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show all sequences of 2.3.1.191

Steady-state kinetics and mechanism of LpxD, the N-acyltransferase of lipid A biosynthesis

Bartling, C.M.; Raetz, C.R.; Biochemistry 47, 5290-5302 (2008)

Data extracted from this reference:

Activating Compound
Activating Compound
Commentary
Organism
Structure
additional information
LpxD does not require the presence of a detergent for catalytic activity because the critical micelle concentrations of its substrates are likely to be above 0.1 mM
Escherichia coli
Cloned(Commentary)
Commentary
Organism
overexpression in Escherichia coli
Escherichia coli
Engineering
Amino acid exchange
Commentary
Organism
D232A
mutation causes a 10fold reduction in kcat and a striking increase in the KM for both substrates
Escherichia coli
F41A
mutation increases the KM for UDP-3-O-((3R)-3-hydroxymyristoyl)-a-D-glucosamine 30fold and kcat 5fold
Escherichia coli
K194A
mutation has little effect on activity
Escherichia coli
K46A
mutation causes 3fold increase in KM((3R)-3-hydroxymyristoyl-[acyl-carrier protein]) and has no effect on kcat
Escherichia coli
N233A
mutation causes a 10fold reduction in kcat and a striking increase in the KM for both substrates
Escherichia coli
N240A
causes less than a 2fold reduction in specific activity, when assayed at substrate concentrations at 2fold above KM with the purified proteins
Escherichia coli
N44A
causes less than a 2fold reduction in specific activity, when assayed at substrate concentrations at 2fold above KM with the purified proteins
Escherichia coli
Q236A
mutation has little effect on activity
Escherichia coli
Q32A
causes less than a 2fold reduction in specific activity, when assayed at substrate concentrations at 2fold above KM with the purified proteins
Escherichia coli
R293A
KM((3R)-3-hydroxymyristoyl-[acyl-carrier protein]) increases 23fold compared to wild-type with little effect on kcat
Escherichia coli
Inhibitors
Inhibitors
Commentary
Organism
Structure
(3R)-3-hydroxylauroyl-methylphosphopantetheine
competitive inhibitor with respect to UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine and an uncompetitive inhibitor with respect to (3R)-3-hydroxymyristoyl-[acyl-carrier protein]
Escherichia coli
(3R)-3-hydroxylauroylmethylphosphopantetheine
uncompetitive inhibitor against (3R)-3-hydroxymyristoyl-[acyl-carrier protein] and a competitive inhibitor against UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
Escherichia coli
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
-
Escherichia coli
acyl-carrier protein
competitive inhibitor with respect to (3R)-3-hydroxymyristoyl-[acyl-carrier protein] and a noncompetitive inhibitor with respect to UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
Escherichia coli
Ca2+
inhibition is overcome by the addition of excess (3R)-3-hydroxymyristoyl-[acyl-carrier protein]
Escherichia coli
Mg2+
-
Escherichia coli
additional information
divalent cations inhibit (3R)-3-hydroxymyristoyl-[acyl-carrier protein]-dependent acylation but not (3R)-3-hydroxylauroylmethylphosphopantetheine-dependent acylation, indicating that the acidic recognition helix of (3R)-3-hydroxymyristoyl-[acyl-carrier protein] contributes to binding; Na+ and K+ ions do not inhibit LpxD activity
Escherichia coli
UDP-2-N-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
noncompetitive inhibitor against both substrates
Escherichia coli
KM Value [mM]
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
additional information
-
additional information
steady-state kinetic analysis
Escherichia coli
0.00084
-
UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
pH 7.5, 30°C, mutant enzyme H239A
Escherichia coli
0.0017
-
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
pH 7.5, 30°C, mutant enzyme H276A
Escherichia coli
0.0032
-
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
pH 7.5, 30°C, wild-type enzyme
Escherichia coli
0.0034
-
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
pH 7.5, 30°C, mutant enzyme H239A
Escherichia coli
0.0035
-
