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Information on EC 2.3.1.191 - UDP-3-O-(3-hydroxyacyl)glucosamine N-acyltransferase and Organism(s) Escherichia coli and UniProt Accession P21645
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2.3.1.191 UDP-3-O-(3-hydroxyacyl)glucosamine N-acyltransferaseIUBMB Comments
The enzyme catalyses a step of lipid A biosynthesis. LpxD from Escherichia coli prefers (3R)-3-hydroxytetradecanoyl-[acyl-carrier protein] , but it does not have an absolute specificity for 14-carbon hydroxy fatty acids, as it can transfer other fatty acids, including odd-chain fatty acids, if they are available to the organism .
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Word Map
- 2.3.1.191
- acyltransferases
- lps
- francisella
- trachomatis
-
chlamydia
- lipopolysaccharide
- udp-n-acetylglucosamine
-
homotrimer
- drug development
The taxonomic range for the selected organisms is: Escherichia coli
The enzyme appears in selected viruses and cellular organisms
The enzyme appears in selected viruses and cellular organisms
Reaction Schemes
+
=
+
+
a UDP-3-O-[(3R)-3-hydroxyacyl]-alpha-D-glucosamine
=
a UDP-2-N,3-O-bis[(3R)-3-hydroxyacyl]-alpha-D-glucosamine
+
Synonyms
eclpxd, lpxd1, lpxd2, ctlpxd, pa3646, udp-3-o-acyl-glucosamine n-acyltransferase, udp-3-o-(r-3-hydroxyacyl)-glucosamine acyltransferase, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
LpxD
PATHWAY SOURCE
PATHWAYS
BRENDA
-
-, -, -, -, -, -, -
SYSTEMATIC NAME
IUBMB Comments
(3R)-3-hydroxyacyl-[acyl-carrier protein]:UDP-3-O-[(3R)-3-hydroxyacyl]-alpha-D-glucosamine N-acyltransferase
The enzyme catalyses a step of lipid A biosynthesis. LpxD from Escherichia coli prefers (3R)-3-hydroxytetradecanoyl-[acyl-carrier protein] [3], but it does not have an absolute specificity for 14-carbon hydroxy fatty acids, as it can transfer other fatty acids, including odd-chain fatty acids, if they are available to the organism [5].
SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
(R,S)-3-hydroxymyristoyl-[acyl-carrier protein] + UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
UDP-2,3-bis((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine + holo-[acyl-carrier protein]
wild-type LpxD prefers (R,S)-3-hydroxymyristoyl-[acyl-carrier protein] over (R,S)-3-hydroxypalmitoyl-[acyl-carrier protein] by a factor of 3, whereas the M290A mutant has the opposite selectivity
-
-
?
(R,S)-3-hydroxypalmitoyl-[acyl-carrier protein] + UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
UDP-2,3-bis((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine + holo-[acyl-carrier protein]
wild-type LpxD prefers (R,S)-3-hydroxymyristoyl-[acyl-carrier protein] over (R,S)-3-hydroxypalmitoyl-[acyl-carrier protein] by a factor of 3, whereas the M290A mutant has the opposite selectivity
-
-
?
(3R)-3-hydroxymyristoyl-[acyl-carrier protein] + UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
UDP-2,3-bis((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine + holo-[acyl-carrier protein]
(3R)-3-hydroxymyristoyl-[acyl-carrier protein] + UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
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
UDP-2,3-bis[O-(3R)-3-hydroxymyristoyl]-alpha-D-glucosamine + a holo-[acyl-carrier protein]
-
-
-
-
ir
(3R)-3-hydroxymyristoyl-[acyl-carrier protein] + UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
UDP-2,3-bis((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine + holo-[acyl-carrier protein]
-
since only (R)-3-hydroxymyristate is found at the 2,3,2, and 3 positions of Escherichia coli lipid A, it is reassuring that both Escherichia coli acyltransferases display extraordinary specificity for (R)-3-hydroxymyristoyl-[acyl-carrier protein]
-
-
?
(3R)-3-hydroxymyristoyl-[acyl-carrier protein] + UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
UDP-2,3-bis((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine + holo-[acyl-carrier protein]
-
myristoyl-[acyl-carrier protein] does not serve as an acyl donor for the overproduced UDP-3-O-((R)-3-hydroxymyristoyl)-GlcN N-acyltransferase
-
-
?
