Information on EC 2.7.1.130 - tetraacyldisaccharide 4'-kinase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
2.7.1.130
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RECOMMENDED NAME
GeneOntology No.
tetraacyldisaccharide 4'-kinase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
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) = 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)
show the reaction diagram
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} = 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}
show the reaction diagram
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-
-
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
phospho group transfer
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-
-
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
lipid IVA biosynthesis
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Lipopolysaccharide biosynthesis
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Metabolic pathways
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lipid A biosynthesis
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SYSTEMATIC NAME
IUBMB Comments
ATP:2,3,2',3'-tetrakis(3-hydroxytetradecanoyl)-D-glucosaminyl-beta-D-1,6-glucosaminyl-beta-phosphate 4'-O-phosphotransferase
Involved with EC 2.3.1.129 (acyl-[acyl-carrier- protein]---UDP-N-acetylglucosamine O-acyltransferase) and EC 2.4.1.182 (lipid-A-disaccharide synthase) in the biosynthesis of the phosphorylated glycolipid, lipid A, in the outer membrane of Escherichia coli.
CAS REGISTRY NUMBER
COMMENTARY hide
107309-06-8
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
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kinase LpxK is a member of the P-loop containing nucleoside triphosphate hydrolase superfamily. The active site Walker A (P-loop) and Walker B (Mg2+-binding) motifs are common to all P-loop kinase family members
metabolism
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the sixth step in the lipid A biosynthetic pathway involves phosphorylation of the tetraacyldisaccharide-1-phosphate (DSMP) intermediate by the cytosol-facing inner membrane kinase LpxK
physiological function
additional information
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
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)
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)
show the reaction diagram
ATP + 2,3-bis(3-hydroxytetradecanoyl)-D-glucosaminyl-(beta-D-1,6-)-2,3-bis(3-hydroxytetradecanoyl)-D-glucosaminyl beta-phosphate
ADP + 2,3,2',3'-tetrakis(3-hydroxytetradecanoyl)-D-glucosaminyl-1,6-beta-D-glucosamine 1,4'-bisphosphate
show the reaction diagram
ATP + 2,3-bis[3-(tetradecanoyl)tetradecanoyl]-D-glucosaminyl-(beta-D-1,6-)-2,3-bis(3-hydroxytetradecanoyl)-D-glucosaminyl beta-phosphate
ADP + 2,3,2',3'-tetrakis[3-(tetradecanoyl)tetradecanoyl]-D-glucosaminyl-1,6-beta-D-glucosamine 1,4'-bisphosphate
show the reaction diagram
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-
-
?
ATP + 2-(3-hydroxytetradecanoyl)-D-glucosaminyl-(beta-D-1,6-)-2,3-bis(3-hydroxytetradecanoyl)-D-glucosaminyl beta-phosphate
ADP + 2,2',3'-tris(3-hydroxytetradecanoyl)-D-glucosaminyl-1,6-beta-D-glucosamine 1,4'-bisphosphate
show the reaction diagram
-
-
-
?
ATP + 2-(3-hydroxytetradecanoyl)-D-glucosaminyl-(beta-D-1,6-)-2-(3-hydroxytetradecanoyl)-D-glucosaminyl beta-phosphate
ADP + 2,2'-bis(3-hydroxytetradecanoyl)-D-glucosaminyl-1,6-beta-D-glucosamine 1,4'-bisphosphate
show the reaction diagram
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-
-
?
ATP + 3-aza-2,3-bis(3-hydroxytetradecanoyl)-D-glucosaminyl-(beta-D-1,6-)-2,3-bis(3-hydroxytetradecanoyl)-D-glucosaminyl beta-phosphate
ADP + 3-aza-2,3,2',3'-tetrakis(3-hydroxytetradecanoyl)-D-glucosaminyl-1,6-beta-D-glucosamine 1,4'-bisphosphate
show the reaction diagram
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-
-
?
CTP + 2,3-bis(3-hydroxytetradecanoyl)-D-glucosaminyl-(beta-D-1,6-)-2,3-bis(3-hydroxytetradecanoyl)-D-glucosaminyl beta-phosphate
CDP + 2,3,2',3'-tetrakis(3-hydroxytetradecanoyl)-D-glucosaminyl-1,6-beta-D-glucosamine 1,4'-bisphosphate
show the reaction diagram
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approx. 50% of activity with ATP
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?
