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Found 2 structures.
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| a-D-GlcpNAc3NA-(1---P---P---5)-U | Show graphically |
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Structure type: monomer
Trivial name: UDP-2-acetamido-3-amino-2,3-dideoxy-α-D-glucuronic acid, UDP-GlcNAc(3NH2)A, UDP-3-amino-GlcNAcA
The heteropolymeric O-antigen of the lipopolysaccharide from Pseudomonas aeruginosa serogroup O5 as well as the band-A trisaccharide from Bordetella pertussis contain the di-N-acetylated mannosaminuronic acid derivative, β-D-ManNAc3NAcA (2,3-diacetamido-2,3-dideoxy-β-D-mannuronic acid). The biosynthesis of the precursor for this sugar is proposed to require five steps, through which UDP-α-D-GlcNAc (UDP-N-acetyl-α-D-glucosamine) is converted via four steps into UDP-α-D-GlcNAc3NAcA (UDP-2,3-diacetamido-2,3-dideoxy-α-D-glucuronic acid), and this intermediate compound is then epimerized by WbpI (P. aeruginosa), or by its orthologue, WlbD (B. pertussis), to form UDP-α-D-ManNAc3NAcA (UDP-2,3-diacetamido-2,3-dideoxy-α-D-mannuronic acid). UDP-α-D-GlcNAc3NAcA, the proposed substrate for WbpI and WlbD, was obtained through chemical synthesis. His6-WbpI and His6-WlbD were overexpressed and then purified by affinity chromatography using FPLC. Capillary electrophoresis was used to analyse reactions with each enzyme, and revealed that both enzymes used UDP-α-D-GlcNAc3NAcA as a substrate, and reacted optimally in sodium phosphate buffer (pH 6.0). Neither enzyme utilized UDP-α-D-GlcNAc, UDP-α-D-GlcNAcA (UDP-2-acetamido-2,3-dideoxy-α-D-glucuronic acid) or UDP-α-D-GlcNAc3NAc (UDP-2,3-diacetamido-2,3-dideoxy-α-D-glucose) as substrates. His6-WbpI or His6-WlbD reactions with UDP-α-D-GlcNAc3NAcA produce a novel peak with an identical retention time, as shown by capillary electrophoresis. To unambiguously characterize the reaction product, enzyme-substrate reactions were allowed to proceed directly in the NMR tube and conversion of substrate into product was monitored over time through the acquisition of a proton spectrum at regular intervals. Data collected from one- and two-dimensional NMR experiments showed that His6-WbpI catalysed the 2-epimerization of UDP-α-D-GlcNAc3NAcA, converting it into UDP-α-D-ManNAc3NAcA. Collectively, these results provide evidence that WbpI and WlbD are UDP-2,3-diacetamido-2,3-dideoxy-α-D-glucuronic acid 2-epimerases
Lipopolysaccharide, O-antigen, Pseudomonas aeruginosa, 3-diacetamido-2, 2-epimerase, mannosaminuronic acid biosynthesis, sugar-nucleotide metabolism, UDP-2, 3-dideoxy-α-D-glucuronic acid
NCBI PubMed ID: 17346239Pseudomonas aeruginosa and Bordetella pertussis produce lipopolysaccharide (LPS) that contains 2,3-diacetamido-2,3-dideoxy-D-mannuronic acid (D-ManNAc3NAcA). A five-enzyme biosynthetic pathway that requires WbpA, WbpB, WbpE, WbpD and WbpI has been proposed for the production of this sugar in P. aeruginosa, based on analysis of genes present in the B-band LPS biosynthesis cluster. In the analogous B. pertussis cluster, homologs of wbpB-I are present but a putative dehydrogenase was missing; therefore, the biosynthetic mechanism for UDP-D-ManNAc3NAcA was unclear. Non-polar knockout mutants of each P. aeruginosa gene were constructed. Complementation analysis of the mutants demonstrated that B-band LPS production was restored to P. aeruginosa knockout mutants when the relevant B. pertussis genes were supplied in trans. Thus, the genes that encode the putative oxidase, transaminase, N-acetyltransferase, and epimerase enzymes in B. pertussis are functional homologs of those in P. aeruginosa. Two candidate dehydrogenase genes were located by searching the B. pertussis genome; these have 80% identity to P. aeruginosa wbpO (serotype O6) and 32% identity to wbpA (serotype O5). These genes, wbpO1629 and wbpO3150, were shown to complement a wbpA knockout in P. aeruginosa. Capillary electrophoresis was used to characterize the enzymatic activities of purified WbpO1629 and WbpO3150, and mass spectrometry analysis confirmed that the two enzymes are dehydrogenases capable of converting UDP-D-GlcNAc, to a lesser extent UDP-D-GalNAc, and to a much lesser extent, UDP-D-Glc. Together, these results suggest that B. pertussis produces UDP-D-ManNAc3NAcA through the same pathway proposed in P. aeruginosa, despite differences in the genomic context of the genes involved
