Literature summary for 2.7.1.130 extracted from

Emptage, R.P.; Pemble, C.W.; York, J.D.; Raetz, C.R.; Zhou, P.
Mechanistic characterization of the tetraacyldisaccharide-1-phosphate 4-kinase LpxK involved in lipid A biosynthesis (2013), Biochemistry, 52, 2280-2290.

Search for more literature for 2.7.1.130

Cloned(Commentary)

Cloned (Comment)	Organism
recombinant expression of wild-type and mutant enzymes in Escherichia coli strain C41(DE3) membranes	Aquifex aeolicus

Crystallization (Commentary)

Crystallization (Comment)	Organism
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, 20°C, 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	Aquifex aeolicus

Crystallization (Comment)

Organism

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, 20°C, 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

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
D260A	site-directed mutagenesis, the mutant shows altered activity compared to the wild-type enzyme	Aquifex aeolicus
D99A	site-directed mutagenesis, the mutant shows altered activity compared to the wild-type enzyme	Aquifex aeolicus
D99E	site-directed mutagenesis, the mutant shows altered activity compared to the wild-type enzyme	Aquifex aeolicus
D99N	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
E100D	site-directed mutagenesis, the mutant shows altered activity compared to the wild-type enzyme	Aquifex aeolicus
E100Q	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
K51A	site-directed mutagenesis, the mutant shows altered activity compared to the wild-type enzyme	Aquifex aeolicus
S49A	site-directed mutagenesis, the mutant shows altered activity compared to the wild-type enzyme	Aquifex aeolicus
S53A	site-directed mutagenesis, the mutant shows altered activity compared to the wild-type enzyme	Aquifex aeolicus
T52A	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

Inhibitors

Inhibitors	Comment	Organism
Ca2+	-	Aquifex aeolicus
Cu2+	-	Aquifex aeolicus
Fe3+	-	Aquifex aeolicus
Ni2+	-	Aquifex aeolicus
Zn2+	-	Aquifex aeolicus

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
additional information	-	additional information	bi-substrate kinetics, steady-state kinetics	Aquifex aeolicus
0.0011	-	ATP	pH 8.5, 30°C, recombinant mutant D99N	Aquifex aeolicus
0.0012	-	ATP	pH 8.5, 30°C, recombinant wild-type enzyme	Aquifex aeolicus
0.0013	-	ATP	pH 8.5, 30°C, recombinant mutant H261A	Aquifex aeolicus
0.0014	-	ATP	pH 8.5, 30°C, recombinant mutant D139N	Aquifex aeolicus
0.0015	-	ATP	pH 8.5, 30°C, recombinant mutant K51A	Aquifex aeolicus
0.0016	-	ATP	pH 8.5, 30°C, recombinant mutant D260A	Aquifex aeolicus
0.0017	-	ATP	pH 8.5, 30°C, recombinant mutant Y74A	Aquifex aeolicus
0.0019	-	ATP	pH 8.5, 30°C, recombinant mutant E100A	Aquifex aeolicus
0.002	-	ATP	pH 8.5, 30°C, recombinant mutant E100D	Aquifex aeolicus
0.0022	-	ATP	pH 8.5, 30°C, recombinant mutant T52A	Aquifex aeolicus
0.0023	-	ATP	pH 8.5, 30°C, recombinant mutant D99E	Aquifex aeolicus
0.0027	-	ATP	pH 8.5, 30°C, recombinant mutant S49A	Aquifex aeolicus
0.0028	-	ATP	pH 8.5, 30°C, recombinant mutant D99A	Aquifex aeolicus
0.0029	-	ATP	pH 8.5, 30°C, recombinant mutant E100Q	Aquifex aeolicus
0.0032	-	ATP	pH 8.5, 30°C, recombinant mutant D138N	Aquifex aeolicus
0.004	-	ATP	pH 8.5, 30°C, recombinant mutant D138A	Aquifex aeolicus
0.005	-	ATP	pH 8.5, 30°C, recombinant mutant D139A	Aquifex aeolicus
0.006	-	ATP	pH 8.5, 30°C, recombinant mutant S53A	Aquifex aeolicus

Localization

Localization	Comment	Organism	GeneOntology No.	Textmining
inner membrane	cytosol-facing, membrane binding through a hydrophobic lower face assisted by surrounding basic residues of the N-terminal core domain	Aquifex aeolicus	-	-

Metals/Ions

Metals/Ions	Comment	Organism
Cl-	structural basis for anion inhibition of LpxK, modeling, overview	Aquifex aeolicus
Co2+	activates	Aquifex aeolicus
Mg2+	required, activates, optimal activity is observed at an equimolar ratio of ATP to Mg2+, with a decrease in activity at higher amounts of the divalent cation	Aquifex aeolicus
Mn2+	activates	Aquifex aeolicus

