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Information on EC 6.3.1.20 - lipoate-protein ligase and Organism(s) Escherichia coli and UniProt Accession P32099
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6.3.1.20 lipoate-protein ligaseIUBMB Comments
Requires Mg2+. This enzyme participates in lipoate salvage, and is responsible for lipoylation in the presence of exogenous lipoic acid . The enzyme attaches lipoic acid to the lipoyl domains of certain 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) . Lipoylation is essential for the function of these enzymes. The enzyme can also use octanoate instead of lipoate.
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Word Map
- 6.3.1.20
-
lipoylation
- ligases
-
octanoyl-acyl
-
lipoate-dependent
- apicoplast
-
octanoylation
- 2-oxoacids
- lipoyl-amp
-
octanoic
-
bioorthogonal
- lipoyltransferase
- h-protein
-
octanoyltransferase
-
13-amino
- biotechnology
- synthesis
- molecular biology
- 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
hide(Overall reactions are displayed. Show all >>)
Synonyms
lipoic acid ligase, lipoate protein ligase, lpla1, lipoate-protein ligase, lipoate ligase, lipoate-protein ligase a, lipoic acid protein ligase, lipoyl ligase, oslpla, lipl1, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
ATP + (R)-lipoate + a [lipoyl-carrier protein]-L-lysine = a [lipoyl-carrier protein]-N6-(lipoyl)lysine + AMP + diphosphate
LplA structures are determined: LplA/lipoyl-AMP binary complex and LplA/octyl-5'-AMP/apoH-protein ternary complex. These structures represent a post-lipoate adenylation stage and a pre-lipoate transfer stage, respectively. Three large scale conformational changes in the Escherichia coli LplA structure upon completion of the lipoate adenylation reaction are found, which enable LplA to accommodate apoprotein for the second reaction
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Diels-Alder cycloaddition
-
inverse-electron-demand Diels-Alder cycloaddition
SYSTEMATIC NAME
IUBMB Comments
[lipoyl-carrier protein]-L-lysine:lipoate ligase (AMP-forming)
Requires Mg2+. This enzyme participates in lipoate salvage, and is responsible for lipoylation in the presence of exogenous lipoic acid [7]. The enzyme attaches lipoic acid to the lipoyl domains of certain 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]. Lipoylation is essential for the function of these enzymes. The enzyme can also use octanoate instead of lipoate.
CAS REGISTRY NUMBER
COMMENTARY
139639-26-2
-
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
ATP + DL-lipoic acid + protein
AMP + diphosphate + DL-lipoyl-protein
-
-
-
?
ATP + (R)-lipoate + a [lipoyl-carrier protein]-L-lysine
a [lipoyl-carrier protein]-N6-(lipoyl)lysine + AMP + diphosphate
-
-
-
-
?
ATP + (R)-lipoic acid + Escherichia coli apoH-protein
diphosphate + AMP + Escherichia coli (R)-lipoyl-apoH-protein
-
-
-
-
?
ATP + 5-[[(1R,2R,4R)-bicyclo[2.2.1]hept-5-ene-2-carbonyl]amino]pentanoic acid + a [lipoyl-carrier protein]-L-lysine
?
-
-
-
-
?
ATP + 5-[[(1R,2S,4R)-bicyclo[2.2.1]hept-5-ene-2-carbonyl]amino]pentanoic acid + a [lipoyl-carrier protein]-L-lysine
?
-
-
-
-
?
ATP + 6-([[(1R,4R)-bicyclo[2.2.1]hept-5-en-2-yl]methyl]amino)hexanoic acid + a [lipoyl-carrier protein]-L-lysine
?
-
-
-
-
?
ATP + 6-thio-octanoic acid
diphosphate + 6-thio-octanoyl-AMP
-
at 326% of the rate with DL-lipoic acid
-
-
?
ATP + 6-[[(1R,2R,4R)-bicyclo[2.2.1]hept-5-ene-2-carbonyl]amino]hexanoic acid + a [lipoyl-carrier protein]-L-lysine
?
