Any feedback?
Please rate this page
(literature.php)
(0/150)

BRENDA support

Literature summary extracted from

  • Larkin, A.; Olivier, N.B.; Imperiali, B.
    Structural analysis of WbpE from Pseudomonas aeruginosa PAO1: a nucleotide sugar aminotransferase involved in O-antigen assembly (2010), Biochemistry, 49, 7227-7237.
    View publication on PubMedView publication on EuropePMC

Application

EC Number Application Comment Organism
2.3.1.201 synthesis the enzymes WbpA, WbpB, WbpE, WbpD and WbpI which act stepwise manner starting from UDP-GlcNAc can be combined in vitro to generate UDP-ManNAc(3NAc)A in a single reaction vessel Pseudomonas aeruginosa PAO1

Cloned(Commentary)

EC Number Cloned (Comment) Organism
2.3.1.201 expression in Escherichia coli Pseudomonas aeruginosa PAO1
2.6.1.98 gene wbpE, expression of N-terminal His6-tagged WbpE in Escherichia coli BL21-CodonPlus(DE3)RIL Pseudomonas aeruginosa

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
2.6.1.98 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 Pseudomonas aeruginosa

Protein Variants

EC Number Protein Variants Comment Organism
2.6.1.98 D156A site-directed mutagenesis, the mutant shows only slightly affected activity and slight loss of binding affinity compared to the wild-type Pseudomonas aeruginosa
2.6.1.98 H308A site-directed mutagenesis, the mutant shows only slightly affected activity and slight loss of binding affinity compared to the wild-type Pseudomonas aeruginosa
2.6.1.98 K185A site-directed mutagenesis, the activity of the catalytic site mutant is completely abolished Pseudomonas aeruginosa
2.6.1.98 N227A site-directed mutagenesis, the mutant shows only slightly affected activity and slight loss of binding affinity compared to the wild-type Pseudomonas aeruginosa
2.6.1.98 Q159A site-directed mutagenesis, the mutant shows only slightly affected activity and slight loss of binding affinity compared to the wild-type Pseudomonas aeruginosa
2.6.1.98 R229A site-directed mutagenesis, the mutant shows only slightly affected activity and slight loss of binding affinity compared to the wild-type Pseudomonas aeruginosa
2.6.1.98 S180A site-directed mutagenesis, the mutant shows only slightly affected activity and slight loss of binding affinity compared to the wild-type Pseudomonas aeruginosa
2.6.1.98 T60A site-directed mutagenesis, the mutant shows only slightly affected activity and slight loss of binding affinity compared to the wild-type Pseudomonas aeruginosa
2.6.1.98 Y309A site-directed mutagenesis, the mutant shows only slightly affected activity and slight loss of binding affinity compared to the wild-type 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 30°C, pH 7.0 Pseudomonas aeruginosa PAO1

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
2.3.1.201 Pseudomonas aeruginosa PAO1
-
-
-
2.6.1.98 Pseudomonas aeruginosa Q9HZ76 gene wbpE
-

Purification (Commentary)

EC Number Purification (Comment) Organism
2.3.1.201 recombinant enzyme Pseudomonas aeruginosa PAO1
2.6.1.98 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 Pseudomonas aeruginosa

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2.3.1.201 acetyl-CoA + UDP-2-acetamido-3-amino-2,3-dideoxy-alpha-D-glucuronate
-
 Pseudomonas aeruginosa PAO1 CoA + UDP-2,3-diacetamido-2,3-dideoxy-alpha-D-glucuronate
-
 ?
2.3.1.201 additional information no substrate: UDP-GlcNAcA, UDP-GlcNAc, and UDP-2-acetamido-4-amino-2,4,6-trideoxy-D-glucosamine Pseudomonas aeruginosa PAO1 ?
-
 ?
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
2.6.1.98 UDP-2-acetamido-3-amino-2,3-dideoxy-alpha-D-glucuronate + 2-oxoglutarate WbpE product is UDP-2-acetamido-3-amino-2,3-dideoxy-D-glucuronic acid Pseudomonas aeruginosa UDP-2-acetamido-2-deoxy-alpha-D-ribo-hex-3-uluronate + L-glutamate
-
 r

Synonyms

EC Number Synonyms Comment Organism
2.6.1.98 WbpE
-
 Pseudomonas aeruginosa

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
2.3.1.201 30
-
-
 Pseudomonas aeruginosa PAO1

Temperature Range [°C]

EC Number Temperature Minimum [°C] Temperature Maximum [°C] Comment Organism
2.3.1.201 4 65
-
 Pseudomonas aeruginosa PAO1

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
2.3.1.201 7
-
-
 Pseudomonas aeruginosa PAO1

pH Range

EC Number pH Minimum pH Maximum Comment Organism
2.3.1.201 5.5 10
-
 Pseudomonas aeruginosa PAO1

Cofactor

EC Number Cofactor Comment Organism Structure
2.6.1.98 pyridoxal 5'-phosphate
-
 Pseudomonas aeruginosa

General Information

EC Number General Information Comment Organism
2.6.1.98 evolution 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 Pseudomonas aeruginosa
2.6.1.98 metabolism 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 Pseudomonas aeruginosa
2.6.1.98 additional information WbpE nucleotide sugar-binding site structure, overview Pseudomonas aeruginosa
2.6.1.98 physiological function 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 Pseudomonas aeruginosa

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
2.3.1.201 48.3
-
 UDP-2-acetamido-3-amino-2,3-dideoxy-alpha-D-glucuronate 30°C, pH 7.0 Pseudomonas aeruginosa PAO1