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
pH 7.5, 30°C, mutant enzyme F41A
Escherichia coli
0.0036
-
UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
pH 7.5, 30°C, mutant enzyme R293A
Escherichia coli
0.0038
-
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
pH 7.5, 30°C, mutant enzyme K194A
Escherichia coli
0.0042
-
UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
pH 7.5, 30°C, mutant enzyme Q236A
Escherichia coli
0.005
-
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
pH 7.5, 30°C, mutant enzyme Y47A
Escherichia coli
0.0056
-
UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
pH 7.5, 30°C, mutant enzyme K194A
Escherichia coli
0.0063
-
UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
pH 7.5, 30°C, mutant enzyme H276A
Escherichia coli
0.0071
-
UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
pH 7.5, 30°C, mutant enzyme K46A
Escherichia coli
0.0078
-
UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
pH 7.5, 30°C, mutant enzyme Y47A
Escherichia coli
0.012
-
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
pH 7.5, 30°C, mutant enzyme K46A
Escherichia coli
0.013
-
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
pH 7.5, 30°C, mutant enzyme N233A; pH 7.5, 30°C, mutant enzyme Q236A
Escherichia coli
0.028
-
UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
pH 7.5, 30°C, mutant enzyme N233A
Escherichia coli
0.047
-
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
pH 7.5, 30°C, mutant enzyme D232A
Escherichia coli
0.067
-
UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
pH 7.5, 30°C, mutant enzyme D232A
Escherichia coli
0.073
-
UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
pH 7.5, 30°C, mutant enzyme F41A
Escherichia coli
0.074
-
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
pH 7.5, 30°C, mutant enzyme R293A
Escherichia coli
Molecular Weight [Da]
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
35880
-
3 * 35880, electrospray-ionization/time-of-flight mass spectrometry
Escherichia coli
35881
-
3 * 35881, calculated from sequence
Escherichia coli
108000
-
gel filtration
Escherichia coli
Natural Substrates/ Products (Substrates)
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
(3R)-3-hydroxymyristoyl-[acyl-carrier protein] + UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
Escherichia coli
third step of lipid A biosynthesis
UDP-2,3-bis(3-hydroxymyristoyl)-alpha-D-glucosamine + holo-[acyl-carrier protein]
-
-
?
Organism
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
Escherichia coli
-
-
-
Purification (Commentary)
Commentary
Organism
purification of untagged EcLpxD and an active N-terminally His6-tagged LpxD variant to near homogeneityrecombinant enzyme
Escherichia coli
Specific Activity [micromol/min/mg]
Specific Activity Minimum [µmol/min/mg]
Specific Activity Maximum [µmol/min/mg]
Commentary
Organism
7.14
-
specific activity in the absence of Ca2+
Escherichia coli
8.9
-
-
Escherichia coli
Substrates and Products (Substrate)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
(3R)-3-hydroxymyristoyl-[acyl-carrier protein] + UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
third step of lipid A biosynthesis
696264
Escherichia coli
UDP-2,3-bis(3-hydroxymyristoyl)-alpha-D-glucosamine + holo-[acyl-carrier protein]
-
-
-
?
(3R)-3-hydroxymyristoyl-[acyl-carrier protein] + UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
compulsory ordered mechanism in which (3R)-3-hydroxymyristoyl-[acyl-carrier protein] binds prior to UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine. The product, UDP-2,3-diacylglucosamine, dissociates prior to acyl-carrier protein
696264
Escherichia coli
UDP-2,3-bis(3-hydroxymyristoyl)-alpha-D-glucosamine + holo-[acyl-carrier protein]
-
-
-
?
additional information
R-3-hydroxylauroyl-methylphosphopantetheine is a very poor substrate. The specific activity, measured at either 0.01 mM or 1 mM (3R)-3-hydroxylauroylmethylphosphopantetheine as the acyl donor, is more than 100fold lower than with 0.01 mM (3R)-3-hydroxymyristoyl-[acyl-carrier protein]
696264
Escherichia coli
?
