Application | Comment | Organism |
---|---|---|
drug development | the enzyme structure provides a structural template for designing antibiotics and knowledge of catalysis at the membrane interface | Aquifex aeolicus |
Cloned (Comment) | Organism |
---|---|
gene lpxK, complementation of an Escherichia coli lpxK chromosomal knockout mutant strain W3110 with expression of Aquifex aeolicus gene lpxK, recombinant expression of wild-type and mutant enzymes in Escherichia coli strain C41(DE3) membranes | Aquifex aeolicus |
Crystallization (Comment) | Organism |
---|---|
purified enzyme LpxK in complex with lipid IVA, X-ray diffraction structure determination and analysis at 3.5 A | Aquifex aeolicus |
Protein Variants | Comment | Organism |
---|---|---|
D138A | site-directed mutagenesis, the mutant shows altered activity compared to the wild-type enzyme | Aquifex aeolicus |
D138N | site-directed mutagenesis, the mutant shows altered activity compared to the wild-type enzyme | Aquifex aeolicus |
D139A | site-directed mutagenesis, the mutant shows altered activity compared to the wild-type enzyme | Aquifex aeolicus |
D139N | site-directed mutagenesis, the mutant shows altered activity compared to the wild-type enzyme | Aquifex aeolicus |
E100A | site-directed mutagenesis, the mutant shows altered activity compared to the wild-type enzyme | Aquifex aeolicus |
E172A | site-directed mutagenesis, the mutant shows altered activity compared to the wild-type enzyme | Aquifex aeolicus |
H143A | site-directed mutagenesis, the mutant shows altered activity compared to the wild-type enzyme | Aquifex aeolicus |
H261A | site-directed mutagenesis, the mutant shows altered activity compared to the wild-type enzyme | Aquifex aeolicus |
additional information | steady-state kinetic analysis of multiple point mutants of the lipid-binding pocket pinpoints critical residues involved in substrate binding, and construction of two N-terminal helix truncated forms of LpxK, one in which amino acids 2-12 are removed, DELTA12LpxK, and another in which amino acids 2-29 are removed, DELTA29LpxK | Aquifex aeolicus |
N43A | site-directed mutagenesis, the mutant shows altered activity compared to the wild-type enzyme | Aquifex aeolicus |
Q142A | site-directed mutagenesis, the mutant shows altered activity compared to the wild-type enzyme | Aquifex aeolicus |
R119A | site-directed mutagenesis, the mutant shows altered activity compared to the wild-type enzyme | Aquifex aeolicus |
R171A | site-directed mutagenesis, the mutant shows altered activity compared to the wild-type enzyme | Aquifex aeolicus |
R72A | site-directed mutagenesis, the mutant shows altered activity compared to the wild-type enzyme | Aquifex aeolicus |
Y74A | site-directed mutagenesis, the mutant shows altered activity compared to the wild-type enzyme | Aquifex aeolicus |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.007 | - |
(2-N,3-O-bis[(3R)-3-hydroxytetradecanoyl]-beta-D-glucosaminyl)-(1->6)-(2-N,3-O-bis[(3R)-3-hydroxytetradecanoyl]-alpha-D-glucosaminyl phosphate) | pH 8.5, 30°C, recombinant wild-type enzyme | Aquifex aeolicus | |