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	Aquifex aeolicus

Reaction

Reaction	Comment	Organism	Reaction ID
ATP + 2-deoxy-2-([(3R)-3-hydroxyacyl]amino)-3-O-[(3R)-3-hydroxyacyl]-beta-D-glucopyranosyl-(1->6)-2-deoxy-3-O-[(3R)-3-hydroxyacyl]-2-([(3R)-3-hydroxyacyl]amino)-1-O-phospho-alpha-D-glucopyranose = ADP + 2-deoxy-2-([(3R)-3-hydroxyacyl]amino)-3-O-[(3R)-3-hydroxyacyl]-4-O-phospho-beta-D-glucopyranosyl-(1->6)-2-deoxy-3-O-[(3R)-3-hydroxyacyl]-2-([(3R)-3-hydroxyacyl]amino)-1-O-phospho-alpha-D-glucopyranose	ping-pong type mechanism with bi-substrate kinetics	Aquifex aeolicus	a

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	the interaction of residue D99 with H261 acts to increase the pKa of the imidazole moiety, which in turn serves as the catalytic base to deprotonate the 4'-hydroxyl of the DSMP substrate. The LpxK enzyme activity in vitro requires the presence of a detergent micelle and formation of a ternary LpxK-ATP/Mg2+-DSMP complex	Aquifex aeolicus	?	-	?

Synonyms

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

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
30	-	assay at	Aquifex aeolicus

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
0.00035	-	ATP	pH 8.5, 30°C, recombinant mutant D99N	Aquifex aeolicus
0.00048	-	ATP	pH 8.5, 30°C, recombinant mutant D139A	Aquifex aeolicus
0.00048	-	ATP	pH 8.5, 30°C, recombinant mutant E100Q	Aquifex aeolicus
0.00083	-	ATP	pH 8.5, 30°C, recombinant mutant D138A	Aquifex aeolicus
0.0013	-	ATP	pH 8.5, 30°C, recombinant mutant K51A	Aquifex aeolicus
0.0013	-	ATP	pH 8.5, 30°C, recombinant mutant T52A	Aquifex aeolicus
0.0015	-	ATP	pH 8.5, 30°C, recombinant mutant D99A	Aquifex aeolicus
0.0027	-	ATP	pH 8.5, 30°C, recombinant mutant E100D	Aquifex aeolicus
0.0029	-	ATP	pH 8.5, 30°C, recombinant mutant E100A	Aquifex aeolicus
0.0046	-	ATP	pH 8.5, 30°C, recombinant mutant H261A	Aquifex aeolicus
0.0076	-	ATP	pH 8.5, 30°C, recombinant mutant D139N	Aquifex aeolicus
0.022	-	ATP	pH 8.5, 30°C, recombinant mutant Y74A	Aquifex aeolicus
0.05	-	ATP	pH 8.5, 30°C, recombinant mutant D99E	Aquifex aeolicus
0.057	-	ATP	pH 8.5, 30°C, recombinant mutant D138N	Aquifex aeolicus
0.073	-	ATP	pH 8.5, 30°C, recombinant mutant D260A	Aquifex aeolicus
0.47	-	ATP	pH 8.5, 30°C, recombinant mutant S53A	Aquifex aeolicus
3.9	-	ATP	pH 8.5, 30°C, recombinant wild-type enzyme	Aquifex aeolicus
3.9	-	ATP	pH 8.5, 30°C, recombinant mutant S49A	Aquifex aeolicus

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
additional information	-	pH-dependence of active site point mutants, overview	Aquifex aeolicus
7.5	8	recombinant mutant D99A	Aquifex aeolicus
8	-	recombinant mutant H261A	Aquifex aeolicus

Cofactor

Cofactor	Comment	Organism	Structure
ATP	the LpxK enzyme activity in vitro requires the presence of a detergent micelle and formation of a ternary LpxK-ATP/Mg2+-DSMP complex	Aquifex aeolicus

General Information

General Information	Comment	Organism
evolution	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	Aquifex aeolicus
metabolism	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	Aquifex aeolicus
additional information	apparent steady-state kinetic parameters for LpxK activity support the formation of a ternary LpxK-ATP/Mg2+-DSMP complex. In its closed catalytically competent form, the C-terminal domain of LpxK undergoes a hinge motion to close around the nucleotide substrate upon binding. Active sites of ATP-bound enzyme LpxK are in the open form, modeling, overview	Aquifex aeolicus
physiological function	in the reaction catalyzed by LpxK in Kdo2-lipid A biosynthesis, enzyme LpxK is responsible for the phosphorylation of the 4'-hydroxyl of tetraacyldisaccharide-1-phosphate (DSMP)	Aquifex aeolicus