of and nuclear Overhauser NMR were with of the and the of was used as an In of WbpB and Pseudomonas that WbpB contains common to the and an the of the of predicted similarity to putative and WbpE was predicted to have similarity to a putative enzyme in of cell identified the same is a The was the other were also identified as of UDP-d-ManNAc3NAcA WbpB, WbpE, WbpD, and WbpI were purified to and consistent with their expected structures (Fig. with His6-WbpB that the was as This was when the was in with and an of was His6-WbpB also To this the was in the of and of of His6-WbpB were from a into of His6-WbpE is with most used with the of and an to of His6-WbpE was used for of from of the proteins were for the was or to of the proteins and to (9Miller W.L. Wenzel C.Q. Daniels C. Larocque S. Brisson J.R. Lam J.S. J. Biol. Chem. 2004; 279: 37551-37558Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, E.L. McNally D.J. Rejzek M. Miller W.L. Kannathasan V.S. Preston A. Maskell D.J. Field R.A. Brisson J.R. Lam J.S. Biochem. J. 2007; 405: 123-130Crossref PubMed Scopus (17) Google Scholar). of to a reaction product from His6-WbpB reactions all because product were when with one was and was identified as the reaction with additional (Fig. In the reaction between His6-WbpE and a product could be (Fig. when His6-WbpB and His6-WbpE were to a reaction two were and a of the substrate (Fig. This is thought to be which is the predicted substrate for reactions were in with both His6-WbpB and His6-WbpE reactions to produce the putative substrate in with and also because is a required for by CE, these reactions the of and an of the putative UDP-d-GlcNAc3NA that was in the and reactions (Fig. reactions with and produced that were identical to from the reactions with WbpB, WbpE, and WbpD reactions with and or and were by NMR of the reaction the same putative UDP-d-GlcNAc3NA the substrate by chromatography, were and of these were by and the of to the The were shown to two one that with and one that of the these were and to NMR In the of the putative product could be to by the acetyl-CoA were and of each were with and the of UDP-d-GlcNAc3NAcA. by that with the UDP-d-GlcNAc3NAcA were and further by NMR spectroscopy. with using the enzymatically UDP-d-GlcNAc3NAcA the production of a this was in reactions enzymes. to a reaction using UDP-d-GlcNAc3NAcA as a that the with UDP-d-ManNAc3NAcA of WbpB, WbpE, and WbpD spectrometry was used to the same reaction by only His6-WbpB or the of an ion or that of the In the reaction both His6-WbpB and His6-WbpE demonstrated the of three and (Fig. with and ions and as as a ion (Fig. The ion was in reactions that because only and were A consisting of UDP-d-GlcNAc3NAcA in reaction a (Fig. NMR of and purified product of the coupled reaction was in of and by NMR (Fig. The NMR one for (Fig. and of was used to for and as as to (Fig. and a which was by and using a Using a was to be its (Fig. In in was to (11Westman E.L. McNally D.J. Rejzek M. Miller W.L. Kannathasan V.S. Preston A. Maskell D.J. Field R.A. Brisson J.R. Lam J.S. Biochem. J. 2007; 405: 123-130Crossref PubMed Scopus (17) Google Scholar). the of mass as as these NMR the product of the reaction was to be the of TOCSY, NOESY, and the purified reaction product of the WbpD reaction was to be The of was by of and as as that were identical to for (11Westman E.L. McNally D.J. Rejzek M. Miller W.L. Kannathasan V.S. Preston A. Maskell D.J. Field R.A. Brisson J.R. Lam J.S. Biochem. J. 2007; 405: 123-130Crossref PubMed Scopus (17) Google Scholar, M. Kannathasan V.S. C. Preston A. E.L. Lam J.S. Maskell D.J. Field R.A. Chem. PubMed Scopus Google for the of the WbpE reaction product and WbpD reaction product and and in a of the five required for the UDP-d-ManNAc3NAcA biosynthesis pathway in P. aeruginosa have been overexpressed and In this the of His6-WbpB and His6-WbpE was because these were required for the synthesis of the substrate for which been its was (8Wenzel C.Q. Daniels C. Keates R.A. Brewer D. Lam J.S. Mol. Microbiol. 