-
-
-
-
Subunits
Subunits
Commentary
Organism
homotrimer
3 * 35880, electrospray-ionization/time-of-flight mass spectrometry; 3 * 35881, calculated from sequence
Escherichia coli
Temperature Optimum [°C]
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
30
-
assay at
Escherichia coli
Turnover Number [1/s]
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
additional information
-
additional information
steady-state kinetic analysis
Escherichia coli
0.032
-
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
pH 7.5, 30°C, mutant enzyme H239A
Escherichia coli
0.032
-
UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
pH 7.5, 30°C, mutant enzyme H239A
Escherichia coli
0.73
-
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
pH 7.5, 30°C, mutant enzyme H276A
Escherichia coli
0.73
-
UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
pH 7.5, 30°C, mutant enzyme H276A
Escherichia coli
1.4
-
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
pH 7.5, 30°C, mutant enzyme N233A
Escherichia coli
1.4
-
UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
pH 7.5, 30°C, mutant enzyme N233A
Escherichia coli
1.9
-
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
pH 7.5, 30°C, mutant enzyme D232A
Escherichia coli
1.9
-
UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
pH 7.5, 30°C, mutant enzyme D232A
Escherichia coli
4.1
-
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
pH 7.5, 30°C, mutant enzyme F41A
Escherichia coli
4.1
-
UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
pH 7.5, 30°C, mutant enzyme F41A
Escherichia coli
5.7
-
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
pH 7.5, 30°C, mutant enzyme K194A
Escherichia coli
5.7
-
UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
pH 7.5, 30°C, mutant enzyme K194A
Escherichia coli
8.9
-
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
pH 7.5, 30°C, mutant enzyme R293A
Escherichia coli
8.9
-
UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
pH 7.5, 30°C, mutant enzyme R293A
Escherichia coli
12
-
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
pH 7.5, 30°C, mutant enzyme Q236A
Escherichia coli
12
-
UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
pH 7.5, 30°C, mutant enzyme Q236A
Escherichia coli
17
-
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
pH 7.5, 30°C, mutant enzyme K46A
Escherichia coli
17
-
UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
pH 7.5, 30°C, mutant enzyme K46A
Escherichia coli
18
-
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
pH 7.5, 30°C, mutant enzyme Y47A
Escherichia coli
18
-
UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
pH 7.5, 30°C, mutant enzyme Y47A
Escherichia coli
23
-
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
pH 7.5, 30°C, wild-type enzyme
Escherichia coli
23
-
UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
pH 7.5, 30°C, wild-type enzyme
Escherichia coli
pH Optimum
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
7.5
-
assay at
Escherichia coli
Ki Value [mM]
Ki Value [mM]
Ki Value maximum [mM]
Inhibitor
Commentary
Organism
Structure
0.0043
-
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
pH 7.5, 30°C
Escherichia coli
0.006
-
UDP-2-N-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
pH 7.5, 30°C, noncompetitive inhibition against (R)-3-hydroxymyristoyl-[acyl-carrier protein]
Escherichia coli
0.0094
-
UDP-2-N-(R-3-hydroxymyristoyl)-alpha-D-glucosamine
pH 7.5, 30°C, noncompetitive inhibition against UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
Escherichia coli
0.048
-
acyl-carrier protein
pH 7.5, 30°C, competitive inhibition with respect to (R)-3-hydroxymyristoyl-[acyl-carrier protein]
Escherichia coli
0.139
-
acyl-carrier protein
pH 7.5, 30°C, noncompetitive inhibitionr with respect to UDP-3-O-(R-3-hydroxymyristoyl)-alpha-D-glucosamine
Escherichia coli
0.39
-
(3R)-3-hydroxylauroyl-methylphosphopantetheine
pH 7.5, 30°C, competitive inhibition with respect to UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
Escherichia coli
0.69
-
(3R)-3-hydroxylauroyl-methylphosphopantetheine
pH 7.5, 30°C, uncompetitive inhibition with respect to (3R)-3-hydroxymyristoyl-[acyl-carrier protein]
Escherichia coli
IC50 Value
IC50 Value
IC50 Value Maximum
Commentary
Organism
Inhibitor
Structure
0.42
-
pH 7.5, 30°C
Escherichia coli
