A pyridoxal 5'-phosphate protein. This enzyme participates in the biosynthetic pathway for UDP-alpha-D-ManNAc3NAcA (UDP-2,3-diacetamido-2,3-dideoxy-alpha-D-mannuronic acid), an important precursor of B-band lipopolysaccharide. The enzymes from Pseudomonas aeruginosa serotype O5 and Thermus thermophilus form a complex with the previous enzyme in the pathway, EC 1.1.1.335 (UDP-N-acetyl-2-amino-2-deoxyglucuronate oxidase).
WbpE is a member of the broad class of fold type I aspartate-aminotransferase enzymes, which harness the powerful electron-sink properties of PLP to carry out a wide variety of transformations, including transaminations, eliminations, decarboxylations, and racemizations. The general mechanism of this class of enzymes has been worked out in great detail, and is divided into two discrete half reactions that cycle between the PMP and PLP forms of the cofactor, overview
the biosynthetic pathway of 2,3-diacetamido-2,3-dideoxy-D-mannuronic acid production as part of the B-band LPS production requires the five enzymes WbpA, WbpB, WbpE, WbpD, and WbpI
the biosynthetic pathway of 2,3-diacetamido-2,3-dideoxy-D-mannuronic acid production as part of the B-band LPS production requires the five enzymes WbpA, WbpB, WbpE, WbpD, and WbpI
the central carbohydrate of the Pseudomonas aeruginosa PAO1 (O5) B-band O-antigen, UDP-2,3-diacetamido-2,3-dideoxy-D-mannuronic acid or ManNAc(3NAc)A, is critical for virulence and is produced in a stepwise manner by the five enzymes in the Wbp pathway, WbpA, WbpB, WbpE, WbpD and WbpI, overview
the biosynthetic pathway begins with WbpA, catalyzing the C6-oxidation of UDP-GlcNAc to give the corresponding UDP-N-acetyl-D-glucosaminuronic acid. The C3-dehydrogenase WbpB, aminotransferase WbpE, and acetyltransferase WbpD sequentially convert UDP-GlcNAcA into UDP-2,3-diacetamido-2,3-dideoxy-D-glucuronic acid. Finally, the C2-epimerase WbpI modifies UDP-GlcNAc(3NAc)A to give the final UDP-ManNAc(3NAc)A
WbpE, a nucleotide sugar aminotransferase involved in O-antigen assembly, is a pyridoxal 5'-phosphate-dependent aminotransferse responsible for the conversion of UDP-2-acetamido-2-deoxy-3-oxo-D-glucuronic acid and L-glutamate to UDP-2-acetamido-3-amino-2,3-dideoxy-D-glucuronic acid and 2-oxoglutarate, respectively
a strain lacking PorR activity expresses the conventional O-antigen of orphyromonas gingivalis strain W50 (O-LPS), but lacks A-LPS, i.e. a different O-antigen consisting of an anionic polysaccharide (APS) repeat unit. PorE is a homolog of WbpE
a strain lacking PorR activity expresses the conventional O-antigen of orphyromonas gingivalis strain W50 (O-LPS), but lacks A-LPS, i.e. a different O-antigen consisting of an anionic polysaccharide (APS) repeat unit. PorE is a homolog of WbpE
Bordetella pertussis genes wbpO1629 and wbpO3150 complement a wbpA knockout of Pseudomonas aeruginosa. B-band LPS production is restored to Pseudomonas aeruginosa knockout mutants when the relevant Bordetella pertussis genes are supplied in trans
transaminase WbpE product is UDP-2-acetamido-3-amino-2,3-dideoxy-alpha-D-glucuronate. Cofactor pyridoxal 5'-phosphate is converted to pyridoxamine phosphate during the reaction
WbpB and WbpE are a dehydrogenase/aminotransferase pair that converts UDP-N-acetyl-D-glucosaminuronate, UDP-GlcNAcA, to UDP-2-acetamido-3-amino-2,3-dideoxy-D-glucuronate, UDP-GlcNAc(3NH2)A, in a coupled reaction via a unique NAD+ recycling pathway, large-scale coupled reaction WpbB/WpbE, overview. WbpA, WbpB, WbpE, WbpD and WbpI can be combined in vitro to generate UDP-ManNAc(3NAc)A in a single reaction vessel. Addition of 0.2 mM NAD+, for WbpB, and 0.1 mM pyridoxal 5'-phosphate, for WbpE, to the reaction mixture aids in achieving complete turnover of substrate, which implies that the heterologously expressed proteins are not saturated with cofactor due to the limiting intracellular levels of both NAD+ and pyridoxal 5'-phosphate in Escherichia coli. WbpB/WbpE substrate specificity, overview
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Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
purifed recombinant detagged WbpE in complex with the cofactor pyridoxal 5'-phosphate and product UDP-2-acetamido-3-amino-2,3-dideoxy-D-glucuronic acid as the external aldimine, mixing of 0.0015 ml of protein solution containing 10 mg/ml protein in 25 mM HEPES, pH 8.0, 100 mM NaCl, 0.5% glycerol, and 0.5 mM UDP-2-acetamido-3-amino-2,3-dideoxy-D-glucuronic acid, with 0.0015 ml of reservoir solution containing 0.1 M Bis-Tris, pH 5.5, 0.2 M ammonium sulfate and 25% PEG 3350 for the wild-type enzyme, and 0.1 M Bis-Tris, pH 5.5, 0.2 M ammonium acetate, 10 mM SrCl2 and 25% PEG 3350 for the sselenomethionine-labeled enzyme, X-ray diffraction structure determination and analysis at 1.9 A resolution, molecular replacement
recombinant N-terminal His6-tagged WbpE from Escherichia coli BL21-CodonPlus(DE3)RIL by nickel affinity chromatography, the N-terminal His6-tag is removed by proteolysis with TEV protease over the course of three days while stirring in dialysis buffer, followed by gel filtration
construction of a nonpolar knockout of each of wbpA, wbpB, wbpE, wbpD, and wbpI genes. Expression of Bordetella pertussis genes wbpO1629 and wbpO3150 complements a wbpA knockout of Pseudomonas aeruginosa. B-band LPS production is restored to Pseudomonas aeruginosa knockout mutants when the relevant Bordetella pertussis genes are supplied in trans
Biosynthesis of a rare di-N-acetylated sugar in the lipopolysaccharides of both Pseudomonas aeruginosa and Bordetella pertussis occurs via an identical scheme despite different gene clusters
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
Biosynthesis of UDP-GlcNAc(3NAc)A by WbpB, WbpE, and WbpD: enzymes in the Wbp pathway responsible for O-antigen assembly in Pseudomonas aeruginosa PAO1