-
-
-
-
?
ATP + 6-[[(1S,2R,4S)-bicyclo[2.2.1]hept-5-ene-2-carbonyl]amino]hexanoic acid + a [lipoyl-carrier protein]-L-lysine
?
ATP + 8-methyl-lipoic acid
diphosphate + 8-methyl-lipoyl-AMP
-
at 73% of the rate with DL-lipoic acid
-
-
?
ATP + 8-[[(1R,2S,4R)-bicyclo[2.2.1]hept-5-ene-2-carbonyl]amino]octanoic acid + a [lipoyl-carrier protein]-L-lysine
?
-
-
-
-
?
ATP + D-lipoic acid
diphosphate + D-lipoyl-AMP
-
at 83% of the rate with DL-lipoic acid
-
-
?
ATP + dihydro-DL-lipoic acid
diphosphate + dihydro-DL-lipoyl-AMP
-
at 80% of the rate with DL-lipoic acid
-
-
?
ATP + DL-lipoic acid + protein
diphosphate + AMP + DL-lipoyl-protein
-
-
-
-
?
ATP + L-lipoic acid
diphosphate + L-lipoyl-AMP
-
at 36% of the rate with DL-lipoic acid
-
-
?
ATP + lipoate + apoprotein
AMP + diphosphate + protein N6-(lipoyl)lysine
-
protein lipoylation, salvage pathway
-
-
?
ATP + octanoate + pyruvate dehydrogenase subunit E2
diphosphate + AMP + octanoyl-pyruvate dehydrogenase subunit E2
-
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 + selenolipoic acid
diphosphate + selenolipoyl-AMP
-
at 12% of the rate with DL-lipoic acid
-
-
?
lipoic acid + ATP + apoprotein
diphosphate + AMP + N6-(lipoyl)-lysine
-
lipoic acid is (R)-5-(1,2-dithiolan-3-yl)pentanoic acid, also called 6,8-dithiooctanoic acid or thioctic acid
-
-
?
ATP + 6-[[(1S,2R,4S)-bicyclo[2.2.1]hept-5-ene-2-carbonyl]amino]hexanoic acid + a [lipoyl-carrier protein]-L-lysine
?
-
-
-
-
?
ATP + 6-[[(1S,2R,4S)-bicyclo[2.2.1]hept-5-ene-2-carbonyl]amino]hexanoic acid + a [lipoyl-carrier protein]-L-lysine
?
-
6-[[(1S,2R,4S)-bicyclo[2.2.1]hept-5-ene-2-carbonyl]amino]hexanoic acid-LAP-eDHFR labeled with tetrazine-fluorescein
-
-
?
ATP + octanoic acid
diphosphate + octanoyl-AMP
-
at 13% of the rate with DL-lipoic acid
-
-
?
additional information
?
-
enzyme LplA is able to ligate with high specificity an alkyl azide to a target protein previously fused with a 13 aa recognition sequence for LplA, ligase-acceptor peptide (LAP)
-
-
?
additional information
?
-
-
LipB protein utilizes lipoyl groups generated via endogenous, LipA mediated biosynthesis and causes the accumulation of aberrantly modified octanoyl-proteins in lipoate-deficient cells
-
-
?
additional information
?
-
-
LplA potein attaches octanoate to the dehydrogenase and LipA protein then converts the octanoate to lipoate
-
-
?
additional information
?
-
-
LplA's natural protein substrates have a conserved beta-hairpin structure
-
-
?
additional information
?
