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Literature summary for 2.7.1.130 extracted from

  • Emptage, R.P.; Tonthat, N.K.; York, J.D.; Schumacher, M.A.; Zhou, P.
    Structural basis of lipid binding for the membrane-embedded tetraacyldisaccharide-1-phosphate 4-kinase LpxK (2014), J. Biol. Chem., 289, 24059-24068.
    View publication on PubMedView publication on EuropePMC

Application

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(Commentary)

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 (Commentary)

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

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 [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

Localization Comment Organism GeneOntology No. Textmining
membrane the enzyme is an integral membrane protein Aquifex aeolicus 16020
-

Natural Substrates/ Products (Substrates)

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

Organism UniProt Comment Textmining
Aquifex aeolicus O67572 gene lpxK
-

Purification (Commentary)

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 and Products (Substrate)

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

Synonyms Comment Organism
LpxK
-
Aquifex aeolicus
membrane-bound tetraacyldisaccharide-1-phosphate 4'-kinase
-
Aquifex aeolicus
tetraacyldisaccharide-1-phosphate 4-kinase
-
Aquifex aeolicus

Turnover Number [1/s]

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

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