(3R)-3-hydroxymyristoyl-[acyl-carrier protein] + UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
UDP-2,3-bis(3-hydroxymyristoyl)-alpha-D-glucosamine + holo-[acyl-carrier protein]
-
third step of lipid A biosynthesis
-
-
?
(3R)-3-hydroxymyristoyl-[acyl-carrier protein] + UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
UDP-2,3-bis(3-hydroxymyristoyl)-alpha-D-glucosamine + holo-[acyl-carrier protein]
-
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
-
-
?
additional information
?
-
ordered-sequential reaction mechanism. Acyl-ACP binds first to free LpxD forming a binary complex. ACP associates with the ACP-recognition domain and the acyl-4'-phosphopantetheine group packs into the hydrophobic N-channel. UDP-acyl-GlcN binds next, which initiates acyl transfer. In the ternary product complex, the 4'-phosphopantetheine arm of hydrolysed-acyl-ACP completely encloses the reaction chamber, blocking UDP-diacyl-GlcN from leaving. By moving the 4'-phosphopantetheine group towards Met 290, the catalytic chamber opens up. This motion drives the eventual release of UDP-diacyl-GlcN and triggers conformational changes downstream of helix-II leading to holo-ACP dissociation
-
-
?
additional information
?
-
-
both LpxA and LpxD, from Escherichia coli are also able to incorporate odd-chain fatty acids into lipid A when grown in the presence of 1% propionic acid. When grown on 1% propionic acid lipid A also contains the odd-chain fatty acids tridecanoic acid (C13), pentadecanoic acid (C15), hydroxy tridecanoic acid (C13OH), and hydroxy pentadecanoic acid (C15OH). Escherichia coli lipid A acyltransferases do not have an absolute specificity for 14-carbon hydroxy fatty acids but can transfer fatty acids differing by one carbon unit if the fatty acid substrates are available
-
-
?
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]
-
-
?
additional information
?
-
-
fluorescent enzyme assay, method optimization, overview
-
-
?
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
(3R)-3-hydroxymyristoyl-[acyl-carrier protein] + UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
UDP-2,3-bis((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine + holo-[acyl-carrier protein]
-
since only (R)-3-hydroxymyristate is found at the 2,3,2, and 3 positions of Escherichia coli lipid A, it is reassuring that both Escherichia coli acyltransferases display extraordinary specificity for (R)-3-hydroxymyristoyl-[acyl-carrier protein]
-
-
?
(3R)-3-hydroxymyristoyl-[acyl-carrier protein] + UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
UDP-2,3-bis(3-hydroxymyristoyl)-alpha-D-glucosamine + holo-[acyl-carrier protein]
-
third step of lipid A biosynthesis
-
-
?
(3R)-3-hydroxymyristoyl-[acyl-carrier protein] + UDP-3-O-[(3R)-3-hydroxymyristoyl]-alpha-D-glucosamine
UDP-2,3-bis[O-(3R)-3-hydroxymyristoyl]-alpha-D-glucosamine + a holo-[acyl-carrier protein]
-
-
-
-
ir
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
(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]
(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
4-(2-chlorophenyl)-3-hydroxy-7,7-dimethyl-2-phenyl-7,8-dihydro-2H-pyrazolo[3,4-b]quinolin-5(6H)-one
-
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
Ca2+
-
inhibition is overcome by the addition of excess (3R)-3-hydroxymyristoyl-[acyl-carrier protein]
RJPXD33
-
i.e. TNLYMLPKWDIP, peptide inhibitor, binds to both UDP-N-acetylglucosamine acyltransferase (LpxA, EC 2.3.1.129) and UDP-3-O-(acyl)-glucosamine acyltransferase. Comparison with binding to LpxA suggests overlap with the acyl-phosphopantetheine arm of acyl-ACP, thereby inhibiting acyl-ACP from binding to LpxD. RJPXD33 binds to LpxD without the prior binding of other ligands
UDP-2-N-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
-
noncompetitive inhibitor against both substrates
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
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.004 - 0.012
UDP-3-O-[(3R)-3-hydroxymyristoyl]-alpha-D-glucosamine
-
pH 8.0, 25°C, recombinant His6-tagged LpxD
additional information
additional information
-
steady-state kinetic analysis
-
0.0017
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
-
pH 7.5, 30°C, mutant enzyme H276A
0.0032
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
-
pH 7.5, 30°C, wild-type enzyme
0.0034
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
-
pH 7.5, 30°C, mutant enzyme H239A
0.0035