0.007 | - |
(2-N,3-O-bis[(3R)-3-hydroxytetradecanoyl]-beta-D-glucosaminyl)-(1->6)-(2-N,3-O-bis[(3R)-3-hydroxytetradecanoyl]-alpha-D-glucosaminyl phosphate) | pH 8.5, 30°C, recombinant mutant D138A | Aquifex aeolicus | |
0.007 | - |
(2-N,3-O-bis[(3R)-3-hydroxytetradecanoyl]-beta-D-glucosaminyl)-(1->6)-(2-N,3-O-bis[(3R)-3-hydroxytetradecanoyl]-alpha-D-glucosaminyl phosphate) | pH 8.5, 30°C, recombinant mutant D138N | Aquifex aeolicus | |
0.009 | - |
(2-N,3-O-bis[(3R)-3-hydroxytetradecanoyl]-beta-D-glucosaminyl)-(1->6)-(2-N,3-O-bis[(3R)-3-hydroxytetradecanoyl]-alpha-D-glucosaminyl phosphate) | pH 8.5, 30°C, recombinant mutant N43A | Aquifex aeolicus | |
0.0097 | - |
(2-N,3-O-bis[(3R)-3-hydroxytetradecanoyl]-beta-D-glucosaminyl)-(1->6)-(2-N,3-O-bis[(3R)-3-hydroxytetradecanoyl]-alpha-D-glucosaminyl phosphate) | pH 8.5, 30°C, recombinant mutant R119A | Aquifex aeolicus | |
0.014 | - |
(2-N,3-O-bis[(3R)-3-hydroxytetradecanoyl]-beta-D-glucosaminyl)-(1->6)-(2-N,3-O-bis[(3R)-3-hydroxytetradecanoyl]-alpha-D-glucosaminyl phosphate) | pH 8.5, 30°C, recombinant mutant D139N | Aquifex aeolicus | |
0.015 | - |
(2-N,3-O-bis[(3R)-3-hydroxytetradecanoyl]-beta-D-glucosaminyl)-(1->6)-(2-N,3-O-bis[(3R)-3-hydroxytetradecanoyl]-alpha-D-glucosaminyl phosphate) | pH 8.5, 30°C, recombinant mutant H261A | Aquifex aeolicus | |
0.0174 | - |
(2-N,3-O-bis[(3R)-3-hydroxytetradecanoyl]-beta-D-glucosaminyl)-(1->6)-(2-N,3-O-bis[(3R)-3-hydroxytetradecanoyl]-alpha-D-glucosaminyl phosphate) | pH 8.5, 30°C, recombinant mutant E100A | Aquifex aeolicus | |
0.018 | - |
(2-N,3-O-bis[(3R)-3-hydroxytetradecanoyl]-beta-D-glucosaminyl)-(1->6)-(2-N,3-O-bis[(3R)-3-hydroxytetradecanoyl]-alpha-D-glucosaminyl phosphate) | pH 8.5, 30°C, recombinant mutant E172A | Aquifex aeolicus | |
0.028 | - |
(2-N,3-O-bis[(3R)-3-hydroxytetradecanoyl]-beta-D-glucosaminyl)-(1->6)-(2-N,3-O-bis[(3R)-3-hydroxytetradecanoyl]-alpha-D-glucosaminyl phosphate) | pH 8.5, 30°C, recombinant mutant Q142A | Aquifex aeolicus | |
0.028 | - |
(2-N,3-O-bis[(3R)-3-hydroxytetradecanoyl]-beta-D-glucosaminyl)-(1->6)-(2-N,3-O-bis[(3R)-3-hydroxytetradecanoyl]-alpha-D-glucosaminyl phosphate) | pH 8.5, 30°C, recombinant mutant R72A | Aquifex aeolicus | |
0.046 | - |
(2-N,3-O-bis[(3R)-3-hydroxytetradecanoyl]-beta-D-glucosaminyl)-(1->6)-(2-N,3-O-bis[(3R)-3-hydroxytetradecanoyl]-alpha-D-glucosaminyl phosphate) | pH 8.5, 30°C, recombinant mutant D139A | Aquifex aeolicus | |
0.048 | - |
(2-N,3-O-bis[(3R)-3-hydroxytetradecanoyl]-beta-D-glucosaminyl)-(1->6)-(2-N,3-O-bis[(3R)-3-hydroxytetradecanoyl]-alpha-D-glucosaminyl phosphate) | pH 8.5, 30°C, recombinant mutant Y74A | Aquifex aeolicus | |
0.05 | - |
(2-N,3-O-bis[(3R)-3-hydroxytetradecanoyl]-beta-D-glucosaminyl)-(1->6)-(2-N,3-O-bis[(3R)-3-hydroxytetradecanoyl]-alpha-D-glucosaminyl phosphate) | pH 8.5, 30°C, recombinant mutant H143A | Aquifex aeolicus | |
0.128 | - |
(2-N,3-O-bis[(3R)-3-hydroxytetradecanoyl]-beta-D-glucosaminyl)-(1->6)-(2-N,3-O-bis[(3R)-3-hydroxytetradecanoyl]-alpha-D-glucosaminyl phosphate) | pH 8.5, 30°C, recombinant mutant R171A | Aquifex aeolicus |
Localization | Comment | Organism | GeneOntology No. | Textmining |
