Literature summary for 2.6.1.98 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.

Cloned(Commentary)

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

Crystallization (Commentary)

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

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

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/ Products (Substrates)

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

Organism	UniProt	Comment	Textmining
Pseudomonas aeruginosa	Q9HZ76	gene wbpE	-

Purification (Commentary)

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 and Products (Substrate)

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

Synonyms	Comment	Organism
WbpE	-	Pseudomonas aeruginosa

Cofactor

Cofactor	Comment	Organism	Structure
pyridoxal 5'-phosphate	-	Pseudomonas aeruginosa

General Information

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