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Found 1 structure.
Displayed structure 1
| a-D-GlcpNAcA-(1---P---P---5)-U | Show graphically |
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Structure type: monomer
Trivial name: UDP-2-acetamido-2,3-dideoxy-α-D-glucuronic acid, UDP-N-acetyl-D-glucuronic acid, UDP-N-acetyl-α-D-glucosaminuronic acid, UDP-GlcNAcA, UDP-D-GlcNAcA, UDP-D-GlcNAc
Compound class: nucleoside diphosphate sugar
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 PAK (serotype O6) produces a single, polar, glycosylated flagellum composed of a-type flagellin. To determine whether or not flagellin glycosylation in this serotype requires O-antigen genes, flagellin was isolated from the wild type, three O-antigen deficient mutants wbpL, wbpO and wbpP, and a wbpO mutant complemented with a plasmid containing a wild-type copy of wbpO. Flagellin from the wbpO mutant was smaller (42 kDa) than that of the wild-type (45 kDa), or other mutants strains, and exhibited an altered isoelectric point (pI 4.8) when compared to PAK flagellin (pI 4.6). These differences were due to the truncation of the glycan moiety in the wbpO-flagellin. Thus, flagellin glycosylation in P. aeruginosa PAK apparently requires a functional WbpO but not WbpP. Since WbpP was previously proposed to catalyze a metabolic step in the biosynthesis of B-band O antigen that precedes the action of WbpO, these results prompted us to reevaluate the two-step pathway catalyzed by WbpO and WbpP. Results from WbpO-WbpP coupled-enzymatic assays showed that either WbpO or WbpP is capable of initiating the two-step pathway; however, the kinetic parameters favored the WbpO reaction to occur first, converting UDP-N-acetyl-D-glucosamine to UDP-N-acetyl-D-glucuronic acid prior to the conversion to UDP-N-acetyl-D-galacturonic acid by WbpP. This is the first report to show that a C4 epimerase could utilize UDP-N-acetyl-hexuronic acid as a substrate.
biosynthesis, O-antigen, Pseudomonas aeruginosa, glycosylation, Flagellin
NCBI PubMed ID: 18065759Pseudomonas 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: 19282284We have identified an operon and characterized the functions of two genes from the severe food-poisoning bacterium, Bacillus cereus subsp. cytotoxis NVH 391-98, that are involved in the synthesis of a unique UDP-sugar, UDP-2-acetamido-2-deoxyxylose (UDP-N-acetyl-xylosamine, UDP-XylNAc). UGlcNAcDH encodes a UDP-N-acetyl-glucosamine 6-dehydrogenase, converting UDP-N-acetylglucosamine (UDP-GlcNAc) to UDP-N-acetyl-glucosaminuronic acid (UDP-GlcNAcA). The second gene in the operon, UXNAcS, encodes a distinct decarboxylase not previously described in the literature, which catalyzes the formation of UDP-XylNAc from UDP-GlcNAcA in the presence of exogenous NAD(+). UXNAcS is specific and cannot utilize UDP-glucuronic acid and UDP-galacturonic acid as substrates. UXNAcS is active as a dimer with catalytic efficiency of 7 mM(-1) s(-1). The activity of UXNAcS is completely abolished by NADH but unaffected by UDP-xylose. A real-time NMR-based assay showed unambiguously the dual enzymatic conversions of UDP-GlcNAc to UDP-GlcNAcA and subsequently to UDP-XylNAc. From the analyses of all publicly available sequenced genomes, it appears that UXNAcS is restricted to pathogenic Bacillus species, including Bacillus anthracis and Bacillus thuringiensis. The identification of UXNAcS provides insight into the formation of UDP-XylNAc. Understanding the metabolic pathways involved in the utilization of this amino-sugar may allow the development of drugs to combat and eradicate the disease.