2005; 57: 1288-1303Crossref PubMed Scopus (24) Google Scholar). and were chromatography, was the that was required to these proteins for of an such as the of of the five proteins used in this His6-WbpB was for to required the of and high that are with His6-WbpE was also to in this was to by into the with in this in the of was as as the was into of of acetyl-CoA to has also been shown to (8Wenzel C.Q. Daniels C. Keates R.A. Brewer D. Lam J.S. Mol. Microbiol. 2005; 57: 1288-1303Crossref PubMed Scopus (24) Google Scholar). was used to the enzymatic reactions produced of UDP-d-GlcNAc3NAcA with acetyl-CoA in the WbpD reactions the of that be consistent with the of this we used a to this and other reactions for products that have with other compounds the of enzyme reaction has been using liquid to the of the in these including proteins, and that to MALDI-TOF is to the of and a direct and for reactions that quantities of This the of a substrate and product with a mass with His6-WbpB and His6-WbpE demonstrated the production of a by and an ion consistent with the mass of UDP-d-GlcNAc3NA was identified by This that His6-WbpB and His6-WbpE were the expected and for the second and step in the proposed UDP-d-ManNAc3NAcA biosynthesis an ion consistent with the mass of was detected by and only in the reaction both His6-WbpB and This direct of the oxidation of and that WbpB and WbpE have to to ions consistent with the mass of and UDP-d-GlcNAc3NA were only in reactions His6-WbpB and His6-WbpE were both is to the of WbpB as the and WbpE the The of reaction when WbpE was with by both and MS, demonstrated that WbpE could as a This in reactions with both WbpE be using a product by WbpB to produce because in the by MS, ions consistent with and UDP-d-GlcNAc3NA were our that WbpB is an that first to the of and WbpE is an that to produce The acid of WbpB and WbpE also this because WbpB contains putative and a for the of WbpE a high of to proteins of the B. pertussis was shown to a that they have the same (7Westman E.L. Preston A. Field R.A. Lam J.S. J. Bacteriol. 2008; 190: 6060-6069Crossref PubMed Scopus (26) Google Scholar). has been shown to as a structural and A. C. A. D. and J. WbpB is a enzyme that be UDP-2-acetamido-2-deoxy-d-glucuronic acid and the enzyme the of or as WbpE also and be acid with in to His6-WbpB and His6-WbpE by that the UDP-d-GlcNAc3NA was and the that an ion consistent with UDP-d-GlcNAc3NAcA was from NMR and of this by which was shown to be (11Westman E.L. McNally D.J. Rejzek M. Miller W.L. Kannathasan V.S. Preston A. Maskell D.J. Field R.A. Brisson J.R. Lam J.S. Biochem. J. 2007; 405: 123-130Crossref PubMed Scopus (17) Google further that the product of reaction is UDP-d-GlcNAc3NAcA. with that WbpD to the hexapeptide acyltransferase superfamily of proteins and acetyl-CoA as a (8Wenzel C.Q. Daniels C. Keates R.A. Brewer D. Lam J.S. Mol. Microbiol. 2005; 57: 1288-1303Crossref PubMed Scopus (24) Google our of the enzymatic of WbpD for the first This enzyme be the UDP-2-acetamido-3-amino-2,3-dideoxy-d-glucuronic acid N-acetyltransferase WbpB, WbpE, and WbpD to the of a group forming UDP-d-GlcNAc3NAcA. or are such as and and the biosynthesis of these has been J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar, A. A. P. P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, A. S. A. P. C. P. Biochem. J. 2008; PubMed Scopus (36) Google Scholar). The of requires the the transaminase, and the genes A. A. P. P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). and are to WbpE and WbpD, in of acid required and conserved the other a group via of A. A. P. P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) and similarity to WbpB, the required for the of The biosynthesis of is to the pathway in this study, and as the genes that encode transaminase, and A. S. A. P. C. P. Biochem. J. 2008; PubMed Scopus (36) Google Scholar). and are also to WbpE and WbpD, only similarity to WbpD all the other share The of requires an and a J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar). similarity to WbpE, similarity is detected between and In all these the product could be purified to and and