Ca2+
1.41
-
pH 7.5, 30°C
Escherichia coli
Mg2+
Activating Compound (protein specific)
Activating Compound
Commentary
Organism
Structure
additional information
LpxD does not require the presence of a detergent for catalytic activity because the critical micelle concentrations of its substrates are likely to be above 0.1 mM
Escherichia coli
Cloned(Commentary) (protein specific)
Commentary
Organism
overexpression in Escherichia coli
Escherichia coli
Engineering (protein specific)
Amino acid exchange
Commentary
Organism
D232A
mutation causes a 10fold reduction in kcat and a striking increase in the KM for both substrates
Escherichia coli
F41A
mutation increases the KM for UDP-3-O-((3R)-3-hydroxymyristoyl)-a-D-glucosamine 30fold and kcat 5fold
Escherichia coli
K194A
mutation has little effect on activity
Escherichia coli
K46A
mutation causes 3fold increase in KM((3R)-3-hydroxymyristoyl-[acyl-carrier protein]) and has no effect on kcat
Escherichia coli
N233A
mutation causes a 10fold reduction in kcat and a striking increase in the KM for both substrates
Escherichia coli
N240A
causes less than a 2fold reduction in specific activity, when assayed at substrate concentrations at 2fold above KM with the purified proteins
Escherichia coli
N44A
causes less than a 2fold reduction in specific activity, when assayed at substrate concentrations at 2fold above KM with the purified proteins
Escherichia coli
Q236A
mutation has little effect on activity
Escherichia coli
Q32A
causes less than a 2fold reduction in specific activity, when assayed at substrate concentrations at 2fold above KM with the purified proteins
Escherichia coli
R293A
KM((3R)-3-hydroxymyristoyl-[acyl-carrier protein]) increases 23fold compared to wild-type with little effect on kcat
Escherichia coli
IC50 Value (protein specific)
IC50 Value
IC50 Value Maximum
Commentary
Organism
Inhibitor
Structure
0.42
-
pH 7.5, 30°C
Escherichia coli
Ca2+
1.41
-
pH 7.5, 30°C
Escherichia coli
Mg2+
Inhibitors (protein specific)
Inhibitors
Commentary
Organism
Structure
(3R)-3-hydroxylauroyl-methylphosphopantetheine
competitive inhibitor with respect to UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine and an uncompetitive inhibitor with respect to (3R)-3-hydroxymyristoyl-[acyl-carrier protein]
Escherichia coli
(3R)-3-hydroxylauroylmethylphosphopantetheine
uncompetitive inhibitor against (3R)-3-hydroxymyristoyl-[acyl-carrier protein] and a competitive inhibitor against UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
Escherichia coli
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
-
Escherichia coli
acyl-carrier protein
competitive inhibitor with respect to (3R)-3-hydroxymyristoyl-[acyl-carrier protein] and a noncompetitive inhibitor with respect to UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
Escherichia coli
Ca2+
inhibition is overcome by the addition of excess (3R)-3-hydroxymyristoyl-[acyl-carrier protein]
Escherichia coli
Mg2+
-
Escherichia coli
additional information
divalent cations inhibit (3R)-3-hydroxymyristoyl-[acyl-carrier protein]-dependent acylation but not (3R)-3-hydroxylauroylmethylphosphopantetheine-dependent acylation, indicating that the acidic recognition helix of (3R)-3-hydroxymyristoyl-[acyl-carrier protein] contributes to binding; Na+ and K+ ions do not inhibit LpxD activity
Escherichia coli
UDP-2-N-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
noncompetitive inhibitor against both substrates
Escherichia coli
Ki Value [mM] (protein specific)
Ki Value [mM]
Ki Value maximum [mM]
Inhibitor
Commentary
Organism
Structure
0.0043
-
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
pH 7.5, 30°C
Escherichia coli
0.006
-
UDP-2-N-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
pH 7.5, 30°C, noncompetitive inhibition against (R)-3-hydroxymyristoyl-[acyl-carrier protein]
Escherichia coli
0.0094
-
UDP-2-N-(R-3-hydroxymyristoyl)-alpha-D-glucosamine
pH 7.5, 30°C, noncompetitive inhibition against UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
Escherichia coli
0.048
-
acyl-carrier protein
pH 7.5, 30°C, competitive inhibition with respect to (R)-3-hydroxymyristoyl-[acyl-carrier protein]
Escherichia coli
0.139
-
acyl-carrier protein
pH 7.5, 30°C, noncompetitive inhibitionr with respect to UDP-3-O-(R-3-hydroxymyristoyl)-alpha-D-glucosamine
