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Literature summary extracted from

  • Larkin, A.; Imperiali, B.
    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 (2009), Biochemistry, 48, 5446-5455.
    View publication on PubMedView publication on EuropePMC

Activating Compound

EC Number Activating Compound Comment Organism Structure
1.1.1.335 2-oxoglutarate required as oxidant for NAD+ recycling, product of 2-oxoglutarate reduction is 2-hydroxyglutarate Pseudomonas aeruginosa

Application

EC Number Application Comment Organism
1.1.1.335 synthesis enzyme WbpB and the related enzymes of the B-band O-antigen pathway of Pseudomonas aeruginosa lipopolysaccharide, WbpA, 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 Pseudomonas aeruginosa

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.1.1.335 expression in Escherichia coli Pseudomonas aeruginosa
2.3.1.201 expressed in Escherichia coli BL21-CodonPlus(DE3) RIL cells Pseudomonas aeruginosa
2.6.1.98 gene wpbE, overexpression of N-terminally T7- and C-terminally His6-tagged WpbE in Escherichia coli strain BL21-CodonPlus(DE3)RIL Pseudomonas aeruginosa

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
2.3.1.201 0.107
-
 UDP-2-acetamido-3-amino-2,3-dideoxy-alpha-D-glucuronate in 50 mM HEPES (pH 8.0), 2.5 mM dithiothreitol, and 2 mM MgCl2, at 30°C Pseudomonas aeruginosa

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
1.1.1.335 38271
-
 x * 38271, calculated for recombinant enzyme including His- and T7 tags Pseudomonas aeruginosa

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
2.6.1.98 UDP-2-acetamido-3-amino-2,3-dideoxy-alpha-D-glucuronate + 2-oxoglutarate Pseudomonas aeruginosa
-
 UDP-2-acetamido-2-deoxy-alpha-D-ribo-hex-3-uluronate + L-glutamate
-
 r

Organism

EC Number Organism UniProt Comment Textmining
1.1.1.335 Pseudomonas aeruginosa G3XD23
-
-
2.3.1.201 Pseudomonas aeruginosa
-
 serotype O5
-
2.6.1.98 Pseudomonas aeruginosa
-
 gene wbpE
-

Purification (Commentary)

EC Number Purification (Comment) Organism
2.3.1.201 Ni-NTA agarose resin column chromatography, gel filtration Pseudomonas aeruginosa
2.6.1.98 recombinant His-tagged WpbE from Escherichia coli strain BL21-CodonPlus(DE3)RIL by nickel affinity chromatography Pseudomonas aeruginosa

Storage Stability

EC Number Storage Stability Organism
1.1.1.335 -20°C, stable for at least 3 months Pseudomonas aeruginosa

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.1.1.335 UDP-2-acetamido-2-deoxy-alpha-D-glucuronate + NAD+ isolated enzyme EWbpB in presence of UDP-2-acetamido-2-deoxy-alpha-D-glucuronate and NAD+ does not show enzymic activity. Upon the addition of WbpE and L-glutamate to the reaction, complete turnover of the starting material and the formation of UDP-2-acetamido-3-amino-2,3-dideoxy-D-glucuronic acid are observed. No turnover is observed in the presence of UDP-N-acetyl-D-glucosamine or UDP-UDP-N-acetyl-D-galactosamine, and only minimal turnover is observed when UDP-D-glucuronic acid is used as the nucleotide sugar substrate. Enzyme WbpB prefers the glucopyranose configuration of the sugar as well as the presence of both the carboxylate at the C'' carbon and the acetylated amine at the C'' position. WbpE is specific for L-glutamate as the amine donor. Presence of 2-ketoglutarate is required as oxidant for NAD+ recycling Pseudomonas aeruginosa UDP-2-acetamido-2-deoxy-alpha-D-ribo-hex-3-uluronate + NADH + H+
-
 ?
2.3.1.201 acetyl-CoA + UDP-2-acetamido-3-amino-2,3-dideoxy-alpha-D-glucuronate
-
 Pseudomonas aeruginosa CoA + UDP-2,3-diacetamido-2,3-dideoxy-alpha-D-glucuronate
-
 ?
2.3.1.201 additional information no activity with UDP-GlcNAcA, UDP-GlcNAc, and UDP-2-acetamido-4-amino-2,4,6-trideoxy-D-glucosamine Pseudomonas aeruginosa ?
-
 ?
2.6.1.98 additional information 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 Pseudomonas aeruginosa ?
-
 ?
2.6.1.98 UDP-2-acetamido-3-amino-2,3-dideoxy-alpha-D-glucuronate + 2-oxoglutarate
-
 Pseudomonas aeruginosa UDP-2-acetamido-2-deoxy-alpha-D-ribo-hex-3-uluronate + L-glutamate
-
 r

Subunits

EC Number Subunits Comment Organism
1.1.1.335 ? x * 38271, calculated for recombinant enzyme including His- and T7 tags Pseudomonas aeruginosa

Synonyms

EC Number Synonyms Comment Organism
1.1.1.335 WbpB
-
 Pseudomonas aeruginosa
2.3.1.201 UDP-2-acetamido-3-amino-2,3-dideoxy-glucuronate N-acetyltransferase
-
 Pseudomonas aeruginosa
2.3.1.201 WbpD
-
 Pseudomonas aeruginosa
2.6.1.98 aminotransferase WbpE
-
 Pseudomonas aeruginosa
2.6.1.98 WbpE
-
 Pseudomonas aeruginosa

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.1.1.335 30
-
 coupled reaction of enzymes WbpB/WbpE Pseudomonas aeruginosa
2.3.1.201 30
-
-
 Pseudomonas aeruginosa
2.6.1.98 30
-
 assay at Pseudomonas aeruginosa

Temperature Range [°C]

EC Number Temperature Minimum [°C] Temperature Maximum [°C] Comment Organism
2.3.1.201 4 65
-
 Pseudomonas aeruginosa

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
2.3.1.201 48.3
-
 UDP-2-acetamido-3-amino-2,3-dideoxy-alpha-D-glucuronate in 50 mM HEPES (pH 8.0), 2.5 mM dithiothreitol, and 2 mM MgCl2, at 30°C Pseudomonas aeruginosa

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.1.1.335 8
-
 coupled reaction of enzymes WbpB/WbpE Pseudomonas aeruginosa
2.3.1.201 7
-
-
 Pseudomonas aeruginosa
2.6.1.98 7 8 assay at Pseudomonas aeruginosa

pH Range

EC Number pH Minimum pH Maximum Comment Organism
2.3.1.201 5.5 10
-
 Pseudomonas aeruginosa

Cofactor

EC Number Cofactor Comment Organism Structure
1.1.1.335 NAD+
-
 Pseudomonas aeruginosa
1.1.1.335 NADH
-
 Pseudomonas aeruginosa
2.3.1.201 NAD+
-
 Pseudomonas aeruginosa
2.3.1.201 pyridoxal 5'-phosphate
-
 Pseudomonas aeruginosa
2.6.1.98 pyridoxal 5'-phosphate
-
 Pseudomonas aeruginosa

General Information

EC Number General Information Comment Organism
1.1.1.335 physiological function enzyme WbpB is part of the B-band O-antigen pathway of Pseudomonas aeruginosa lipopolysaccharide. Proteins WbpB and WbpE are a dehydrogenase/aminotransferase pair that converts UDP-GlcNAcA to UDP-GlcNAc(3NH2)A in a coupled reaction via a NAD+ recycling pathway Pseudomonas aeruginosa
2.6.1.98 metabolism 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 Pseudomonas aeruginosa