reactions were therefore used to the product in a reaction J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar, A. A. P. P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, A. S. A. P. C. P. Biochem. J. 2008; PubMed Scopus (36) Google Scholar). was as we reactions with the WbpB and WbpE transaminase, to our this is the first report of the of a using the mass spectrometry between WbpB and WbpE was by the that was detected in is that with to the This is the because the was also to an ion consistent with when only WbpB was used in a reaction. is that WbpE WbpB through direct or the of WbpE the of WbpB by the reactions products have been using a of the could be because of with the substrate and of the product A. S. A. P. C. P. Biochem. J. 2008; PubMed Scopus (36) Google Scholar). A. S. A. P. C. P. Biochem. J. 2008; PubMed Scopus (36) Google Scholar) that to product This that the enzyme the group was of with that could in the of J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar). to between WbpB and WbpE are A to that for the of a group was to create a or group in and Y. Y. Y. Y.A. Wang L. L. J. Bacteriol. 2007; 189: PubMed Scopus Google Scholar). In this is to by a an then the of to which is further by an to Y. Y. Y. Y.A. Wang L. L. J. Bacteriol. 2007; 189: PubMed Scopus Google Scholar). WbpB, WbpE, and WbpD only similarity to these proteins, because both the substrate and are These the of and as a required to an group a The synthesis of UDP-d-GlcNAc3NA has been by enzymatic or This required the of three purified and a of using a that only from each of the In such a is to an for all because that one of the reactions be to one or of the other In of UDP-d-ManNAc3NAcA as is to produce LPS in P. aeruginosa the reaction. of and NMR of the purified to the of UDP-d-GlcNAc3NA were used to UDP-d-GlcNAc3NAcA from reactions using and UDP-d-GlcNAc3NAcA was produced by the required and an of M. Kannathasan V.S. C. Preston A. E.L. Lam J.S. Maskell D.J. Field R.A. Chem. PubMed Scopus Google Scholar). The enzymatic required reactions that were in a Following the was used to UDP-d-GlcNAc3NAcA a of the of substrate versus of purified from both and NMR identified the of the product of WbpD reaction to be demonstrating that this enzymatic synthesis a the synthesis This is the first that in of the UDP-d-ManNAc3NAcA biosynthesis pathway has been and these have importance beyond LPS biosynthesis in P. B. pertussis proteins and have been shown to have the same as WbpB, WbpE, and WbpD, (7Westman E.L. Preston A. Field R.A. Lam J.S. J. Bacteriol. 2008; 190: 6060-6069Crossref PubMed Scopus (26) Google Scholar). this also the of as a UDP-2-acetamido-2-deoxy-d-glucuronic acid as a acid transaminase, and as a UDP-2-acetamido-3-amino-2,3-dideoxy-d-glucuronic acid The to enzymatically synthesize UDP-d-GlcNAc3NAcA and UDP-d-ManNAc3NAcA additional B. pertussis is to Bordetella and Bordetella organisms also produce and as of the LPS A. J. R. J. B. Maskell D.J. 1999; PubMed Google Scholar, M. D. FEMS Microbiol. Lett. PubMed Scopus Google Scholar). is thought to be from UDP-d-ManNAc3NAcA by the of the cluster Preston A. Rejzek M. Field R.A. Maskell D.J. J. Mol. Biol. 2007; PubMed Scopus Google further from enzymatic production of the predicted UDP-d-GlcNAc3NA also be to the to enzyme or other as for UDP-d-GlcNAc3NAcA Rejzek M. Field R.A. J. Biol. Chem. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). In we have the and of and which has been and from CE, MS, and NMR the of the WbpB, WbpE, and WbpD as a UDP-2-acetamido-2-deoxy-d-glucuronic acid acid transaminase, and UDP-2-acetamido-3-amino-2,3-dideoxy-d-glucuronic acid of these three in to for the synthesis and of UDP-d-GlcNAc3NA or which were by purified UDP-d-GlcNAc3NAcA and the This the of in of the UDP-d-ManNAc3NAcA biosynthesis pathway from P. Rejzek for a of UDP-d-GlcNAc3NAcA.

Récupéré en direct depuis OpenAlex et désinversé. Les résumés ne sont pas conservés dans cette base de données : les index inversés représentent 8,6 Go des 9,3 Go de texte de la base, et le serveur dispose de 13 Go libres.