Escherichia coli
0.39
-
(3R)-3-hydroxylauroyl-methylphosphopantetheine
pH 7.5, 30°C, competitive inhibition with respect to UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
Escherichia coli
0.69
-
(3R)-3-hydroxylauroyl-methylphosphopantetheine
pH 7.5, 30°C, uncompetitive inhibition with respect to (3R)-3-hydroxymyristoyl-[acyl-carrier protein]
Escherichia coli
KM Value [mM] (protein specific)
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
additional information
-
additional information
steady-state kinetic analysis
Escherichia coli
0.00084
-
UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
pH 7.5, 30°C, mutant enzyme H239A
Escherichia coli
0.0017
-
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
pH 7.5, 30°C, mutant enzyme H276A
Escherichia coli
0.0032
-
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
pH 7.5, 30°C, wild-type enzyme
Escherichia coli
0.0034
-
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
pH 7.5, 30°C, mutant enzyme H239A
Escherichia coli
0.0035
-
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
pH 7.5, 30°C, mutant enzyme F41A
Escherichia coli
0.0036
-
UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
pH 7.5, 30°C, mutant enzyme R293A
Escherichia coli
0.0038
-
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
pH 7.5, 30°C, mutant enzyme K194A
Escherichia coli
0.0042
-
UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
pH 7.5, 30°C, mutant enzyme Q236A
Escherichia coli
0.005
-
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
pH 7.5, 30°C, mutant enzyme Y47A
Escherichia coli
0.0056
-
UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
pH 7.5, 30°C, mutant enzyme K194A
Escherichia coli
0.0063
-
UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
pH 7.5, 30°C, mutant enzyme H276A
Escherichia coli
0.0071
-
UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
pH 7.5, 30°C, mutant enzyme K46A
Escherichia coli
0.0078
-
UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
pH 7.5, 30°C, mutant enzyme Y47A
Escherichia coli
0.012
-
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
pH 7.5, 30°C, mutant enzyme K46A
Escherichia coli
0.013
-
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
pH 7.5, 30°C, mutant enzyme N233A; pH 7.5, 30°C, mutant enzyme Q236A
Escherichia coli
0.028
-
UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
pH 7.5, 30°C, mutant enzyme N233A
Escherichia coli
0.047
-
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
pH 7.5, 30°C, mutant enzyme D232A
Escherichia coli
0.067
-
UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
pH 7.5, 30°C, mutant enzyme D232A
Escherichia coli
0.073
-
UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
pH 7.5, 30°C, mutant enzyme F41A
Escherichia coli
0.074
-
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
pH 7.5, 30°C, mutant enzyme R293A
Escherichia coli
Molecular Weight [Da] (protein specific)
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
35880
-
3 * 35880, electrospray-ionization/time-of-flight mass spectrometry
Escherichia coli
35881
-
3 * 35881, calculated from sequence
Escherichia coli
108000
-
gel filtration
Escherichia coli
Natural Substrates/ Products (Substrates) (protein specific)
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
(3R)-3-hydroxymyristoyl-[acyl-carrier protein] + UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
Escherichia coli
third step of lipid A biosynthesis
UDP-2,3-bis(3-hydroxymyristoyl)-alpha-D-glucosamine + holo-[acyl-carrier protein]
-
-
?
Purification (Commentary) (protein specific)
Commentary
Organism
purification of untagged EcLpxD and an active N-terminally His6-tagged LpxD variant to near homogeneityrecombinant enzyme
Escherichia coli
Specific Activity [micromol/min/mg] (protein specific)
Specific Activity Minimum [µmol/min/mg]
Specific Activity Maximum [µmol/min/mg]
Commentary
Organism
7.14
-
specific activity in the absence of Ca2+
Escherichia coli
8.9
-
-
Escherichia coli
Substrates and Products (Substrate) (protein specific)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
(3R)-3-hydroxymyristoyl-[acyl-carrier protein] + UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
third step of lipid A biosynthesis
696264
Escherichia coli
UDP-2,3-bis(3-hydroxymyristoyl)-alpha-D-glucosamine + holo-[acyl-carrier protein]
-
-
-
?
