Information on EC 2.7.7.63 - lipoate-protein ligase

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

EC NUMBER
COMMENTARY hide
2.7.7.63
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RECOMMENDED NAME
GeneOntology No.
lipoate-protein ligase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
ATP + lipoate = diphosphate + lipoyl-AMP
show the reaction diagram
-
-
-
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lipoyl-AMP + apoprotein = protein N6-(lipoyl)lysine + AMP
show the reaction diagram
SYSTEMATIC NAME
IUBMB Comments
ATP:lipoate adenylyltransferase
Requires Mg2+. Both 6S- and 6R-lipoates can act as substrates but there is a preference for the naturally occurring R-form. Selenolipoate, i.e. 5-(1,2-diselenolan-3-yl)pentanoic acid, and 6-sulfanyloctanoate can also act as substrates, but more slowly [2]. This enzyme is responsible for lipoylation in the presence of exogenous lipoic acid [7]. Lipoylation is essential for the function of several key enzymes involved in oxidative metabolism, including pyruvate dehydrogenase (E2 domain), 2-oxoglutarate dehydrogenase (E2 domain), the branched-chain 2-oxoacid dehydrogenases and the glycine cleavage system (H protein) [6]. This enzyme attaches lipoic acid to the lipoyl domains of these proteins, converting apoproteins into holoproteins. It is likely that an alternative pathway, involving EC 2.3.1.181, lipoyl(octanoyl) transferase and EC 2.8.1.8, lipoyl synthase, is the normal route for lipoylation [7].
CAS REGISTRY NUMBER
COMMENTARY hide
139639-26-2
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
-
-
Manually annotated by BRENDA team
Japonica type
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
metabolism
physiological function
-
LplA1 plays a pivotal role during the development of the erythrocytic stages (asexual parasite growth) of Plasmodium falciparum
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
ATP + (R)-lipoic acid + Escherichia coli apoH-protein
diphosphate + AMP + Escherichia coli (R)-lipoyl-apoH-protein
show the reaction diagram
-
-
-
-
?
ATP + 6-thio-octanoic acid
diphosphate + 6-thio-octanoyl-AMP
show the reaction diagram
-
at 326% of the rate with DL-lipoic acid
-
-
?
ATP + 8-methyl-lipoic acid
diphosphate + 8-methyl-lipoyl-AMP
show the reaction diagram
-
at 73% of the rate with DL-lipoic acid
-
-
-
ATP + D-lipoic acid
diphosphate + D-lipoyl-AMP
show the reaction diagram
ATP + dihydro-DL-lipoic acid
diphosphate + dihydro-DL-lipoyl-AMP
show the reaction diagram
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at 80% of the rate with DL-lipoic acid
-
-
-
ATP + DL-lipoic acid
diphosphate + DL-lipoyl-AMP
show the reaction diagram
-
-
-
-
?
ATP + DL-lipoic acid + protein
AMP + DL-lipoyl-protein
show the reaction diagram
-
-
-
?
ATP + DL-lipoic acid + protein
diphosphate + AMP + DL-lipoyl-protein
show the reaction diagram
-
-
-
-
?
ATP + L-lipoic acid
diphosphate + L-lipoyl-AMP
show the reaction diagram
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at 36% of the rate with DL-lipoic acid
-
-
-
ATP + lipoate + apoprotein
AMP + diphosphate + protein N6-(lipoyl)lysine
show the reaction diagram
ATP + lipoate + biotin
AMP + diphosphate + ?
show the reaction diagram
-
-
-
-
?
ATP + lipoate + H-protein
AMP + diphosphate + ?
show the reaction diagram
-
-
-
-
?
ATP + lipoate + LplA acceptor peptide 1
?
show the reaction diagram
-
-
-
-
?
ATP + lipoate + LplA acceptor peptide 2
?
show the reaction diagram
-
-
-
-
?
ATP + octanoate + pyruvate dehydrogenase subunit E2
diphosphate + AMP + octanoyl-pyruvate dehydrogenase subunit E2
show the reaction diagram
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lipoate-protein ligase attaches octanoate to the dehydrogenase subunit and sulfur insertion protein LipA, then converts octanoate to lipoate. LipA acts on both octanoate and octanoyl-proteins
-
-
?
ATP + octanoic acid
diphosphate + octanoyl-AMP
show the reaction diagram
ATP + selenolipoic acid
diphosphate + selenolipoyl-AMP
show the reaction diagram
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at 12% of the rate with DL-lipoic acid
-
-
?
