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1.3.1.98: UDP-N-acetylmuramate dehydrogenase

This is an abbreviated version!
For detailed information about UDP-N-acetylmuramate dehydrogenase, go to the full flat file.

Word Map on EC 1.3.1.98

Reaction

UDP-N-acetyl-alpha-D-muramate
+
NADP+
=
UDP-N-acetyl-3-O-(1-carboxyvinyl)-alpha-D-glucosamine
 +
NADPH
 +
H+

Synonyms

alr5066, EC 1.1.1.158, More, MurB, MurBAb, PA2977, PaMurB, reductase, uridine diphosphoacetylpyruvoylglucosamine, type I UNAGEP reductase, UDP-GlcNAc-enoylpyruvate reductase, UDP-N-acetylenolpyruvoylglucosamine reductase, UDP-N-acetylenolpyruvyl glucosamine reductase, UDP-N-acetylenolpyruvylglucosamine reductase, UDP-N-acetylglucosamine-enoylpyruvate reductase, UDP-N-acetylmuramate dehydrogenase, UNAGEP reductase, uridine diphospho-N-acetylglucosamine-enolpyruvate reductase, uridine-5'-diphospho-N-acetyl-2-amino-2-deoxy-3-O-lactylglucose:NADP-oxidoreductase, VspiD_010100018130

ECTree

     1 Oxidoreductases
         1.3 Acting on the CH-CH group of donors
             1.3.1 With NAD+ or NADP+ as acceptor
                1.3.1.98 UDP-N-acetylmuramate dehydrogenase

Crystallization

Crystallization on EC 1.3.1.98 - UDP-N-acetylmuramate dehydrogenase

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CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
S229A mutant and wild-type MurB
-
enzyme bound to FAD and K+, hanging drop vapor diffusion method
sequence alignment with Escherichia coli structures, PDB entries 2Q85 and 2MBR, and modeling of structure as well as docking with N-acetylpyruvyl glucosamine
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
by sitting-drop vapour diffusion
-
purified recombinant His-tagged enzyme, 20 mg/ml protein in 20 mM HEPES, pH 7.5 5 mM 2-mercaptoethanol or DTT, 20°C, hanging drop vapour diffusion method, mixing with reservoir solution containing 0.1 M sodium cacodylate, pH 6.5, 15% PEG 8000, and 0.5 M (NH4)2SO4, 4 days, yellow plate-shaped crystals cryoprotected in 10% PEG 8000, 0.55 M (NH4)2SO4, 0.1 M sodium cacodylate, pH 6.5, 5 mM BME or DTT with 15% 2-methyl 2,4-pentanediol, X-ray diffraction structure determination and analysis at 2.3 A resolution, altered crystallization conditions were optimized using the purified selenomethionine-labeled MurB at 20 mg/ml, in complex with 10 mM enolpyruvyl-UDP-N-acetylglucosamine, in 1-2 weeks on grease-coated microbridges by mixing 5 ml Se-Met MurB and 5 ml reservoir solution containing 0.1 M sodium cacodylate, 9.75% PEG 8000, 0.55 M (NH4)2SO4, 20% DMSO, and 5 mM 2-mercaptoethanol and producing larger crystals, overview
-
purified recombinant MurB, hanging drop-vapor diffusion method, 0.001 ml of protein solution containing 20 mg/ml protein in 20 mM HEPES-NaOH, pH 7.5, is mixed with 0.001 ml of reservoir solution containing 100 mM MES-NaOH, pH 6.5, 18% w/v PEG 8000 and 200 mM CaCl2, equilibration against 1 ml of mother liquor, Se-Met substituted crystals are grown under the same conditions, while the substrate-complex crystals are grown in the presence of 25 mM substrate UDPGlcNAc, crystals appear within 8 h, X-ray difraction structure determination and analysis at 1.15-1.7 A resolution