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Literature summary for 2.3.1.191 extracted from

  • Bartling, C.M.; Raetz, C.R.
    Steady-state kinetics and mechanism of LpxD, the N-acyltransferase of lipid A biosynthesis (2008), Biochemistry, 47, 5290-5302.
    View publication on PubMedView publication on EuropePMC
Search for more literature for 2.3.1.191

Activating Compound

Activating Compound Comment 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)

Cloned (Comment) Organism
overexpression in Escherichia coli Escherichia coli

Protein Variants

Protein Variants Comment 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 Comment 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 Comment 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 Escherichia coli
0.013
-
 (3R)-3-hydroxymyristoyl-[acyl-carrier protein] 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] Comment 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 Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
(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 UniProt Comment Textmining
Escherichia coli
-
-
-

Purification (Commentary)

Purification (Comment) 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] Comment Organism
7.14
-
 specific activity in the absence of Ca2+ Escherichia coli
8.9
-
-
 Escherichia coli

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
(3R)-3-hydroxymyristoyl-[acyl-carrier protein] + UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine third step of lipid A biosynthesis 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 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] Escherichia coli ?
-
 ?

Subunits

Subunits Comment Organism
homotrimer 3 * 35880, electrospray-ionization/time-of-flight mass spectrometry Escherichia coli
homotrimer 3 * 35881, calculated from sequence Escherichia coli

Synonyms

Synonyms Comment Organism
EcLpxD
-
 Escherichia coli

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
30
-
 assay at Escherichia coli

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment 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 Comment Organism
7.5
-
 assay at Escherichia coli

Ki Value [mM]

Ki Value [mM] Ki Value maximum [mM] Inhibitor Comment 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 Comment Organism Inhibitor Structure
0.42
-
 pH 7.5, 30°C Escherichia coli Ca2+
1.41
-
 pH 7.5, 30°C Escherichia coli Mg2+

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment 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