biosynthesis, gene, Gene Expression Regulation, Uridine Diphosphate Sugars, Bacillus cereus, Uridine diphosphate Xylose
NCBI PubMed ID: 20529859Summary The major component of the outer leaflet of the outer membrane of Gram-negative bacteria is lipopolysaccharide (LPS). The outermost domain of LPS is a polysaccharide called O antigen. Pseudomonas aeruginosa establishes biofilms on wet surfaces in a wide range of habitats and mutations in O-antigen biosynthesis genes affect bacterial adhesion and the structure of these biofilms. The P. aeruginosa O6 O antigen contains a 2-acetamido-2-deoxy-d-galacturonamide (d-GalNAcAN) residue. O-antigen biosynthesis in this serotype requires the wbpS gene, which encodes a protein with conserved domains of the glutamine-dependent amidotransferase family. Replacement of conserved amino acids in the N-terminal glutaminase conserved domain of WbpS inhibited O-antigen biosynthesis under restricted-ammonia conditions, but not in rich media; suggesting that this domain functions to provide ammonia for O-antigen biosynthesis under restricted-ammonia conditions, by hydrolysis of glutamine. Escherichia coli O121 also produces a d-GalNAcAN-containing O antigen, and possesses a homologue of wbpS called wbqG. An E. coli O121 wbqG mutant was cross-complemented by providing wbpS in trans, and vice versa, showing that these two genes are functionally interchangeable. The E. coli O121 wbqG mutant O antigen contains 2-acetamido-2-deoxy-d-galacturonate (d-GalNAcA), instead of d-GalNAcAN, demonstrating that wbqG is specifically required for biosynthesis of the carboxamide in this sugar.
biosynthesis, O-antigen, Pseudomonas aeruginosa, Biofilm
NCBI PubMed ID: 20192967In 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: 206045442,3-Diacetamido-2,3-dideoxy-d-mannuronic acid (ManNAc3NAcA) is an unusual dideoxy sugar first identified nearly 30 years ago in the lipopolysaccharide of Pseudomonas aeruginosa O:3a,d. It has since been observed in other organisms, including Bordetella pertussis, the causative agent of whooping cough. Five enzymes are required for the biosynthesis of UDP-ManNAc3NAcA starting from UDP-N-acetyl-d-glucosamine. Here we describe a structural study of WlbA, the NAD-dependent dehydrogenase that catalyzes the second step in the pathway, namely, the oxidation of the C-3' hydroxyl group on the UDP-linked sugar to a keto moiety and the reduction of NAD(+) to NADH. This enzyme has been shown to use α-ketoglutarate as an oxidant to regenerate the oxidized dinucleotide. For this investigation, three different crystal structures were determined: the enzyme with bound NAD(H), the enzyme in a complex with NAD(H) and α-ketoglutarate, and the enzyme in a complex with NAD(H) and its substrate (UDP-N-acetyl-d-glucosaminuronic acid). The tetrameric enzyme assumes an unusual quaternary structure with the dinucleotides positioned quite closely to one another. Both α-ketoglutarate and the UDP-linked sugar bind in the WlbA active site with their carbon atoms (C-2 and C-3', respectively) abutting the re face of the cofactor. They are positioned approximately 3 A from the nicotinamide C-4. The UDP-linked sugar substrate adopts a highly unusual curved conformation when bound in the WlbA active site cleft. Lys 101 and His 185 most likely play key roles in catalysis.
biosynthesis, Pseudomonas, Bordetella, Bordetella pertussis, crystal structure, Enzymes, dehydrogenase, WlbA
NCBI PubMed ID: 20690587The O antigen is an essential component of the lipopolysaccharides on the surface of Gram-negative bacteria and its variation provides a major basis for serotyping schemes. The Escherichia coli O-antigen form O180 was first designated in 2004, and O180 strains were found to contain virulence factors and cause diarrhea. Different O-antigen forms are almost entirely due to genetic variations in the O-antigen gene clusters. In this study, the chemical structure and gene cluster of E. coli O180 O antigen were investigated. A tetrasaccharide repeating unit with the following structure: -4)-b-D-ManpNAc3NAcA-(1-2)-a-L-Rhap(I)-(1-3)-b-L-Rhap(II)-(1-4)-a-D-GlcpNAc-(1- was identified in the E. coli O180 O antigen, including the residue D-ManpNAc3NAcA (2,3-diacetamido-2,3-dideoxy-D-mannopyranuronic acid) that had not been hitherto identified in E. coli. Genes in the O-antigen gene cluster were assigned functions based on their similarities with those from available databases, and five genes involved in the synthesis of UDP-D-ManpNAc3NAcA (the nucleotide-activated form of D-ManpNAc3NAcA) were identified. The gnaA gene, encoding the enzyme involved in the initial step of the UDP-D-ManpNAc3NAcA biosynthetic pathway, was cloned and the enzyme product was expressed, purified and assayed for its activity. GnaA was characterized using capillary electrophoresis and electrospray ionization mass spectrometry and identified as a UDP-GlcNAc 6-dehydrogenase. The kinetic and physicochemical parameters of GnaA also were determined.
biosynthesis, O-antigen, Escherichia coli O180, UDP-D-ManNAc3NAcA
NCBI PubMed ID: 22730467| New query | Export IDs | Home | Help |
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