DL-lipoyladenylate + protein
adenylate + DL-lipoyl-protein
show the reaction diagram
-
-
-
-
?
lipoic acid + ATP + apoprotein
diphosphate + AMP + N6-(lipoyl)-lysine
show the reaction diagram
octanoic acid + ATP
diphosphate + octanoyl-AMP
show the reaction diagram
-
-
-
-
?
octanoyl adenylate + protein
adenylate + octanoyl-protein
show the reaction diagram
-
-
-
-
?
octanoyl-ACP + lipoyl protein
octanoylated lipoyl protein + ACP
show the reaction diagram
additional information
?
-
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
ATP + lipoate + apoprotein
AMP + diphosphate + protein N6-(lipoyl)lysine
show the reaction diagram
ATP + lipoate + H-protein
AMP + diphosphate + ?
show the reaction diagram
-
-
-
-
?
lipoic acid + ATP + apoprotein
diphosphate + AMP + N6-(lipoyl)-lysine
show the reaction diagram
additional information
?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Ca2+
-
may substitute for Mg2+, LPL-B
Cu2+
-
may substitute for Mg2+, LPL-A
Fe3+
-
may substitute for Mg2+, LPL-B
MgCl2
assay in the presence of; assay in the presence of
Zn2+
-
may substitute for Mg2+, both LPL-A and LPL-B
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
6-seleno-octanoate
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0.28 mM, complete inhibition, reactivation by reducing agents
8-bromooctanoate
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inhibition of isoform LipL1 activity, in vitro growth arrest of Plasmodium falciparum
8-thiooctanoate
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0.28 mM, 50% inhibition, reactivation by reducing agents
Cu2+
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0.13 mM, 50% inhibition
additional information
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
ATP
assay in the presence of; assay in the presence of
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1.6
6-thio-octanoate
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pH 7.0, 30C
0.0011 - 0.0155
apoH-protein
0.0019 - 0.295
ATP
0.0016 - 0.0017
DL-lipoic acid
0.2
LplA acceptor peptide 1
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-
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13.32
LplA acceptor peptide 2
-
-
-
0.004 - 0.2143
Octanoic acid
0.00055 - 0.0089
R-lipoic acid
0.013
selenolipoic acid
-
pH 7.0, 30C
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.028 - 0.258
apoH-protein
0.0084 - 0.16
ATP
0.22
LplA acceptor peptide 2
Escherichia coli
-
-
-
0.0042 - 0.04
Octanoic acid
0.006 - 0.219
R-lipoic acid
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0000165
LplA acceptor peptide 2
Escherichia coli
-
-
40096
0.186 - 2.55
Octanoic acid
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
27.6
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pH 7.0, 30C
additional information
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Vmax of mutant S221P is 0.0025 nmol/min; Vmax of mutant V19L is 0.016 nmol/min; Vmax of wild type enzyme is 0.024 nmol/min
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.5 - 8
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more than 70% of maximum activity within this range
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0
-
assay at
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.8
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isoelectric focusing
6
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calculated
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
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developing, moderate expression, Northern blot
Manually annotated by BRENDA team
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northern blot
Manually annotated by BRENDA team
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less abundant, Northern blot
Manually annotated by BRENDA team
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developing seed, increasing level at 3 to 5 days after pollination followed by decreasing for 15 days, Northern blot
Manually annotated by BRENDA team
additional information
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
UNIPROT
Thermoplasma acidophilum (strain ATCC 25905 / DSM 1728 / JCM 9062 / NBRC 15155 / AMRC-C165)
Thermoplasma acidophilum (strain ATCC 25905 / DSM 1728 / JCM 9062 / NBRC 15155 / AMRC-C165)
Thermoplasma acidophilum (strain ATCC 25905 / DSM 1728 / JCM 9062 / NBRC 15155 / AMRC-C165)
Thermoplasma acidophilum (strain ATCC 25905 / DSM 1728 / JCM 9062 / NBRC 15155 / AMRC-C165)
Thermoplasma acidophilum (strain ATCC 25905 / DSM 1728 / JCM 9062 / NBRC 15155 / AMRC-C165)
Thermoplasma acidophilum (strain ATCC 25905 / DSM 1728 / JCM 9062 / NBRC 15155 / AMRC-C165)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
11000
SDS-PAGE, molecular weight of HisCTD fusion protein
32000
SDS-PAGE, molecular weight of HisLplA fusion protein
41100
-
LplA-LplB complex, gel filtration
41700
-
gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
heterodimer
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1 * 30800 + 1 * 11200, LplA-LplB complex, gel filtration
monomer
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1 * 38000, SDS-PAGE
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
alone and in complex with lipoic acid
-
structural models of LplA1 and LplA2 based on Escherichia coli (PDB: 1X2H) and Streptococcus pneumoniae (PDB: 1VQZ) LplA proteins, conserved lipoyl-AMP binding pocket, LplA1: regions of clustered negatively charged residues, overall electrostatic potential of -13, LplA2: overall electrostatic potential of -5