(3R)-3-hydroxymyristoyl-[acyl-carrier protein] + UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
compulsory ordered mechanism in which (3R)-3-hydroxymyristoyl-[acyl-carrier protein] binds prior to UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine. The product, UDP-2,3-diacylglucosamine, dissociates prior to acyl-carrier protein
696264
Escherichia coli
UDP-2,3-bis(3-hydroxymyristoyl)-alpha-D-glucosamine + holo-[acyl-carrier protein]
-
-
-
?
additional information
R-3-hydroxylauroyl-methylphosphopantetheine is a very poor substrate. The specific activity, measured at either 0.01 mM or 1 mM (3R)-3-hydroxylauroylmethylphosphopantetheine as the acyl donor, is more than 100fold lower than with 0.01 mM (3R)-3-hydroxymyristoyl-[acyl-carrier protein]
696264
Escherichia coli
?
-
-
-
-
Subunits (protein specific)
Subunits
Commentary
Organism
homotrimer
3 * 35880, electrospray-ionization/time-of-flight mass spectrometry; 3 * 35881, calculated from sequence
Escherichia coli
Temperature Optimum [°C] (protein specific)
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
30
-
assay at
Escherichia coli
Turnover Number [1/s] (protein specific)
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
additional information
-
additional information
steady-state kinetic analysis
Escherichia coli
0.032
-
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
pH 7.5, 30°C, mutant enzyme H239A
Escherichia coli
0.032
-
UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
pH 7.5, 30°C, mutant enzyme H239A
Escherichia coli
0.73
-
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
pH 7.5, 30°C, mutant enzyme H276A
Escherichia coli
0.73
-
UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
pH 7.5, 30°C, mutant enzyme H276A
Escherichia coli
1.4
-
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
pH 7.5, 30°C, mutant enzyme N233A
Escherichia coli
1.4
-
UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
pH 7.5, 30°C, mutant enzyme N233A
Escherichia coli
1.9
-
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
pH 7.5, 30°C, mutant enzyme D232A
Escherichia coli
1.9
-
UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
pH 7.5, 30°C, mutant enzyme D232A
Escherichia coli
4.1
-
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
pH 7.5, 30°C, mutant enzyme F41A
Escherichia coli
4.1
-
UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
pH 7.5, 30°C, mutant enzyme F41A
Escherichia coli
5.7
-
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
pH 7.5, 30°C, mutant enzyme K194A
Escherichia coli
5.7
-
UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
pH 7.5, 30°C, mutant enzyme K194A
Escherichia coli
8.9
-
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
pH 7.5, 30°C, mutant enzyme R293A
Escherichia coli
8.9
-
UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
pH 7.5, 30°C, mutant enzyme R293A
Escherichia coli
12
-
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
pH 7.5, 30°C, mutant enzyme Q236A
Escherichia coli
12
-
UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
pH 7.5, 30°C, mutant enzyme Q236A
Escherichia coli
17
-
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
pH 7.5, 30°C, mutant enzyme K46A
Escherichia coli
17
-
UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
pH 7.5, 30°C, mutant enzyme K46A
Escherichia coli
18
-
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
pH 7.5, 30°C, mutant enzyme Y47A
Escherichia coli
18
-
UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
pH 7.5, 30°C, mutant enzyme Y47A
Escherichia coli
23
-
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
pH 7.5, 30°C, wild-type enzyme
Escherichia coli
23
-
UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
pH 7.5, 30°C, wild-type enzyme
Escherichia coli
pH Optimum (protein specific)
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
7.5
-
assay at
Escherichia coli
KCat/KM [mM/s]
kcat/KM Value [1/mMs-1]
kcat/KM Value Maximum [1/mMs-1]
Substrate
Commentary
Organism
Structure
9.4
-
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
pH 7.5, 30°C, mutant enzyme H239A
Escherichia coli
10.7
-
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
pH 7.5, 30°C, mutant enzyme N233A
Escherichia coli
40.4
-
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
pH 7.5, 30°C, mutant enzyme D232A
Escherichia coli
120.3
-
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