-
-
two-step protein labeling by using lipoic acid ligase with norbornene substrates and subsequent inverse-electron demand Diels-Alder reaction, identification of a potential candidate for use as a norbornene-bearing substrate for LplAW37V-mediated peptide labeling. The norbornene moiety is highly stable in the presence of nucleophiles, and can be efficiently coupled to the 13-aa LAP-tag and further modified with tetrazine fluorophore conjugates in inverse-electron-demand Diels-Alder cycloaddition. The rigid compounds 4-[(3aS,4R,7S,7aS)-1,3-dioxo-1,3,3a,4,7,7a-hexahydro-2H-4,7-methanoisoindol-2-yl]butanoic acid, 5-[(3aS,4R,7S,7aS)-1,3-dioxo-1,3,3a,4,7,7a-hexahydro-2H-4,7-methanoisoindol-2-yl]pentanoic acid, and 6-[(3aS,4R,7S,7aS)-1,3-dioxo-1,3,3a,4,7,7a-hexahydro-2H-4,7-methanoisoindol-2-yl]hexanoic acid are not accepted as substrates at all, likely due to less flexibility and steric hindrance. Derivatives with shorter (4-[[(1R,2S,4R)-bicyclo[2.2.1]hept-5-ene-2-carbonyl]amino]butanoic acid, 5-[[(1R,2S,4R)-bicyclo[2.2.1]hept-5-ene-2-carbonyl]amino]pentanoic acid, and 4-[[(1R,2S,4R)-bicyclo[2.2.1]hept-5-ene-2-carbonyl]amino]butanoic acid) and longer (9-[[(1R,2S,4R)-bicyclo[2.2.1]hept-5-ene-2-carbonyl]amino]nonanoic acid) aliphatic chains are not accepted by LplAW37V, thus indicating the necessity of a particular chain length to fit the dimensions of the active site of LplAW37V
-
-
?
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
ATP + (R)-lipoate + a [lipoyl-carrier protein]-L-lysine
a [lipoyl-carrier protein]-N6-(lipoyl)lysine + AMP + diphosphate
-
-
-
-
?
ATP + lipoate + apoprotein
AMP + diphosphate + protein N6-(lipoyl)lysine
-
protein lipoylation, salvage pathway
-
-
?
lipoic acid + ATP + apoprotein
diphosphate + AMP + N6-(lipoyl)-lysine
-
lipoic acid is (R)-5-(1,2-dithiolan-3-yl)pentanoic acid, also called 6,8-dithiooctanoic acid or thioctic acid
-
-
?
additional information
?
-
-
LipB protein utilizes lipoyl groups generated via endogenous, LipA mediated biosynthesis and causes the accumulation of aberrantly modified octanoyl-proteins in lipoate-deficient cells
-
-
?
additional information
?
-
-
LplA potein attaches octanoate to the dehydrogenase and LipA protein then converts the octanoate to lipoate
-
-
?
additional information
?
-
-
LplA's natural protein substrates have a conserved beta-hairpin structure
-
-
?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Co2+
Fe2+
Mg2+
Mn2+
Ni2+
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
6-seleno-octanoate
-
0.28 mM, complete inhibition, reactivation by reducing agents
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pI VALUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
metabolism
-
The LipB octanoyltransferase catalyzes the first step of lipoic acid synthesis in Escherichia coli, transfer of the octanoyl moiety from octanoyl-acyl carrier protein to the lipoyl domains of the E2 subunits of the 2-oxoacid dehydrogenases of aerobic metabolism.