GTP + 2,3-bis(3-hydroxytetradecanoyl)-D-glucosaminyl-(beta-D-1,6-)-2,3-bis(3-hydroxytetradecanoyl)-D-glucosaminyl beta-phosphate
GDP + 2,3,2',3'-tetrakis(3-hydroxytetradecanoyl)-D-glucosaminyl-1,6-beta-D-glucosamine 1,4'-bisphosphate
show the reaction diagram
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approx. 50% of activity with ATP
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?
UTP + 2,3-bis(3-hydroxytetradecanoyl)-D-glucosaminyl-(beta-D-1,6-)-2,3-bis(3-hydroxytetradecanoyl)-D-glucosaminyl beta-phosphate
UDP + 2,3,2',3'-tetrakis(3-hydroxytetradecanoyl)-D-glucosaminyl-1,6-beta-D-glucosamine 1,4'-bisphosphate
show the reaction diagram
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approx. 50% of activity with ATP
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?
additional information
?
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NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
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)
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)
show the reaction diagram
ATP + 2,3-bis(3-hydroxytetradecanoyl)-D-glucosaminyl-(beta-D-1,6-)-2,3-bis(3-hydroxytetradecanoyl)-D-glucosaminyl beta-phosphate
ADP + 2,3,2',3'-tetrakis(3-hydroxytetradecanoyl)-D-glucosaminyl-1,6-beta-D-glucosamine 1,4'-bisphosphate
show the reaction diagram
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involved with EC 2.3.1.129 and 2.4.1.182 in the biosynthesis of the phosphorylated glycolipid, lipid A, in the outer membrane
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?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
ATP
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the LpxK enzyme activity in vitro requires the presence of a detergent micelle and formation of a ternary LpxK-ATP/Mg2+-DSMP complex
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Cl-
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structural basis for anion inhibition of LpxK, modeling, overview
Co2+
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activates
Mn2+
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activates
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Nonidet P-40
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inactivation by preincubation
octylglucoside
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inactivation by preincubation
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
cardiolipin
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other phospholipids including phosphatidylethanolamine, phosphatidylserine, phosphatidylcholine and phosphatidylglycerol are not as effective as cardiolipin
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.007 - 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)
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0.0011 - 0.006
ATP
additional information
additional information
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bi-substrate kinetics, steady-state kinetics
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00051 - 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)
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0.00035 - 3.9
ATP
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
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assay procedure
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.4
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assay at
7.5 - 8
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recombinant mutant D99A
8
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recombinant mutant H261A
additional information
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pH-dependence of active site point mutants, overview
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
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cytosol-facing, membrane binding through a hydrophobic lower face assisted by surrounding basic residues of the N-terminal core domain
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Manually annotated by BRENDA team
PDB
SCOP
CATH
ORGANISM
UNIPROT
Aquifex aeolicus (strain VF5)
Aquifex aeolicus (strain VF5)
Aquifex aeolicus (strain VF5)
Aquifex aeolicus (strain VF5)
Aquifex aeolicus (strain VF5)
Aquifex aeolicus (strain VF5)
Aquifex aeolicus (strain VF5)
Chromobacterium violaceum (strain ATCC 12472 / DSM 30191 / JCM 1249 / NBRC 12614 / NCIMB 9131 / NCTC 9757)
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
apo- and ADP/Mg2+-bound forms, to a resolution of 1.9 A and 2.2 A, respectively. The enzyme consists of two alpha/beta/alpha sandwich domains connected by a two-stranded beta-sheet linker. The N-terminal domain, which has most structural homology to other family members, is responsible for catalysis at the P-loop and positioning of the disaccharide 1-phosphate substrate for phosphoryl transfer on the inner membrane. The smaller C-terminal domain helps to bind the nucleotide substrate and Mg2+ cation using a 25 hinge motion about its base
purified enzyme LpxK in complex with lipid IVA, X-ray diffraction structure determination and analysis at 3.5 A