Lipopolysaccharide, biosynthesis, Pseudomonas aeruginosa, gene cluster, Bordetella pertussis, WbpA, WbpI
NCBI PubMed ID: 18621892The B-band O-antigen of the lipopolysaccharide found in the opportunistic pathogen Pseudomonas aeruginosa PAO1 (serotype O5) comprises a repeating trisaccharide unit that is critical for virulence and protection from host defense systems. One of the carbohydrates in this repeating unit, the rare diacetylated aminuronic acid derivative 2,3-diacetamido-2,3-dideoxy-β-D-mannuronic acid (ManNAc(3NAc)A), is thought to be produced by five enzymes (WbpA, WbpB, WbpE, WbpD, and WbpI) in a stepwise manner starting from UDP-GlcNAc. Although the genes responsible for the biosynthesis of this sugar are known, only two of the five encoded proteins (WbpA and WbpI) have been thoroughly investigated. In this report, we describe the cloning, overexpression, purification, and biochemical characterization of the three central enzymes in this pathway, WbpB, WbpE, and WbpD. Using a combination of capillary electrophoresis, RP-HPLC, and NMR spectroscopy, we show that WbpB and WbpE are a dehydrogenase/aminotransferase pair that converts UDP-GlcNAcA to UDP-GlcNAc(3NH(2))A in a coupled reaction via a unique NAD(+) recycling pathway. In addition, we confirm that WbpD catalyzes the acetylation of UDP-GlcNAc(3NH(2))A to give UDP-GlcNAc(3NAc)A. Notably, WbpA, WbpB, WbpE, WbpD, and WbpI can be combined in vitro to generate UDP-ManNAc(3NAc)A in a single reaction vessel, thereby providing supplies of this complex glycosyl donor for future studies of lipopolysaccharide assembly. This work completes the biochemical characterization of the enzymes in this pathway and provides novel targets for potential therapeutics to combat infections with drug resistant P. aeruginosa strains.
biosynthesis, lipopolysaccharides, O-antigen, B-band, Pseudomonas aeruginosa, Enzymes, acetylation, Uridine Diphosphate Sugars, WbpA, WbpI, WbpB, WbpE, WbpD
NCBI PubMed ID: 19348502The 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 HPLC 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.
biosynthesis, Pseudomonas aeruginosa, MALDI-TOF mass spectrometry, enzymatic synthesis, Recombinant Proteins
NCBI PubMed ID: 19282284In recent years, the opportunistic pathogen Pseudomonas aeruginosa has emerged as a major source of hospital-acquired infections. Effective treatment has proven increasingly difficult due to the spread of multidrug resistant strains and thus requires a deeper understanding of the biochemical mechanisms of pathogenicity. The central carbohydrate of the P. aeruginosa PAO1 (O5) B-band O-antigen, ManNAc(3NAc)A, has been shown to be critical for virulence and is produced in a stepwise manner by five enzymes in the Wbp pathway (WbpA, WbpB, WbpE, WbpD and WbpI). Herein, we present the crystal structure of the aminotransferase WbpE from P. aeruginosa PAO1 in complex with the cofactor pyridoxal 5'-phosphate (PLP) and product UDP-GlcNAc(3NH2)A as the external aldimine at 1.9 A resolution. We also report the structures of WbpE in complex with PMP alone as well as the PLP internal aldimine, and show that the dimeric structure of WbpE observed in the crystal structure is confirmed by analytical ultracentrifugation. Analysis of these structures reveals that the active site of the enzyme is composed of residues from both subunits. In particular, we show that a key residue (Arg229), which has previously been implicated in direct interactions with the carboxylate moiety of α-ketoglutarate, is also uniquely positioned to bestow specificity for the 6' carboxyl group of GlcNAc(3NH2)A through a salt bridge. This finding is intriguing, because while an analogous basic residue is present in WbpE homologs that do not process C6'-carboxyl-modified saccharides, recent structural studies reveal that this side chain is retracted to accommodate a neutral C-6' carbon. This work represents the first structural analysis of a nucleotide sugar aminotransferase with a bound product modified at the C2', C3', and C6' positions and provides insight into a novel target for treatment of P. aeruginosa infection.
