Cloned (Comment) | Organism |
---|---|
gene wbpE, expression of N-terminal His6-tagged WbpE in Escherichia coli BL21-CodonPlus(DE3)RIL | Pseudomonas aeruginosa |
Crystallization (Comment) | Organism |
---|---|
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 | Comment | Organism |
---|---|---|
D156A | site-directed mutagenesis, the mutant shows only slightly affected activity and slight loss of binding affinity compared to the wild-type | Pseudomonas aeruginosa |
H308A | site-directed mutagenesis, the mutant shows only slightly affected activity and slight loss of binding affinity compared to the wild-type | Pseudomonas aeruginosa |
K185A | site-directed mutagenesis, the activity of the catalytic site mutant is completely abolished | Pseudomonas aeruginosa |
N227A | site-directed mutagenesis, the mutant shows only slightly affected activity and slight loss of binding affinity compared to the wild-type | Pseudomonas aeruginosa |
Q159A | site-directed mutagenesis, the mutant shows only slightly affected activity and slight loss of binding affinity compared to the wild-type | Pseudomonas aeruginosa |
R229A | site-directed mutagenesis, the mutant shows only slightly affected activity and slight loss of binding affinity compared to the wild-type | Pseudomonas aeruginosa |
S180A | site-directed mutagenesis, the mutant shows only slightly affected activity and slight loss of binding affinity compared to the wild-type | Pseudomonas aeruginosa |
T60A | site-directed mutagenesis, the mutant shows only slightly affected activity and slight loss of binding affinity compared to the wild-type | Pseudomonas aeruginosa |
Y309A | site-directed mutagenesis, the mutant shows only slightly affected activity and slight loss of binding affinity compared to the wild-type | Pseudomonas aeruginosa |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
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 | UniProt | Comment | Textmining |
---|---|---|---|
Pseudomonas aeruginosa | Q9HZ76 | gene wbpE | - |
Purification (Comment) | Organism |
---|---|
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 | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
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 | |
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 | Comment | Organism |
---|---|---|
WbpE | - |
Pseudomonas aeruginosa |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
pyridoxal 5'-phosphate | - |
Pseudomonas aeruginosa |
General Information | Comment | Organism |
---|---|---|
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 |
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 |
additional information | WbpE nucleotide sugar-binding site structure, overview | Pseudomonas aeruginosa |
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 |