pH 7.5, 30°C, mutant enzyme R293A
Escherichia coli
429
-
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
pH 7.5, 30°C, mutant enzyme H276A
Escherichia coli
923
-
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
pH 7.5, 30°C, mutant enzyme Q236A
Escherichia coli
1171
-
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
pH 7.5, 30°C, mutant enzyme F41A
Escherichia coli
1417
-
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
pH 7.5, 30°C, mutant enzyme K46A
Escherichia coli
1500
-
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
pH 7.5, 30°C, mutant enzyme K194A
Escherichia coli
3600
-
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
pH 7.5, 30°C, mutant enzyme Y47A
Escherichia coli
7188
-
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
pH 7.5, 30°C, wild-type enzyme
Escherichia coli
KCat/KM [mM/s] (protein specific)
KCat/KM Value [1/mMs-1]
KCat/KM Value Maximum [1/mMs-1]
Substrate
Commentary
Organism
Structure
9.4
-
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
pH 7.5, 30°C, mutant enzyme H239A
Escherichia coli
10.7
-
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
pH 7.5, 30°C, mutant enzyme N233A
Escherichia coli
40.4
-
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
pH 7.5, 30°C, mutant enzyme D232A
Escherichia coli
120.3
-
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
pH 7.5, 30°C, mutant enzyme R293A
Escherichia coli
429
-
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
pH 7.5, 30°C, mutant enzyme H276A
Escherichia coli
923
-
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
pH 7.5, 30°C, mutant enzyme Q236A
Escherichia coli
1171
-
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
pH 7.5, 30°C, mutant enzyme F41A
Escherichia coli
1417
-
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
pH 7.5, 30°C, mutant enzyme K46A
Escherichia coli
1500
-
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
pH 7.5, 30°C, mutant enzyme K194A
Escherichia coli
3600
-
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
pH 7.5, 30°C, mutant enzyme Y47A
Escherichia coli
7188
-
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
pH 7.5, 30°C, wild-type enzyme
Escherichia coli
Other publictions for EC 2.3.1.191
No.
1st author
Pub Med
title
organims
journal
volume
pages
year
Activating Compound
Application
Cloned(Commentary)
Crystallization (Commentary)
Engineering
General Stability
Inhibitors
KM Value [mM]
Localization
Metals/Ions
Molecular Weight [Da]
Natural Substrates/ Products (Substrates)
Organic Solvent Stability
Organism
Oxidation Stability
Posttranslational Modification
Purification (Commentary)
Reaction
Renatured (Commentary)
Source Tissue
Specific Activity [micromol/min/mg]
Storage Stability
Substrates and Products (Substrate)
Subunits
Temperature Optimum [°C]
Temperature Range [°C]
Temperature Stability [°C]
Turnover Number [1/s]
pH Optimum
pH Range
pH Stability
Cofactor
Ki Value [mM]
pI Value
IC50 Value
Activating Compound (protein specific)
Application (protein specific)
Cloned(Commentary) (protein specific)
Cofactor (protein specific)
Crystallization (Commentary) (protein specific)
Engineering (protein specific)
General Stability (protein specific)
IC50 Value (protein specific)
Inhibitors (protein specific)
Ki Value [mM] (protein specific)
KM Value [mM] (protein specific)
Localization (protein specific)
Metals/Ions (protein specific)
Molecular Weight [Da] (protein specific)
Natural Substrates/ Products (Substrates) (protein specific)
Organic Solvent Stability (protein specific)
Oxidation Stability (protein specific)
Posttranslational Modification (protein specific)
Purification (Commentary) (protein specific)
Renatured (Commentary) (protein specific)
Source Tissue (protein specific)
Specific Activity [micromol/min/mg] (protein specific)
Storage Stability (protein specific)
Substrates and Products (Substrate) (protein specific)
Subunits (protein specific)
Temperature Optimum [°C] (protein specific)
Temperature Range [°C] (protein specific)
Temperature Stability [°C] (protein specific)
Turnover Number [1/s] (protein specific)
pH Optimum (protein specific)
pH Range (protein specific)
pH Stability (protein specific)
pI Value (protein specific)
Expression
General Information
General Information (protein specific)
Expression (protein specific)
KCat/KM [mM/s]
KCat/KM [mM/s] (protein specific)
735568
Alshalchi
Expression of the lipopolysacc ...