---|---|---|---|---|
membrane | the enzyme is an integral membrane protein | Aquifex aeolicus | 16020 | - |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
ATP + (2-N,3-O-bis[(3R)-3-hydroxytetradecanoyl]-beta-D-glucosaminyl)-(1->6)-(2-N,3-O-bis[(3R)-3-hydroxytetradecanoyl]-alpha-D-glucosaminyl phosphate) | Aquifex aeolicus | - |
ADP + (2-N,3-O-bis[(3R)-3-hydroxytetradecanoyl]-4-O-phospho-beta-D-glucosaminyl)-(1->6)-(2-N,3-O-bis[(3R)-3-hydroxytetradecanoyl]-alpha-D-glucosaminyl phosphate) | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Aquifex aeolicus | O67572 | gene lpxK | - |
Purification (Comment) | Organism |
---|---|
recombinant wild-type and mutant enzymes from Escherichia coli strain C41(DE3) membranes by solubilization from membranes with Triton X-100, and ultracentrifugation | Aquifex aeolicus |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
ATP + (2-N,3-O-bis[(3R)-3-hydroxytetradecanoyl]-beta-D-glucosaminyl)-(1->6)-(2-N,3-O-bis[(3R)-3-hydroxytetradecanoyl]-alpha-D-glucosaminyl phosphate) | - |
Aquifex aeolicus | ADP + (2-N,3-O-bis[(3R)-3-hydroxytetradecanoyl]-4-O-phospho-beta-D-glucosaminyl)-(1->6)-(2-N,3-O-bis[(3R)-3-hydroxytetradecanoyl]-alpha-D-glucosaminyl phosphate) | - |
? | |
additional information | lipid IVA is bound in the putative lipid-binding pocket on the underside of the N-terminal enzyme domain. The L4 loop, which includes the Walker B motif, appears to contain important residues for binding the lipid substrate, lipid IVA binding structures of wild-type and mutant enzymes, overview | Aquifex aeolicus | ? | - |
? |
Synonyms | Comment | Organism |
---|---|---|
LpxK | - |
Aquifex aeolicus |
membrane-bound tetraacyldisaccharide-1-phosphate 4'-kinase | - |
Aquifex aeolicus |
tetraacyldisaccharide-1-phosphate 4-kinase | - |
Aquifex aeolicus |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.00051 | - |
(2-N,3-O-bis[(3R)-3-hydroxytetradecanoyl]-beta-D-glucosaminyl)-(1->6)-(2-N,3-O-bis[(3R)-3-hydroxytetradecanoyl]-alpha-D-glucosaminyl phosphate) | pH 8.5, 30°C, recombinant mutant D139A | Aquifex aeolicus | |
0.0011 | - |
(2-N,3-O-bis[(3R)-3-hydroxytetradecanoyl]-beta-D-glucosaminyl)-(1->6)-(2-N,3-O-bis[(3R)-3-hydroxytetradecanoyl]-alpha-D-glucosaminyl phosphate) | pH 8.5, 30°C, recombinant mutant D138A | Aquifex aeolicus | |
0.00123 | - |
(2-N,3-O-bis[(3R)-3-hydroxytetradecanoyl]-beta-D-glucosaminyl)-(1->6)-(2-N,3-O-bis[(3R)-3-hydroxytetradecanoyl]-alpha-D-glucosaminyl phosphate) | pH 8.5, 30°C, recombinant mutant E100A | Aquifex aeolicus | |
0.0031 | - |
(2-N,3-O-bis[(3R)-3-hydroxytetradecanoyl]-beta-D-glucosaminyl)-(1->6)-(2-N,3-O-bis[(3R)-3-hydroxytetradecanoyl]-alpha-D-glucosaminyl phosphate) | pH 8.5, 30°C, recombinant mutant H261A | Aquifex aeolicus | |
0.0063 | - |
(2-N,3-O-bis[(3R)-3-hydroxytetradecanoyl]-beta-D-glucosaminyl)-(1->6)-(2-N,3-O-bis[(3R)-3-hydroxytetradecanoyl]-alpha-D-glucosaminyl phosphate) | pH 8.5, 30°C, recombinant mutant D139N | Aquifex aeolicus | |
0.0138 | - |
(2-N,3-O-bis[(3R)-3-hydroxytetradecanoyl]-beta-D-glucosaminyl)-(1->6)-(2-N,3-O-bis[(3R)-3-hydroxytetradecanoyl]-alpha-D-glucosaminyl phosphate) | pH 8.5, 30°C, recombinant mutant R171A | Aquifex aeolicus | |
