Literature summary for 1.3.1.98 extracted from

Chen, M.W.; Lohkamp, B.; Schnell, R.; Lescar, J.; Schneider, G.
Substrate channel flexibility in Pseudomonas aeruginosa MurB accommodates Two distinct substrates (2013), PLoS ONE, 8, e66936 .

Cloned(Commentary)

Cloned (Comment)	Organism
gene murB, recombinant expression of N-terminally His6-tagged enzyme, harboring a TEV protease cleavage site, in Escherichia coli strain BL21(DE3)	Pseudomonas aeruginosa

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified recombinant enzyme MurB complexed with FAD and NADP+ in two crystal forms, hanging drop vapour diffusion method, for crystal type A: mixing of 0.001 ml of protein solution containing 25 mg/ml PaMurB, and 2 mM unbuffered NADPH, with 0.001 ml of reservoir solution containing 0.1 M Bis-Tris propane, pH 7.0, 0.2 M sodium potassium tartrate, and 15% w/v PEG 3350, and equilibration against 0.5 ml reservoir solution, crystals from these drops are cryoprotected with a solution containing additional 15% w/v PEG3350 and 2 mM NADPH before flash freezing, for crystal type B: the protein is mixed with a reservoir solution containing 40 mM potassium phosphate, 20% v/v glycerol, 16% w/v PEG 8000 and 2 mM Tris-buffered NADP+ sodium salt, the crystals are frozen directly without addition of a cryoprotectant, X-ray diffraction structure determination and analysis at 2.2 A and 2.1 A resolution, respectively, molecular replacement using the atomic coordinates of the complex of Escherichia coli MurB with UDP-N-acetylglucosamine enolpyruvate, PDB code 2MBR, stripped of all ligands and water molecules as search model, modeling	Pseudomonas aeruginosa

Crystallization (Comment)

Organism

purified recombinant enzyme MurB complexed with FAD and NADP+ in two crystal forms, hanging drop vapour diffusion method, for crystal type A: mixing of 0.001 ml of protein solution containing 25 mg/ml PaMurB, and 2 mM unbuffered NADPH, with 0.001 ml of reservoir solution containing 0.1 M Bis-Tris propane, pH 7.0, 0.2 M sodium potassium tartrate, and 15% w/v PEG 3350, and equilibration against 0.5 ml reservoir solution, crystals from these drops are cryoprotected with a solution containing additional 15% w/v PEG3350 and 2 mM NADPH before flash freezing, for crystal type B: the protein is mixed with a reservoir solution containing 40 mM potassium phosphate, 20% v/v glycerol, 16% w/v PEG 8000 and 2 mM Tris-buffered NADP+ sodium salt, the crystals are frozen directly without addition of a cryoprotectant, X-ray diffraction structure determination and analysis at 2.2 A and 2.1 A resolution, respectively, molecular replacement using the atomic coordinates of the complex of Escherichia coli MurB with UDP-N-acetylglucosamine enolpyruvate, PDB code 2MBR, stripped of all ligands and water molecules as search model, modeling

Pseudomonas aeruginosa

Metals/Ions

Metals/Ions	Comment	Organism	Structure
K+	PaMurB crystal structure contains a potassium ion in the active site. The potassium ion displays a pentagonal bipyramidal coordination geometry with two main chain oxygen atoms (Ala237, Ser239), one side chain carboxyl oxygen atom from Glu-335, one side chain oxygen atom of Asn-57 and the O7n oxygen atom of the nicotinamide ring as ligands. The potassium ion activates MurB by facilitating substrate orientation and binding	Pseudomonas aeruginosa

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
UDP-N-acetyl-3-O-(1-carboxyvinyl)-alpha-D-glucosamine + NADPH + H+	Pseudomonas aeruginosa	-	UDP-N-acetyl-alpha-D-muramate + NADP+	-	?

Organism

Organism	UniProt	Comment	Textmining
Pseudomonas aeruginosa	Q9HZM7	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant N-terminally His6-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, tag cleavage by TEV protease, followed by a second step of affinity chromatography to remove uncleaved protein and the His6-tagged TEV protease, further purification by preparation scale gel filtration, and ultrafiltration	Pseudomonas aeruginosa

Reaction

Reaction	Comment	Organism	Reaction ID
UDP-N-acetyl-alpha-D-muramate + NADP+ = UDP-N-acetyl-3-O-(1-carboxyvinyl)-alpha-D-glucosamine + NADPH + H+	the nicotinamide ring of NADP+ stacks against the si face of the isoalloxazine ring of FAD, suggesting an unusual mode of hydride transfer to flavin. Both substrates share the binding site located between two lobes of the substrate-binding domain III, consistent with a ping pong mechanism with sequential substrate binding, structural analysis of the first half-reaction, overview	Pseudomonas aeruginosa	a

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
UDP-N-acetyl-3-O-(1-carboxyvinyl)-alpha-D-glucosamine + NADPH + H+	-	Pseudomonas aeruginosa	UDP-N-acetyl-alpha-D-muramate + NADP+	-	?
UDP-N-acetyl-3-O-(1-carboxyvinyl)-alpha-D-glucosamine + NADPH + H+	structural analysis of the first half-reaction, hydride transfer from NADPH to FAD using the crystal structure of the enzyme in ternary complex with FAD and NADP+, overview. Enzyme MurB is a type I UNAGEP reductase	Pseudomonas aeruginosa	UDP-N-acetyl-alpha-D-muramate + NADP+	-	?

Subunits

Subunits	Comment	Organism
More	three-dimensional structure analysis and comparison, overview	Pseudomonas aeruginosa

Synonyms

Synonyms	Comment	Organism
MurB	-	Pseudomonas aeruginosa
PA2977	-	Pseudomonas aeruginosa
PaMurB	-	Pseudomonas aeruginosa
type I UNAGEP reductase	-	Pseudomonas aeruginosa
UNAGEP reductase	-	Pseudomonas aeruginosa

Cofactor

Cofactor	Comment	Organism	Structure
FAD	binding structure, overview	Pseudomonas aeruginosa
NADPH	binding structure, overview. Hydride transfer occurs via the isoalloxazine si face	Pseudomonas aeruginosa