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Information on EC 2.3.1.191 - UDP-3-O-(3-hydroxymyristoyl)glucosamine N-acyltransferase and Organism(s) Escherichia coli and UniProt Accession P21645

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IUBMB Comments
The enzyme catalyses a step of lipid A biosynthesis. LpxD from Escherichia prefers (R,S)-3-hydroxytetradecanoyl-[acyl-carrier protein] over (R,S)-3-hydroxyhexadecanoyl-[acyl-carrier protein] . 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. When grown on 1% propionic acid, lipid A also contains the odd-chain fatty acids tridecanoic acid, pentadecanoic acid, hydroxytridecanoic acid, and hydroxypentadecanoic acid .
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This record set is specific for:
Escherichia coli
UNIPROT: P21645
Word Map
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota
The taxonomic range for the selected organisms is: Escherichia coli
Synonyms
acyl-ACP:UDP-3-O-(3-hydroxyacyl)-GlcN N-acyltransferase, acyltransferase LpxD, CtLpxD, EcLpxD, firA, La0512, LpxD, LpxD1, Lpxd2, PA3646, more
SYNONYM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
acyltransferase LpxD
246
-
EcLpxD
firA
246
gene name
UDP-3-O-(R-3-hydroxyacyl)-glucosamine acyltransferase
246
-
SYSTEMATIC NAME
IUBMB Comments
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]:UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine N-acetyltransferase
The enzyme catalyses a step of lipid A biosynthesis. LpxD from Escherichia prefers (R,S)-3-hydroxytetradecanoyl-[acyl-carrier protein] over (R,S)-3-hydroxyhexadecanoyl-[acyl-carrier protein] [1]. 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. When grown on 1% propionic acid, lipid A also contains the odd-chain fatty acids tridecanoic acid, pentadecanoic acid, hydroxytridecanoic acid, and hydroxypentadecanoic acid [5].
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
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]
show the reaction diagram
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]
show the reaction diagram
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]
show the reaction diagram
(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]
show the reaction diagram
(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]
show the reaction diagram
-
-
-
-
ir
additional information
?
-
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
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]
show the reaction diagram
-
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]
show the reaction diagram
-
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]
show the reaction diagram
-
-
-
-
ir
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
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
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
-
-
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; Na+ and K+ ions do not inhibit LpxD activity
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
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 hide
LITERATURE
IMAGE
0.0017 - 0.074
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
0.00084 - 0.073
UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
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
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.032 - 23
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
0.032 - 23
UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
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
-
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
9.4 - 7188
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.39 - 0.69
(3R)-3-hydroxylauroyl-methylphosphopantetheine
0.0043
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
-
pH 7.5, 30°C
0.048 - 0.139
acyl-carrier protein
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
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.42
Ca2+
Escherichia coli
-
pH 7.5, 30°C
1.41
Mg2+
Escherichia coli
-
pH 7.5, 30°C
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.14
-
specific activity in the absence of Ca2+
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.5
-
assay at
8
-
assay at
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25
-
assay at
30
-
assay at
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
SwissProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
metabolism
physiological function
-
LpxD is essential for survival in Gram-negative bacteria
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
35880
-
3 * 35880, electrospray-ionization/time-of-flight mass spectrometry
35881
-
3 * 35881, calculated from sequence
108000
-
gel filtration
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
homotrimer
-
3 * 35880, electrospray-ionization/time-of-flight mass spectrometry; 3 * 35881, calculated from sequence
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
-
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
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
K194A
-
mutation has little effect on activity
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
Q236A
-
mutation has little effect on activity
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
-
overexpression in Escherichia coli
-
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 hide
LITERATURE
drug development
-
LpxD is a viable target for antimicrobial development
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
Manually annotated by BRENDA team
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
Manually annotated by BRENDA team
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, Escherichia coli (P21645)
Manually annotated by BRENDA team
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
Escherichia coli, Porphyromonas gingivalis
Manually annotated by BRENDA team
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
Manually annotated by BRENDA team
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
Manually annotated by BRENDA team
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
Manually annotated by BRENDA team
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)
Manually annotated by BRENDA team
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
Manually annotated by BRENDA team
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