0.028 | - |
(2-N,3-O-bis[(3R)-3-hydroxytetradecanoyl]-beta-D-glucosaminyl)-(1->6)-(2-N,3-O-bis[(3R)-3-hydroxytetradecanoyl]-alpha-D-glucosaminyl phosphate) | pH 8.5, 30°C, recombinant mutant D138N | Aquifex aeolicus | |
0.031 | - |
(2-N,3-O-bis[(3R)-3-hydroxytetradecanoyl]-beta-D-glucosaminyl)-(1->6)-(2-N,3-O-bis[(3R)-3-hydroxytetradecanoyl]-alpha-D-glucosaminyl phosphate) | pH 8.5, 30°C, recombinant mutant Y74A | Aquifex aeolicus | |
0.036 | - |
(2-N,3-O-bis[(3R)-3-hydroxytetradecanoyl]-beta-D-glucosaminyl)-(1->6)-(2-N,3-O-bis[(3R)-3-hydroxytetradecanoyl]-alpha-D-glucosaminyl phosphate) | pH 8.5, 30°C, recombinant mutant R119A | Aquifex aeolicus | |
0.16 | - |
(2-N,3-O-bis[(3R)-3-hydroxytetradecanoyl]-beta-D-glucosaminyl)-(1->6)-(2-N,3-O-bis[(3R)-3-hydroxytetradecanoyl]-alpha-D-glucosaminyl phosphate) | pH 8.5, 30°C, recombinant mutant H143A | Aquifex aeolicus | |
0.35 | - |
(2-N,3-O-bis[(3R)-3-hydroxytetradecanoyl]-beta-D-glucosaminyl)-(1->6)-(2-N,3-O-bis[(3R)-3-hydroxytetradecanoyl]-alpha-D-glucosaminyl phosphate) | pH 8.5, 30°C, recombinant mutant Q142A | Aquifex aeolicus | |
0.36 | - |
(2-N,3-O-bis[(3R)-3-hydroxytetradecanoyl]-beta-D-glucosaminyl)-(1->6)-(2-N,3-O-bis[(3R)-3-hydroxytetradecanoyl]-alpha-D-glucosaminyl phosphate) | pH 8.5, 30°C, recombinant mutant N43A | Aquifex aeolicus | |
0.39 | - |
(2-N,3-O-bis[(3R)-3-hydroxytetradecanoyl]-beta-D-glucosaminyl)-(1->6)-(2-N,3-O-bis[(3R)-3-hydroxytetradecanoyl]-alpha-D-glucosaminyl phosphate) | pH 8.5, 30°C, recombinant mutant R72A | Aquifex aeolicus | |
0.7 | - |
(2-N,3-O-bis[(3R)-3-hydroxytetradecanoyl]-beta-D-glucosaminyl)-(1->6)-(2-N,3-O-bis[(3R)-3-hydroxytetradecanoyl]-alpha-D-glucosaminyl phosphate) | pH 8.5, 30°C, recombinant mutant E172A | Aquifex aeolicus | |
2.4 | - |
(2-N,3-O-bis[(3R)-3-hydroxytetradecanoyl]-beta-D-glucosaminyl)-(1->6)-(2-N,3-O-bis[(3R)-3-hydroxytetradecanoyl]-alpha-D-glucosaminyl phosphate) | pH 8.5, 30°C, recombinant wild-type enzyme | Aquifex aeolicus |
General Information | Comment | Organism |
---|---|---|
additional information | structural and kinetic studies reveal the molecular basis of lipid binding, overview. The LpxK active site recognizes the lipid's glucosamine/phosphate headgroups and only accommodates disaccharides. Steady-state kinetic analysis of multiple point mutants of the lipid-binding pocket pinpoints critical residues involved in substrate binding, and characterization of N-terminal helix truncation mutants uncovers the role of this substructure as a hydrophobic membrane anchor | Aquifex aeolicus |
physiological function | enzyme LpxK is an essential membrane-bound kinase in the lipid A biosynthetic pathway. In Gram-negative bacteria, lipidA is the hydrophobic anchor of lipopolysaccharide, which makes up the outer leaflet of the asymmetric outer membrane of these organisms. This acylated disaccharide of glucosamine plays an important role in eliciting an immunogenic response to bacterial pathogens and is essential to the survival of the vast majority of these microbes | Aquifex aeolicus |