O-antigen, Pseudomonas aeruginosa, crystal structure, WbpA, WbpI, aminotransferase WbpE
NCBI PubMed ID: 20604544The pathogenic bacteria Pseudomonas aeruginosa and Bordetella pertussis contain in their outer membranes the rare sugar 2,3-diacetamido-2,3-dideoxy-d-mannuronic acid. Five enzymes are required for the biosynthesis of this sugar starting from UDP-N-acetylglucosamine. One of these, referred to as WlbB, is an N-acetyltransferase that converts UDP-2-acetamido-3-amino-2,3-dideoxy-d-glucuronic acid (UDP-GlcNAc3NA) to UDP-2,3-diacetamido-2,3-dideoxy-d-glucuronic acid (UDP-GlcNAc3NAcA). Here we report the three-dimensional structure of WlbB from Bordetella petrii. For this analysis, two ternary structures were determined to 1.43 A resolution: one in which the protein was complexed with acetyl-CoA and UDP and the second in which the protein contained bound CoA and UDP-GlcNAc3NA. WlbB adopts a trimeric quaternary structure and belongs to the LβH superfamily of N-acyltransferases. Each subunit contains 27 beta-strands, 23 of which form the canonical left-handed beta-helix. There are only two hydrogen bonds that occur between the protein and the GlcNAc3NA moiety, one between O(∆1) of Asn 84 and the sugar C-3' amino group and the second between the backbone amide group of Arg 94 and the sugar C-5' carboxylate. The sugar C-3' amino group is ideally positioned in the active site to attack the si face of acetyl-CoA. Given that there are no protein side chains that can function as general bases within the GlcNAc3NA binding pocket, a reaction mechanism is proposed for WlbB whereby the sulfur of CoA ultimately functions as the proton acceptor required for catalysis.
biosynthesis, Pseudomonas, Bordetella, Bordetella pertussis, pertussis, Enzymes, N-acetyltransferase, WlbB
NCBI PubMed ID: 20433200| a-D-GlcpNAc3NAcA-(1---P---P---5)-U | Show graphically |
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Structure type: monomer
Trivial name: UDP-2,3-diacetamido-2,3-dideoxy-α-D-glucuronic acid, UDP-GlcNAc(3NAc)A, UDP-Glc-2,3-diNAcA, UDP-D-GlcNAc3NAcA
The heteropolymeric O-antigen of the lipopolysaccharide from Pseudomonas aeruginosa serogroup O5 as well as the band-A trisaccharide from Bordetella pertussis contain the di-N-acetylated mannosaminuronic acid derivative, β-D-ManNAc3NAcA (2,3-diacetamido-2,3-dideoxy-β-D-mannuronic acid). The biosynthesis of the precursor for this sugar is proposed to require five steps, through which UDP-α-D-GlcNAc (UDP-N-acetyl-α-D-glucosamine) is converted via four steps into UDP-α-D-GlcNAc3NAcA (UDP-2,3-diacetamido-2,3-dideoxy-α-D-glucuronic acid), and this intermediate compound is then epimerized by WbpI (P. aeruginosa), or by its orthologue, WlbD (B. pertussis), to form UDP-α-D-ManNAc3NAcA (UDP-2,3-diacetamido-2,3-dideoxy-α-D-mannuronic acid). UDP-α-D-GlcNAc3NAcA, the proposed substrate for WbpI and WlbD, was obtained through chemical synthesis. His6-WbpI and His6-WlbD were overexpressed and then purified by affinity chromatography using FPLC. Capillary electrophoresis was used to analyse reactions with each enzyme, and revealed that both enzymes used UDP-α-D-GlcNAc3NAcA as a substrate, and reacted optimally in sodium phosphate buffer (pH 6.0). Neither enzyme utilized UDP-α-D-GlcNAc, UDP-α-D-GlcNAcA (UDP-2-acetamido-2,3-dideoxy-α-D-glucuronic acid) or UDP-α-D-GlcNAc3NAc (UDP-2,3-diacetamido-2,3-dideoxy-α-D-glucose) as substrates. His6-WbpI or His6-WlbD reactions with UDP-α-D-GlcNAc3NAcA produce a novel peak with an identical retention time, as shown by capillary electrophoresis. To unambiguously characterize the reaction product, enzyme-substrate reactions were allowed to proceed directly in the NMR tube and conversion of substrate into product was monitored over time through the acquisition of a proton spectrum at regular intervals. Data collected from one- and two-dimensional NMR experiments showed that His6-WbpI catalysed the 2-epimerization of UDP-α-D-GlcNAc3NAcA, converting it into UDP-α-D-ManNAc3NAcA. Collectively, these results provide evidence that WbpI and WlbD are UDP-2,3-diacetamido-2,3-dideoxy-α-D-glucuronic acid 2-epimerases