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
-
pH 7.5, 30°C, mutant enzyme F41A
0.0038
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
-
pH 7.5, 30°C, mutant enzyme K194A
0.005
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
-
pH 7.5, 30°C, mutant enzyme Y47A
0.012
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
-
pH 7.5, 30°C, mutant enzyme K46A
0.013
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
-
pH 7.5, 30°C, mutant enzyme N233A
0.013
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
-
pH 7.5, 30°C, mutant enzyme Q236A
0.047
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
-
pH 7.5, 30°C, mutant enzyme D232A
0.074
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
-
pH 7.5, 30°C, mutant enzyme R293A
0.00084
UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
-
pH 7.5, 30°C, mutant enzyme H239A
0.0036
UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
-
pH 7.5, 30°C, mutant enzyme R293A
0.0042
UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
-
pH 7.5, 30°C, mutant enzyme Q236A
0.0056
UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
-
pH 7.5, 30°C, mutant enzyme K194A
0.0063
UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
-
pH 7.5, 30°C, mutant enzyme H276A
0.0071
UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
-
pH 7.5, 30°C, mutant enzyme K46A
0.0078
UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
-
pH 7.5, 30°C, mutant enzyme Y47A
0.028
UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
-
pH 7.5, 30°C, mutant enzyme N233A
0.067
UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
-
pH 7.5, 30°C, mutant enzyme D232A
0.073
UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
-
pH 7.5, 30°C, mutant enzyme F41A
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
3
UDP-3-O-[(3R)-3-hydroxymyristoyl]-alpha-D-glucosamine
-
pH 8.0, 25°C, recombinant His6-tagged LpxD
additional information
additional information
-
steady-state kinetic analysis
-
0.032
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
-
pH 7.5, 30°C, mutant enzyme H239A
0.73
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
-
pH 7.5, 30°C, mutant enzyme H276A
1.4
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
-
pH 7.5, 30°C, mutant enzyme N233A
1.9
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
-
pH 7.5, 30°C, mutant enzyme D232A
4.1
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
-
pH 7.5, 30°C, mutant enzyme F41A
5.7
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
-
pH 7.5, 30°C, mutant enzyme K194A
8.9
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
-
pH 7.5, 30°C, mutant enzyme R293A
12
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
-
pH 7.5, 30°C, mutant enzyme Q236A
17
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
-
pH 7.5, 30°C, mutant enzyme K46A
18
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
-
pH 7.5, 30°C, mutant enzyme Y47A
23
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
-
pH 7.5, 30°C, wild-type enzyme
0.032
UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
-
pH 7.5, 30°C, mutant enzyme H239A
0.73
UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
-
pH 7.5, 30°C, mutant enzyme H276A
1.4
UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
-
pH 7.5, 30°C, mutant enzyme N233A
1.9
UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
-
pH 7.5, 30°C, mutant enzyme D232A
4.1
UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
-
pH 7.5, 30°C, mutant enzyme F41A
5.7
UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
-
pH 7.5, 30°C, mutant enzyme K194A
8.9
UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
-
pH 7.5, 30°C, mutant enzyme R293A
12
UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
-
pH 7.5, 30°C, mutant enzyme Q236A
17
UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
-
pH 7.5, 30°C, mutant enzyme K46A
18
UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
-
pH 7.5, 30°C, mutant enzyme Y47A
23
UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
-
pH 7.5, 30°C, wild-type enzyme
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
9.4
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
-
pH 7.5, 30°C, mutant enzyme H239A
10.7
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
-
pH 7.5, 30°C, mutant enzyme N233A
40.4
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
-
pH 7.5, 30°C, mutant enzyme D232A
120.3
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
-