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purified LpxK in complex with the ATP analogue AMPPCP in the closed catalytically competent conformation, sitting drop vapor diffusion method, mixing of 0.70 ml well solution with 0.01 ml sample solution, containing four parts of a reservoir solution consisting of 50% v/v MPD and 0.1 M HEPES, pH 7.5, and one part protein solution containing 13 mg/ml enzyme LpxK, 4.3 mM AMP-PCP, 1 mM EDTA, 0.5% w/v DDM, 540 mM NaCl, 14% v/v glycerol, and 35 mM HEPES, pH 8.0, 20C, 1 month, or by microseeding, X-ray diffraction structure determination and analysis at 2.1 A resolution, molecular replacement using ADP-Mg2+ LpxK structure, PDB ID 4EHY, as the search model with all ligands removed, and AMP-PCP is subsequently added to the model. Purified LpxK in complex with ATP in a pre-catalytic binding state, mixing of seventeen parts of a reservoir solution consisting of 60% v/v MPD and 0.1 M HEPES, pH 7.5, and three parts protein solution containing 7.4 mg/ml enzyme LpxK, 10 mM ATP, 1 mM EDTA, 0.35% w/v DDM, 700 mM NaCl, 18.5% v/v glycerol, and 45 mM HEPES, pH 8.0, X-ray diffraction structure determination and analysis at 2.2 A resolution, molecular replacement using LpxK structure, PDB ID 4EHX, as the search model, ATP is subsequently added to the model. Purified LpxK in complex with a chloride anion in an inhibitory conformation of the nucleotide-binding P-loop, mixing of three parts of a reservoir solution consisting of 40% v/v MPD and 0.1 M HEPES, pH 7.5, and one part protein solution containing 8.3 mg/ml LpxK, 4 mM methyl 2-acetamido-2-deoxy-beta-D-glucopyranoside, 0.35% w/v DDM, 625 mM NaCl, 17% v/v glycerol, and 45 mM HEPES, pH 8.0, X-ray diffraction structure determination and analysis at 2.2 A resolution, molecular replacement using the apo enzyme LpxK structure, PDB ID 4EHX, as search model and spherical active site density refines well as a chloride ion
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GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
25% glycerol stabilizes
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phosphoenolpyruvate stabilizes
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
recombinant wild-type and mutant enzymes from Escherichia coli strain C41(DE3) membranes
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recombinant wild-type and mutant enzymes from Escherichia coli strain C41(DE3) membranes by solubilization from membranes with Triton X-100, and ultracentrifugation
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
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
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overexpression of lipid A 4'-kinase structural gene lpxK in Escherichia coli
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recombinant expression of wild-type and mutant enzymes in Escherichia coli strain C41(DE3) membranes
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D138N
site-directed mutagenesis, the mutant shows altered activity compared to the wild-type enzyme
D139N
site-directed mutagenesis, the mutant shows altered activity compared to the wild-type enzyme
D260A
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site-directed mutagenesis, the mutant shows altered activity compared to the wild-type enzyme
D99A
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site-directed mutagenesis, the mutant shows altered activity compared to the wild-type enzyme
D99E
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site-directed mutagenesis, the mutant shows altered activity compared to the wild-type enzyme
D99N
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site-directed mutagenesis, the mutant shows altered activity compared to the wild-type enzyme
E100A
site-directed mutagenesis, the mutant shows altered activity compared to the wild-type enzyme
E100D
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site-directed mutagenesis, the mutant shows altered activity compared to the wild-type enzyme
E100Q
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site-directed mutagenesis, the mutant shows altered activity compared to the wild-type enzyme
E172A
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site-directed mutagenesis, the mutant shows altered activity compared to the wild-type enzyme
G47A
about 43% of wild-type activity
G48A
about 1.8% of wild-type activity
G50A
about 0.1% of wild-type activity
H143A
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site-directed mutagenesis, the mutant shows altered activity compared to the wild-type enzyme
H261A
site-directed mutagenesis, the mutant shows altered activity compared to the wild-type enzyme
N43A
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site-directed mutagenesis, the mutant shows altered activity compared to the wild-type enzyme
Q142A
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site-directed mutagenesis, the mutant shows altered activity compared to the wild-type enzyme
R119A
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site-directed mutagenesis, the mutant shows altered activity compared to the wild-type enzyme
R171A
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site-directed mutagenesis, the mutant shows altered activity compared to the wild-type enzyme
R72A
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site-directed mutagenesis, the mutant shows altered activity compared to the wild-type enzyme
S49A
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site-directed mutagenesis, the mutant shows altered activity compared to the wild-type enzyme
Y74A
site-directed mutagenesis, the mutant shows altered activity compared to the wild-type enzyme
additional information
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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
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
drug development
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the enzyme structure provides a structural template for designing antibiotics and knowledge of catalysis at the membrane interface
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