Information on EC 2.3.1.191 - UDP-3-O-(3-hydroxymyristoyl)glucosamine N-acyltransferase

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The expected taxonomic range for this enzyme is: Bacteria, Eukaryota

EC NUMBER
COMMENTARY hide
2.3.1.191
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RECOMMENDED NAME
GeneOntology No.
UDP-3-O-(3-hydroxymyristoyl)glucosamine N-acyltransferase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
(3R)-3-hydroxytetradecanoyl-[acyl-carrier protein] + UDP-3-O-[(3R)-3-hydroxytetradecanoyl]-alpha-D-glucosamine = UDP-2-N,3-O-bis[(3R)-3-hydroxytetradecanoyl]-alpha-D-glucosamine + a holo-[acyl-carrier protein]
show the reaction diagram
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
lipid IVA biosynthesis
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Lipopolysaccharide biosynthesis
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Metabolic pathways
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lipid A biosynthesis
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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].
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
AtlpxD1; gene AtlpxD1
UniProt
Manually annotated by BRENDA team
AtlpxD1; gene AtlpxD1
UniProt
Manually annotated by BRENDA team
genes lpxD1 and lpxD2
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Manually annotated by BRENDA team
genes lpxD1 and lpxD2
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Manually annotated by BRENDA team
serovar Manilae
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Manually annotated by BRENDA team
serovar Manilae
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Manually annotated by BRENDA team
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SwissProt
Manually annotated by BRENDA team
about half of the LpxD protein is made of hexad repeats (26 repeating hexapeptides). The lpxD gene from Yersinia enterocolitica is sequenced and the deduced amino acid sequence is compared with the lpxD sequences of Escherichia coli and Salmonella typhimurium. The hexapeptide repeat pattern is a very conservative property of these enzymes. Even though the overall homology between the proteins is 58%, the homology in the first residue of each hexapeptide is 100%
SwissProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
metabolism
physiological function
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
(3R)-3-hydroxyarachidonoyl-[acyl-carrier protein] + UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
UDP-2-N-((3R)-3-hydroxyarachidonoyl)-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine + holo-[acyl-carrier protein]
show the reaction diagram
the external layer of the Gram-negative bacterial outer membrane is primarily composed of a protective, selectively permeable lipopolysaccharide. The biosynthesis of lipopolysaccharide relies on UDP-3-O-acyl-glucosamine N-acyltransferase (LpxD), which transfers 3-hydroxy-arachidonic acid from acyl carrier protein to the 2' amine of UDP-3-O-myristoyl glucosamine. CtLpxD is expected to utilize R-3-hydroxyarachidonoyl-[acyl-carrier protein] and UDP-3-O-(myristoyl)-R-D-glucosamine, based on the predominant molecular species of Chlamydia trachomatis lipid A. This proposal is not validated by in vitro assays
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-
?
(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
(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
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-
?
(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
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-
?
additional information
?
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NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
(3R)-3-hydroxyarachidonoyl-[acyl-carrier protein] + UDP-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
UDP-2-N-((3R)-3-hydroxyarachidonoyl)-3-O-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine + holo-[acyl-carrier protein]
show the reaction diagram
P0CD76
the external layer of the Gram-negative bacterial outer membrane is primarily composed of a protective, selectively permeable lipopolysaccharide. The biosynthesis of lipopolysaccharide relies on UDP-3-O-acyl-glucosamine N-acyltransferase (LpxD), which transfers 3-hydroxy-arachidonic acid from acyl carrier protein to the 2' amine of UDP-3-O-myristoyl glucosamine. CtLpxD is expected to utilize R-3-hydroxyarachidonoyl-[acyl-carrier protein] and UDP-3-O-(myristoyl)-R-D-glucosamine, based on the predominant molecular species of Chlamydia trachomatis lipid A. This proposal is not validated by in vitro assays
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-
?
(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
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third step of lipid A biosynthesis
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-
?