Lipopolysaccharide, O-antigen, Pseudomonas aeruginosa, 3-diacetamido-2, 2-epimerase, mannosaminuronic acid biosynthesis, sugar-nucleotide metabolism, UDP-2, 3-dideoxy-α-D-glucuronic acid
NCBI PubMed ID: 17346239Pseudomonas aeruginosa and Bordetella pertussis produce lipopolysaccharide (LPS) that contains 2,3-diacetamido-2,3-dideoxy-D-mannuronic acid (D-ManNAc3NAcA). A five-enzyme biosynthetic pathway that requires WbpA, WbpB, WbpE, WbpD and WbpI has been proposed for the production of this sugar in P. aeruginosa, based on analysis of genes present in the B-band LPS biosynthesis cluster. In the analogous B. pertussis cluster, homologs of wbpB-I are present but a putative dehydrogenase was missing; therefore, the biosynthetic mechanism for UDP-D-ManNAc3NAcA was unclear. Non-polar knockout mutants of each P. aeruginosa gene were constructed. Complementation analysis of the mutants demonstrated that B-band LPS production was restored to P. aeruginosa knockout mutants when the relevant B. pertussis genes were supplied in trans. Thus, the genes that encode the putative oxidase, transaminase, N-acetyltransferase, and epimerase enzymes in B. pertussis are functional homologs of those in P. aeruginosa. Two candidate dehydrogenase genes were located by searching the B. pertussis genome; these have 80% identity to P. aeruginosa wbpO (serotype O6) and 32% identity to wbpA (serotype O5). These genes, wbpO1629 and wbpO3150, were shown to complement a wbpA knockout in P. aeruginosa. Capillary electrophoresis was used to characterize the enzymatic activities of purified WbpO1629 and WbpO3150, and mass spectrometry analysis confirmed that the two enzymes are dehydrogenases capable of converting UDP-D-GlcNAc, to a lesser extent UDP-D-GalNAc, and to a much lesser extent, UDP-D-Glc. Together, these results suggest that B. pertussis produces UDP-D-ManNAc3NAcA through the same pathway proposed in P. aeruginosa, despite differences in the genomic context of the genes involved
Lipopolysaccharide, biosynthesis, Pseudomonas aeruginosa, gene cluster, Bordetella pertussis, WbpA, WbpI
NCBI PubMed ID: 18621892The B-band O-antigen of the lipopolysaccharide found in the opportunistic pathogen Pseudomonas aeruginosa PAO1 (serotype O5) comprises a repeating trisaccharide unit that is critical for virulence and protection from host defense systems. One of the carbohydrates in this repeating unit, the rare diacetylated aminuronic acid derivative 2,3-diacetamido-2,3-dideoxy-β-D-mannuronic acid (ManNAc(3NAc)A), is thought to be produced by five enzymes (WbpA, WbpB, WbpE, WbpD, and WbpI) in a stepwise manner starting from UDP-GlcNAc. Although the genes responsible for the biosynthesis of this sugar are known, only two of the five encoded proteins (WbpA and WbpI) have been thoroughly investigated. In this report, we describe the cloning, overexpression, purification, and biochemical characterization of the three central enzymes in this pathway, WbpB, WbpE, and WbpD. Using a combination of capillary electrophoresis, RP-HPLC, and NMR spectroscopy, we show that WbpB and WbpE are a dehydrogenase/aminotransferase pair that converts UDP-GlcNAcA to UDP-GlcNAc(3NH(2))A in a coupled reaction via a unique NAD(+) recycling pathway. In addition, we confirm that WbpD catalyzes the acetylation of UDP-GlcNAc(3NH(2))A to give UDP-GlcNAc(3NAc)A. Notably, WbpA, WbpB, WbpE, WbpD, and WbpI can be combined in vitro to generate UDP-ManNAc(3NAc)A in a single reaction vessel, thereby providing supplies of this complex glycosyl donor for future studies of lipopolysaccharide assembly. This work completes the biochemical characterization of the enzymes in this pathway and provides novel targets for potential therapeutics to combat infections with drug resistant P. aeruginosa strains.
