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Literature summary for 2.4.1.182 extracted from
- Crystal structure of lipid A disaccharide synthase LpxB from Escherichia coli (2018), Nat. Commun., 9, 377 .
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| gene lpxB, recombinant expression of His6-tagged wild-type and mutant enzymes, coexpression of His6-tagged mutants LpxBR201A, LpxBN316A, and LpxBFN, and pull-down assays | Escherichia coli |
Crystallization (Commentary)
| Crystallization (Comment) | Organism |
|---|---|
| purified soluble recombinant enzymes, LpxB in the apo form and bound to UDP: LpxB7S, SeMet-LpxB7S, LpxB7S with UDP, and mutant LpxB6S (V66S/V68S/L69S/L72S/L75S/L76S), the ligand-bound structure of LpxB is obtained by soaking crystals with 10mM UDP-N-acytlglucosamine (UDP-GlcNAc), X-ray diffraction structure determination and analysis at 1.98-3.43 A resolution | Escherichia coli |
Protein Variants
| Protein Variants | Comment | Organism |
|---|---|---|
| F298E/N316A | site-directed mutagenesis, mutant LpxBFN, the mutant shows 0.0078% of wild-type activity | Escherichia coli |
| L69S | site-directed mutagenesis, the mutation improves solubility and yield of LpxB | Escherichia coli |
| L72S | site-directed mutagenesis, the mutation improves solubility and yield of LpxB | Escherichia coli |
| L72S/L75S | site-directed mutagenesis, the mutant shows 1.74% of wild-type enzyme activity | Escherichia coli |
| L72S/L75S/L76S | site-directed mutagenesis, the mutant shows highly reduced activity compared to wild-type enzyme | Escherichia coli |
| L72S/L76S | site-directed mutagenesis, the mutant shows 32.2% of wild-type enzyme activity | Escherichia coli |
| L75S | site-directed mutagenesis, the mutation improves solubility and yield of LpxB | Escherichia coli |
| L75S/L76S | site-directed mutagenesis, the mutant shows 30.6% of wild-type enzyme activity | Escherichia coli |
| L76S | site-directed mutagenesis, the mutation improves solubility and yield of LpxB | Escherichia coli |
| M207S | site-directed mutagenesis, structure determination as selenomethionine-labeled enzyme | Escherichia coli |
| additional information | genetic knockout and complementation of LpxB | Escherichia coli |
| N316A | site-directed mutagenesis, mutant LpxBFN, the mutant shows 0.34% of wild-type activity | Escherichia coli |
| R201A | site-directed mutagenesis, mutant LpxBFN, the mutant shows 0.0051% of wild-type activity | Escherichia coli |
| V66S | site-directed mutagenesis, the mutation improves solubility and yield of LpxB | Escherichia coli |
| V66S/V68S/L69S | site-directed mutagenesis, the mutant shows highly reduced activity compared to wild-type enzyme | Escherichia coli |
| V66S/V68S/L69S/L72S/L75S/L76S | site-directed mutagenesis, mutant LpxB6S , the mutation improves solubility and yield of LpxB and shows the least aggregation of all mutants on a size exclusion column | Escherichia coli |
| V68S | site-directed mutagenesis, the mutation improves solubility and yield of LpxB | Escherichia coli |
Inhibitors
| Inhibitors | Comment | Organism | Structure |
|---|---|---|---|
| Triton X-100 | the ability of LpxB6S to catalyze the reaction of Triton X-100-solubilized substrates is completely abolished | Escherichia coli |  |
Localization
| Localization | Comment | Organism | GeneOntology No. | Textmining |
|---|---|---|---|---|
| membrane | membrane-associated. LpxB is a membrane surface active enzyme. The hydrophobic patch (V66, V68, L69, L72, L75, and L76) is essential for productive membrane association or substrate binding | Escherichia coli | 16020 | - |
Molecular Weight [Da]
| Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
|---|---|---|---|
| 78900 | - |
dimeric soluble recombinant enzyme, analytical ultracentrifugation | Escherichia coli |
| 84500 | - |
dimeric enzyme, sequence calculation | Escherichia coli |
Natural Substrates/ Products (Substrates)
| Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| UDP-2-N,3-O-bis[(3R)-3-hydroxytetradecanoyl]-alpha-D-glucosamine + 2-N,3-O-bis[(3R)-3-hydroxytetradecanoyl]-alpha-D-glucosaminyl 1-phosphate | Escherichia coli | - |
UDP + 2-N,3-O-bis[(3R)-3-hydroxytetradecanoyl]-beta-D-glucosaminyl-(1->6)-2-N,3-O-bis[(3R)-3-hydroxytetradecanoyl]-alpha-D-glucosaminyl 1-phosphate | - |
? | |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Escherichia coli | P10441 | - |
- |
Reaction
| Reaction | Comment | Organism | Reaction ID |
|---|---|---|---|
| a UDP-2-N,3-O-bis[(3R)-3-hydroxyacyl]-alpha-D-glucosamine + a lipid X = UDP + a lipid A disaccharide | enzyme LpxB catalyzes the nucleophilic attack of the 6'-hydroxyl of lipid X (1) on the anomeric carbon of UDP-DAG (2) with UDP as the leaving group. As for other inverting GT-B enzymes, this reaction is thought to proceed by an SN2 mechanism | Escherichia coli |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| additional information | analysis of UDP binding site structure and ligand-bound structure of LpxB. The GlcNAc moiety is highly flexible or that UDP-GlcNAc is hydrolysed during soaking. The uracil base binds in a hydrophobic pocket formed by L197, P198, P231, and V233, which is on the second Rossmann-fold domain and facing the deep inter-domain cleft. The P231 carbonyl oxygen also hydrogen-bonds with N3 of uracil and the G199 amide nitrogen hydrogen-bonds with the O4 carbonyl of uracil. In addition, two water molecules connect active site residues to uracil: the first water connects the G199 carbonyl oxygen and the V233 amide nitrogen to O4 of uracil, and the second water connects the G261 amide nitrogen to the O2 carbonyl of uracil | Escherichia coli | ? | - |
- |
|
| UDP-2-N,3-O-bis[(3R)-3-hydroxytetradecanoyl]-alpha-D-glucosamine + 2-N,3-O-bis[(3R)-3-hydroxytetradecanoyl]-alpha-D-glucosaminyl 1-phosphate | - |
Escherichia coli | UDP + 2-N,3-O-bis[(3R)-3-hydroxytetradecanoyl]-beta-D-glucosaminyl-(1->6)-2-N,3-O-bis[(3R)-3-hydroxytetradecanoyl]-alpha-D-glucosaminyl 1-phosphate | - |
? | |
Subunits
| Subunits | Comment | Organism |
|---|---|---|
| dimer | 2 * 40000, about, soluble recombinant enzyme, SDS-PAGE | Escherichia coli |
| More | purified recombinant LpxB tends to form large, soluble aggregates | Escherichia coli |
Synonyms
| Synonyms | Comment | Organism |
|---|---|---|
| lipid A disaccharide synthase | - |
Escherichia coli |
| LpxB | - |
Escherichia coli |
General Information
| General Information | Comment | Organism |
|---|---|---|
| evolution | LpxB is among the most highly conserved enzymes in the Raetz pathway | Escherichia coli |
| metabolism | LpxB is a glycosyltransferase in the Raetz (lipid A synthesis) pathway that catalyzes nucleophilic attack of the 6'-hydroxyl of lipid X on the anomeric carbon of UDP-diacyl-glucosamine (UDP-DAG) to form beta(1-6)-tetraacyl-disaccharide 1-phosphate (lipid A disaccharide) | Escherichia coli |
| additional information | Escherichia coli enzyme LpxB has a glycosyltransferase-B family fold but with a highly intertwined, C-terminally swapped dimer comprising four domains. Homology modeling using the UDP-N-acetylglucosamine 2-epimerase structure from Thermus thermophilus strain HB8 (PDB ID 1V4V) as template, structure comparisons, overview. The hydrophobic patch (V66, V68, L69, L72, L75, and L76) is essential for productive membrane association or substrate binding | Escherichia coli |
| physiological function | most Gram-negative bacteria are surrounded by a glycolipid called lipopolysaccharide (LPS), which forms a barrier to hydrophobic toxins and, in pathogenic bacteria, is a virulence factor. During LPS biosynthesis, the membrane-associated glycosyltransferase (LpxB) forms a tetraacylated disaccharide that is further acylated to form the membrane anchor moiety of LPS. LpxB is essential for growth of Escherichia coli and is among the most highly conserved enzymes in the Raetz pathway | Escherichia coli |
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