pH 7.5, 30°C, mutant enzyme R293A
429
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
-
pH 7.5, 30°C, mutant enzyme H276A
923
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
-
pH 7.5, 30°C, mutant enzyme Q236A
1171
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
-
pH 7.5, 30°C, mutant enzyme F41A
1417
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
-
pH 7.5, 30°C, mutant enzyme K46A
1500
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
-
pH 7.5, 30°C, mutant enzyme K194A
3600
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
-
pH 7.5, 30°C, mutant enzyme Y47A
7188
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
-
pH 7.5, 30°C, wild-type enzyme
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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]
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
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
0.69
(3R)-3-hydroxylauroyl-methylphosphopantetheine
-
pH 7.5, 30°C, uncompetitive inhibition with respect to (3R)-3-hydroxymyristoyl-[acyl-carrier protein]
0.048
acyl-carrier protein
-
pH 7.5, 30°C, competitive inhibition with respect to (R)-3-hydroxymyristoyl-[acyl-carrier protein]
0.139
acyl-carrier protein
-
pH 7.5, 30°C, noncompetitive inhibitionr with respect to UDP-3-O-(R-3-hydroxymyristoyl)-alpha-D-glucosamine
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
malfunction
metabolism
physiological function
-
LpxD is essential for survival in Gram-negative bacteria
malfunction
-
enzyme activity of the temperature-sensitive firA mutant RL-25 is reduced to less than 10% of wild-type enzyme
metabolism
-
LpxD catalyzes the third step of lipid A biosynthesis, an acyl-acyl carrier protein (ACP)-dependent transfer of a fatty acyl moiety to a UDP-glucosamine core ring, overview
metabolism
-
quantitative model of the nine enzyme-catalyzed steps of Escherichia coli lipid A biosynthesis. Biosynthesis regulation occurs through regulated degradation of the LpxC and WaaA enzymes. LpxC, EC 3.5.1.108, is the rate-limiting enzyme if pathway regulation is ignored, but LpxK, EC 2.7.1.130, is the rate-limiting enzyme if pathway regulation is present, as it is in real cells
metabolism
the enzyme is involved in lipid A biosynthesis in Gram-negative bacteria
PDB
SCOP
CATH
UNIPROT
ORGANISM
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
homotrimer
homotrimer
-
3 * 35880, electrospray-ionization/time-of-flight mass spectrometry
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
hanging drop/vapor diffusion method. The crystal structure of N-terminally His6-tagged EcLpxD is determined by molecular replacement at 2.6 A resolution, using Chlamydia trachomatis (PDB code: 2IUA) as the model. Comparison of LpxD from Escherichia coli and Chlamydia trachomatis. Attempts to crystallize EcLpxD with UDP-GlcNAc, UDP-3-O-(R-3-hydroxymyristoyl)-R-D-GlcNAc or its product UDP-2,3-diacylglucosamine are unsuccessful
in complex with three forms of acyl carrier protein. Interactions at the interface optimally position acyl carrier protein for acyl delivery and directly involve the pantetheinyl group
enzyme in complex with 4-(2-chlorophenyl)-3-hydroxy-7,7-dimethyl-2-phenyl-7,8-dihydro-2H-pyrazolo[3,4-b]quinolin-5(6H)-one. Enzyme in complex with 3-hydroxy-7,7-dimethyl-2-phenyl-4-(thiophen-2-yl)-7,8-dihydro-2H-pyrazolo[3,4-b]-quinolin-5(6H)-one
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
M290A
wild-type EcLpxD prefers (R,S)-3-hydroxymyristoyl-ACP over (R,S)-3-hydroxypalmitoyl-ACP by a factor of 3, whereas the M290A mutant has the opposite selectivity. Both wild-type and M290A EcLpxD rescue the conditional lethality of Escherichia coli RL25, a temperature-sensitive strain harboring point mutations in lpxD. Complementation with wild-type EcLpxD restores normal lipid A containing only N-linked hydroxymyristate to RL25 at 42°C, as judged by mass spectrometry, whereas the M290A mutant generates multiple lipid A species containing one or two longer hydroxy fatty acids in place of the usual (3R)-3-hydroxymyristate at positions 2 and 20
M292A
wild-type EcLpxD prefers (R,S)-3-hydroxymyristoyl-ACP over (R,S)-3-hydroxypalmitoyl-ACP by a factor of 3, mutant enzyme M292A prefers (R,S)-3-hydroxymyristoyl-ACP over (R,S)-3-hydroxypalmitoyl-ACP by a factor of 2.5
D232A
-
mutation causes a 10fold reduction in kcat and a striking increase in the KM for both substrates
F41A
-