Pseudomonas aeruginosa, Pseudomonas aeruginosa UCBPP-PA14
Arch. Microbiol.
197
135-145
2015
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1
1
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736235
Eshghi
Leptospira interrogans lpxD ho ...
Leptospira interrogans, Leptospira interrogans L495
Infect. Immun.
83
4314-4321
2015
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8
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1
1
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736449
Jenkins
Structural basis for the recog ...
Escherichia coli
J. Biol. Chem.
289
15527-15535
2014
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1
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1
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736880
Masoudi
Chasing acyl carrier protein t ...
Escherichia coli
Nature
505
422-426
2014
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1
1
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1
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1
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1
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1
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1
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737072
Emiola
A complete pathway model for l ...
Escherichia coli
PLoS ONE
10
e0121216
2014
-
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1
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1
1
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735384
Badger
Structure determination of Lpx ...
Acinetobacter baumannii, Acinetobacter baumannii SDF
Acta Crystallogr. Sect. F
69
6-9
2013
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1
1
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2
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736185
Yeom
Pleiotropic effects of acyltra ...
Pseudomonas aeruginosa
Genes Cells
18
682-693
2013
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2
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1
1
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718571
Jenkins
A continuous fluorescent enzym ...
Escherichia coli
Anal. Biochem.
425
21-27
2012
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1
1
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1
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1
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2
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1
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2
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1
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1
1
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2
2
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720944
Li
LPS remodeling is an evolved s ...
Francisella tularensis subsp. novicida, Francisella tularensis subsp. novicida U112
Proc. Natl. Acad. Sci. USA
109
8716-8721
2012
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1
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7
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4
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3
3
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718513
Badger
The structure of LpxD from Pse ...
Pseudomonas aeruginosa
Acta Crystallogr. Sect. F
67
749-752
2011
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1
1
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720913
Li
Pathway for lipid A biosynthes ...
Arabidopsis thaliana, Arabidopsis thaliana Col-0
Proc. Natl. Acad. Sci. USA
108
11387-11392
2011
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1
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1
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1
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696356
Bartling
Crystal structure and acyl cha ...
Escherichia coli
Biochemistry
48
8672-8683
2009
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1
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2
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696264
Bartling
Steady-state kinetics and mech ...
Escherichia coli
Biochemistry
47
5290-5302
2008
1
-
1
-
10
-
8
21
-
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3
1
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1
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1
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2
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3
1
1
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23
1
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7
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2
1
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1
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10
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2
8
7
21
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3
1
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1
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2
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3
1
1
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23
1
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11
11
698587
Bainbridge
Acyl chain specificity of the ...
Escherichia coli, Porphyromonas gingivalis
J. Bacteriol.
190
4549-4558
2008
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2
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694027
Albers
Expression of Legionella pneum ...
Legionella pneumophila
Microbiology
153
3817-3829
2007
-
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700935
Buetow
Structure and reactivity of Lp ...
Chlamydia trachomatis
Proc. Natl. Acad. Sci. USA
104
4321-4326
2007
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1
1
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1
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695627
Vaara
Outer membrane permeability ba ...
Escherichia coli
Antimicrob. Agents Chemother.
43
1459-1462
1999
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-
-
-
-
-
-
-
-
-
-
-
-
1
1
-
-
-
698088
Steeghs
Isolation and characterization ...
Neisseria meningitidis
Gene
190
263-270
1997
-
-
1
-
-
-
-
-
-
-
-
1
-
1
-
-
-
-
-
-
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-
1
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-
-
-
-
-
-
-
-
-
-
-
697921
Vuorio
The novel hexapeptide motif fo ...
Yersinia enterocolitica
FEBS Lett.
337
289-292
1994
-
-
-
-
-
-
-
-
-
-
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1
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-
-
-
-
-
-
-
-
-
-
-
698693
Kelly
Raetz, C.R.; Anderson, M.S.: T ...
Escherichia coli
J. Biol. Chem.
268
19866-19874
1993
-
-
1
-
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2
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1
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1
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