biosynthesis, lipopolysaccharides, O-antigen, B-band, Pseudomonas aeruginosa, Enzymes, acetylation, Uridine Diphosphate Sugars, WbpA, WbpI, WbpB, WbpE, WbpD
NCBI PubMed ID: 19348502In connection with studies on lipopolysaccharide biosynthesis in respiratory pathogens we had a need to access potential biosynthetic intermediate sugar nucleotides. Herein we report the chemical synthesis of uridine 5'-diphospho 2,3-diacetamido-2,3-dideoxy-α-D-glucuronic acid (UDP-Glc-2,3-diNAcA) () from N-acetyl-D-glucosamine in 17 steps and approximately 9% overall yield. This compound has proved invaluable in the elucidation of biosynthetic pathways leading to the formation of 2,3-diacetamido-2,3-dideoxy-D-mannuronic acid-containing polysaccharides.
lipopolysaccharide biosynthesis, Bordetella pertussis, pathway, surface polysaccharide, chemical synthesis, sugar nucleotide
NCBI PubMed ID: 19262941The 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 HPLC 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.
biosynthesis, Pseudomonas aeruginosa, MALDI-TOF mass spectrometry, enzymatic synthesis, Recombinant Proteins
NCBI PubMed ID: 19282284In recent years, the opportunistic pathogen Pseudomonas aeruginosa has emerged as a major source of hospital-acquired infections. Effective treatment has proven increasingly difficult due to the spread of multidrug resistant strains and thus requires a deeper understanding of the biochemical mechanisms of pathogenicity. The central carbohydrate of the P. aeruginosa PAO1 (O5) B-band O-antigen, ManNAc(3NAc)A, has been shown to be critical for virulence and is produced in a stepwise manner by five enzymes in the Wbp pathway (WbpA, WbpB, WbpE, WbpD and WbpI). Herein, we present the crystal structure of the aminotransferase WbpE from P. aeruginosa PAO1 in complex with the cofactor pyridoxal 5'-phosphate (PLP) and product UDP-GlcNAc(3NH2)A as the external aldimine at 1.9 A resolution. We also report the structures of WbpE in complex with PMP alone as well as the PLP internal aldimine, and show that the dimeric structure of WbpE observed in the crystal structure is confirmed by analytical ultracentrifugation. Analysis of these structures reveals that the active site of the enzyme is composed of residues from both subunits. In particular, we show that a key residue (Arg229), which has previously been implicated in direct interactions with the carboxylate moiety of α-ketoglutarate, is also uniquely positioned to bestow specificity for the 6' carboxyl group of GlcNAc(3NH2)A through a salt bridge. This finding is intriguing, because while an analogous basic residue is present in WbpE homologs that do not process C6'-carboxyl-modified saccharides, recent structural studies reveal that this side chain is retracted to accommodate a neutral C-6' carbon. This work represents the first structural analysis of a nucleotide sugar aminotransferase with a bound product modified at the C2', C3', and C6' positions and provides insight into a novel target for treatment of P. aeruginosa infection.
O-antigen, Pseudomonas aeruginosa, crystal structure, WbpA, WbpI, aminotransferase WbpE
NCBI PubMed ID: 20604544The pathogenic bacteria Pseudomonas aeruginosa and Bordetella pertussis contain in their outer membranes the rare sugar 2,3-diacetamido-2,3-dideoxy-d-mannuronic acid. Five enzymes are required for the biosynthesis of this sugar starting from UDP-N-acetylglucosamine. One of these, referred to as WlbB, is an N-acetyltransferase that converts UDP-2-acetamido-3-amino-2,3-dideoxy-d-glucuronic acid (UDP-GlcNAc3NA) to UDP-2,3-diacetamido-2,3-dideoxy-d-glucuronic acid (UDP-GlcNAc3NAcA). Here we report the three-dimensional structure of WlbB from Bordetella petrii. For this analysis, two ternary structures were determined to 1.43 A resolution: one in which the protein was complexed with acetyl-CoA and UDP and the second in which the protein contained bound CoA and UDP-GlcNAc3NA. WlbB adopts a trimeric quaternary structure and belongs to the LβH superfamily of N-acyltransferases. Each subunit contains 27 beta-strands, 23 of which form the canonical left-handed beta-helix. There are only two hydrogen bonds that occur between the protein and the GlcNAc3NA moiety, one between O(∆1) of Asn 84 and the sugar C-3' amino group and the second between the backbone amide group of Arg 94 and the sugar C-5' carboxylate. The sugar C-3' amino group is ideally positioned in the active site to attack the si face of acetyl-CoA. Given that there are no protein side chains that can function as general bases within the GlcNAc3NA binding pocket, a reaction mechanism is proposed for WlbB whereby the sulfur of CoA ultimately functions as the proton acceptor required for catalysis.
biosynthesis, Pseudomonas, Bordetella, Bordetella pertussis, pertussis, Enzymes, N-acetyltransferase, WlbB
NCBI PubMed ID: 20433200| New query | Export IDs | Home | Help |
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