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structural comparison of lipoate-protein ligase with cysteine/lysine dyad acyltransferase LipB shows conserved structural and sequence active-site features, but 4-phosphopantheine-bound octanoic acid recognition is a specific property of cysteine/lysine dyad acyltransferase
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as apo form, in complex with ATP, and as lipoyl-AMP-complex. Lipoyl-AMP is bound deeply in the bifurcated pocket and adopts a U-shaped conformation
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OXIDATION STABILITY
ORGANISM
UNIPROT
LITERATURE
slow oxidation to an inactive form containing at least one intramolecular disulfide bond, reactivation by reducing agent
-
663919
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20C, stable for several months
-
4C, slow oxidation to an inactive form containing at least one intramolecular disulfide bond, reactivation by reducing agent
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
; Ni-NTA column chromatography, ammonium sulfate precipitation gel filtration
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both LPL-A and LPL-B, partial
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Ni2+-NTA-agarose resin column chromatography and POROS HQ 20 column chromatography
-
using His-tag affinity purification; using His-tag affinity purification
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli
expressed in Escherichia coli as a His-tagged fusion protein; expressed in Escherichia coli as a His-tagged fusion protein
from cDNA library in pTrcHis2A for expression and functional complementation in Escherichia coli strain TM137 (lplA/lipB null mutant of strain JK1)
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in Escherichia coli
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LplA1: in pAM401 for complementation in Listeria monocytogenes mutant strains DP-L4263 and 10403S, in ptac85 for IPTG-inducible expression in Escherichia coli wild-type strain JK1 or strain TM131 lacking endogenous LplA, LplA2: from bacterial genome (10403S) in pKSV7 for allelic exchange (homologous recombination), in ptac85 for IPTG-inducible expression in Escherichia coli wild-type strain JK1 or strain TM131 lacking endogenous LplA
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D122A
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D122A mutation results in a marked reduction in the overall, lipoate adenylation, and lipoate transfer reaction activities (0.14, 4, and 4% of those of wild type, respectively)
E116A/E312K/L328F
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mutations allow a LipB knockout strain to grow on a glucose minimal medium
F15S/T101A/S114I
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mutations allow a LipB knockout strain to grow on a glucose minimal medium
F35L/V113I
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mutations allow a LipB knockout strain to grow on a glucose minimal medium
G76S
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substitution in LplA ligase gene, is identical to slr1 selenolipoate restistance mutation
H149A
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mutations does not cause a significant reduction in three reaction activities (overall, lipoate adenylation, and lipoate transfer reaction activities), Km value for ATP and lipoic acid increases to 15 and 5.8fold, respectively, relative to those of wild-type
K133A
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K133A mutation almost completely abolishes the overall reaction activity (0.01% of that of wild type) and causes marked reduction in lipoate adenylation and lipoate transfer activities (0.2 and 2.5% of that of wild type, respectively)
N121A
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N121A affects only the lipoate adenylation activity and consequently the overall reaction activity (1.4 and 0.19% of those of wild-type, respectively) but retains a significant lipoate transfer activity (24.2%)
R140A
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12fold increase in Km-value for apoH-protein
R58L/H79N
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mutations allow a LipB knockout strain to grow on a glucose minimal medium
S221P
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mutant with reduced affinity to octanoic acid; strain FH27, referred as lplA11, mutation allows a LipB knockout strain to grow on a glucose minimal medium
S8T/N63K/F78Y/A110T
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mutations allow a LipB knockout strain to grow on a glucose minimal medium
V19L
-
mutant with reduced affinity to octanoic acid; strain FH26, referred as lplA10, mutation allows a LipB knockout strain to grow on a glucose minimal medium
additional information
Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
reactivation of inactive form containing at least one intramolecular disulfide bond, or of enzyme inactivated by substrate-analogues, by reducing agent
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
drug development
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LplA1, but not LplA2, required for intracellular replication and/or virulence of Listeria monocytogenes
molecular biology
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dependency of pathogen Listeria monocytogenes on LplA1 dictated by availability of host lipoyl substrates as alternative lipoate source
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