(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
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
(3R)-3-hydroxylauroyl-methylphosphopantetheine
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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
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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]
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acyl-carrier protein
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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+
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inhibition is overcome by the addition of excess (3R)-3-hydroxymyristoyl-[acyl-carrier protein]
RJPXD33
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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
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noncompetitive inhibitor against both substrates
additional information
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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
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
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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
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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
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pH 8.0, 25°C, recombinant His6-tagged LpxD
additional information
additional information
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steady-state kinetic analysis
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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
Escherichia coli
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pH 8.0, 25°C, recombinant His6-tagged LpxD
additional information
additional information
Escherichia coli
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steady-state kinetic analysis
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kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
9.4 - 7188
(3R)-3-hydroxymyristoyl-[acyl-carrier protein]
2623
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]
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pH 7.5, 30°C
0.048 - 0.139
acyl-carrier protein
0.006
UDP-2-N-((3R)-3-hydroxymyristoyl)-alpha-D-glucosamine
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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
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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
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pH 7.5, 30°C
1.41
Mg2+
Escherichia coli
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pH 7.5, 30°C
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.14
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specific activity in the absence of Ca2+
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.5
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assay at
8
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assay at
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25
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assay at
30
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assay at
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
UNIPROT
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
35880
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3 * 35880, electrospray-ionization/time-of-flight mass spectrometry
35881
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3 * 35881, calculated from sequence
108000
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gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
homotrimer
additional information
the LpxD domain architecture is consisting of an N-terminal domain, amino acids 2-95, a left-handed beta-helix central domain containing many short parallel beta-sheet segments wrappingthrough consecutive triangular sections, amino acids 101-305, and a C-terminal helix, amino acids 312-340
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
apo forms of LpxD in space groups P21 and P4322, to 2.7 and 2.8 A resolution, respectively. The asymmetric unit contains two protein trimers
hanging drop vapor diffusion. Crystallographic analyses of recombinant Chlamydia trachomatis LpxD in complex with UDP-GlcNAc, which represents a fragment of substrate, and fatty acid extracted from the bacterial expression system, apo-structure at 2.7 A resolution, and two structures with bound UDP-N-acetylglucosamine (UDP-GlcNAc) at 2.2 A and 3.1 A resolution
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
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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
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purified recombinant His6-tagged LpxD, hanging drop vapour diffusion, 0.002 ml of 15 mg/ml LpxD in 10 mM Tris pH 8, 500 mM NaCl and 1 mM DTT, with 50 mM uridine diphosphate-N-acetylglucosamine, is mixed with 0.002 ml of 100 mM Tris pH 8.5, 1.5 M lithium sulfate, 20°C, 3-5 days, X-ray diffraction structure determination and analysis at 1.3 A resolution, molecular replacement
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
purification of untagged EcLpxD and an active N-terminally His6-tagged LpxD variant to near homogeneityrecombinant enzyme
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recombinant His6-tagged LpxD from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and gel filtration
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recombinant N-terminally His6-tagged LpxD from Escherichia coli strain BL21 (DE3) by nickel affinity chromatography and gel filtration
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
by complementation of a temperature-sensitive Escherichia coli lpxD mutant, a meningococcal chromosomal fragment is cloned that carries the lpxD homologue
gene AtlpxD, two AtLpxD isozymes, AtLxpD1 and AtLpxD2, are encoded on chromosome 4, DNA and amino acid sequence determination and analysis, expression as GFP-tagged protein in Arabidopsis thaliana
gene lpxD, expression as His6-tagged protein in Escherichia coli strain BL21(DE3)
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gene lpxD, expression of N-terminally His6-tagged LpxD in Escherichia coli strain BL21 (DE3)
genes lpxD1 and lpxD2, DNA and amino acid sequence determination and analysis, phyylogenetic analysis, the lpxD1gene lies within an essential four-gene operon, lpxD1/fabZ/lpxA/lpxB, while the second gene lpxD2 as a single gene is predicted to be regulated by its own promoter
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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
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
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overexpression in Escherichia coli
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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
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D232A
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mutation causes a 10fold reduction in kcat and a striking increase in the KM for both substrates
F41A
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mutation increases the KM for UDP-3-O-((3R)-3-hydroxymyristoyl)-a-D-glucosamine 30fold and kcat 5fold
K194A
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mutation has little effect on activity
K46A
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mutation causes 3fold increase in KM((3R)-3-hydroxymyristoyl-[acyl-carrier protein]) and has no effect on kcat
M290A
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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
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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
N233A
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mutation causes a 10fold reduction in kcat and a striking increase in the KM for both substrates
N240A
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causes less than a 2fold reduction in specific activity, when assayed at substrate concentrations at 2fold above KM with the purified proteins
N44A
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causes less than a 2fold reduction in specific activity, when assayed at substrate concentrations at 2fold above KM with the purified proteins
Q236A
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mutation has little effect on activity
Q32A
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causes less than a 2fold reduction in specific activity, when assayed at substrate concentrations at 2fold above KM with the purified proteins
R293A
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KM((3R)-3-hydroxymyristoyl-[acyl-carrier protein]) increases 23fold compared to wild-type with little effect on kcat
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
drug development
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LpxD is a viable target for antimicrobial development
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