mutation increases the KM for UDP-3-O-((3R)-3-hydroxymyristoyl)-a-D-glucosamine 30fold and kcat 5fold
K46A
-
mutation causes 3fold increase in KM((3R)-3-hydroxymyristoyl-[acyl-carrier protein]) and has no effect on kcat
N233A
-
mutation causes a 10fold reduction in kcat and a striking increase in the KM for both substrates
N240A
-
causes less than a 2fold reduction in specific activity, when assayed at substrate concentrations at 2fold above KM with the purified proteins
N44A
-
causes less than a 2fold reduction in specific activity, when assayed at substrate concentrations at 2fold above KM with the purified proteins
Q32A
-
causes less than a 2fold reduction in specific activity, when assayed at substrate concentrations at 2fold above KM with the purified proteins
R293A
-
KM((3R)-3-hydroxymyristoyl-[acyl-carrier protein]) increases 23fold compared to wild-type with little effect on kcat
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
purification of untagged EcLpxD and an active N-terminally His6-tagged LpxD variant to near homogeneityrecombinant enzyme
-
recombinant His6-tagged LpxD from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and gel filtration
-
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
LpxD protein modified with an N-terminal His6 tag followed by a one glycine residue linker and the P2A substitution, is constructed and transformed into Escherichia coli Rosetta (DE3)/pLysS
gene lpxD, expression as His6-tagged protein in Escherichia coli strain BL21(DE3)
-
lpxA (lpxAPg) and lpxDPg are cloned and expressed in Escherichia coli strains in which the homologous gene is mutated. Lipid A from strains expressing either of the Porphyromonas gingivalis transferases contains 16-carbon hydroxy fatty acids in addition to the normal Escherichia coli 14-carbon hydroxy fatty acids, demonstrating that these acyltransferases display a relaxed acyl chain length specificity
-
when the wild-type firA gene is cloned into a T7-based expression vector, N-acyltransferase specific activity increases almost 360fold relative to wild-type extracts
-
APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Vaara, M.; Nurminen, M.
Outer membrane permeability barrier in Escherichia coli mutants that are defective in the late acyltransferases of lipid A biosynthesis
Antimicrob. Agents Chemother.
43
1459-1462
1999
Escherichia coli
Bartling, C.M.; Raetz, C.R.
Steady-state kinetics and mechanism of LpxD, the N-acyltransferase of lipid A biosynthesis
Biochemistry
47
5290-5302
2008
Escherichia coli
Bartling, C.M.; Raetz, C.R.
Crystal structure and acyl chain selectivity of Escherichia coli LpxD, the N-acyltransferase of lipid A biosynthesis
Biochemistry
48
8672-8683
2009
Escherichia coli (P21645), Escherichia coli
Bainbridge, B.W.; Karimi-Naser, L.; Reife, R.; Blethen, F.; Ernst, R.K.; Darveau, R.P.
Acyl chain specificity of the acyltransferases LpxA and LpxD and substrate availability contribute to lipid A fatty acid heterogeneity in Porphyromonas gingivalis
J. Bacteriol.
190
4549-4558
2008
Porphyromonas gingivalis, Escherichia coli
Kelly, T.M.; Stachula, S.A.
Raetz, C.R.; Anderson, M.S.: The firA gene of Escherichia coli encodes UDP-3-O-(R-3-hydroxymyristoyl)-glucosamine N-acyltransferase. The third step of endotoxin biosynthesis
J. Biol. Chem.
268
19866-19874
1993
Escherichia coli
Jenkins, R.J.; Dotson, G.D.
A continuous fluorescent enzyme assay for early steps of lipid A biosynthesis
Anal. Biochem.
425
21-27
2012
Escherichia coli
Jenkins, R.J.; Heslip, K.A.; Meagher, J.L.; Stuckey, J.A.; Dotson, G.D.
Structural basis for the recognition of peptide RJPXD33 by acyltransferases in lipid A biosynthesis
J. Biol. Chem.
289
15527-15535
2014
Escherichia coli
Masoudi, A.; Raetz, C.R.; Zhou, P.; Pemble, C.W.
Chasing acyl carrier protein through a catalytic cycle of lipid A production
Nature
505
422-426
2014
Escherichia coli (P21645)
Emiola, A.; George, J.; Andrews, S.S.
A complete pathway model for lipid A biosynthesis in Escherichia coli
PLoS ONE
10
e0121216
2014
Escherichia coli
Ma, X.; Prathapam, R.; Wartchow, C.; Chie-Leon, B.; Ho, C.M.; De Vicente, J.; Han, W.; Li, M.; Lu, Y.; Ramurthy, S.; Shia, S.; Steffek, M.; Uehara, T.
Structural and biological basis of small molecule inhibition of Escherichia coli LpxD acyltransferase essential for lipopolysaccharide biosynthesis
ACS Infect. Dis.
6
1480-1489
2019
Escherichia coli (Q0P6M7)
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