physiological function
-
enzyme LplA naturally catalyzes the ligation of lipoic acid to the free epsilon-amino moiety of a lysine residue in the specific LAP sequence
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
D122A
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)
H149A
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
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
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%)
S72A
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 28 and 2.3fold, respectively, relative to those of wild-type
E116A/E312K/L328F
-
mutations allow a LipB knockout strain to grow on a glucose minimal medium
F15S/T101A/S114I
-
mutations allow a LipB knockout strain to grow on a glucose minimal medium
F35L/V113I
-
mutations allow a LipB knockout strain to grow on a glucose minimal medium
G76S
-
substitution in LplA ligase gene, is identical to slr1 selenolipoate restistance mutation
R58L/H79N
-
mutations allow a LipB knockout strain to grow on a glucose minimal medium
S221P
S8T/N63K/F78Y/A110T
-
mutations allow a LipB knockout strain to grow on a glucose minimal medium
V19L
W37V
-
LplAW37V-mediated surface labeling of HEK293T cells with 6-[[(1S,2R,4S)-bicyclo[2.2.1]hept-5-ene-2-carbonyl]amino]hexanoic acid and tetrazine-TAMRA, overview. Necessity of a particular chain length to fit the dimensions of the active site of LplAW37V
additional information
S221P
-
strain FH27, referred as lplA11, mutation allows a LipB knockout strain to grow on a glucose minimal medium
V19L
-
strain FH26, referred as lplA10, mutation allows a LipB knockout strain to grow on a glucose minimal medium
additional information
enzyme null mutant, normal transport of lipoic acid, but severe defect in incorporation and utilization of exogenously supplied lipoic acid and lipoic acid analogues. Strain is highly resistant to selenolipoate
additional information
-
enzyme null mutant, normal transport of lipoic acid, but severe defect in incorporation and utilization of exogenously supplied lipoic acid and lipoic acid analogues. Strain is highly resistant to selenolipoate
additional information
-
lipB mutant strain, grows well when supplemented with octanoate in place of lipoate
additional information
-
lplA null mutants display no growth defect unless combined with lipA or lipB lipoate synthesis mutations
additional information
-
establishment of an enzyme-mediated two-step labeling protocol suitable for live-cell labeling: construction of a fusion protein LAP-eDHFR-His6, in which eDHFR bears an N-terminal LAP extension and a C-terminal His-tag for purification. In the first step, substrate 6-[[(1S,2R,4S)-bicyclo[2.2.1]hept-5-ene-2-carbonyl]amino]hexanoic acid is coupled to purified recombinant LAP-eDHFR. After removal of excess norbornene substrate with centrifugal filter devices, the modified protein is successfully labeled with tetrazine-fluorescein
additional information
-
the enzyme is used for a two-step labeling procedure for the attachment of various fluorescent probes to a small peptide sequence (13 amino acids) via enzyme-mediated peptide labeling in combination with palladium-catalyzed Sonogashira cross-coupling, method, overview. 4-Iodophenyl derivatives from a small library can be covalently attached to a lysine residue within a specific 13-amino-acid peptide sequence by Escherichia coli lipoic acid ligase A (LplA). The derivatization with 4-iodophenyl subsequently serves as a reactive handle for bioorthogonal transition metal-catalyzed Sonogashira cross-coupling with alkyne-functionalized fluorophores on both the peptide as well as on the protein level
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
4°C, slow oxidation to an inactive form containing at least one intramolecular disulfide bond, reactivation by reducing agent
-
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant N-terminally His- and GST-tagged enzyme LplAW37V from Escherichia coli strain BL21 by nickel and glutathione affinity chromatography to homogeneity
Ni-NTA column chromatography, ammonium sulfate precipitation gel filtration
-
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
gene lplA, recombinant expression of N-terminally His- and GST-tagged enzyme LplAW37V in Escherichia coli strain BL21
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
-
APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
biotechnology
the enzyme lipoic acid ligase (LplA) can be used for PRIME (probe incorporation mediated by enzymes) labeling, a rapid and specific fluorescent labeling method of the protein of interest by LplA
synthesis
synthesis
-
enzyme-mediated two-step labeling protocol suitable for live-cell labeling using lipoic acid ligase with norbornene substrates and subsequent inverse-electron demand Diels-Alder reaction
synthesis
-
the enzyme can be used for a two-step labeling strategy that combines high selectivity of enzyme-mediated labeling with the chemoselectivity of palladium-catalyzed Sonogashira cross-coupling, overview
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Zhao, X.; Miller, J.R.; Jiang, Y.; Marletta, M.A.; Cronan, J.E.
Assembly of the covalent linkage between lipoic acid and its cognate enzymes
Chem. Biol.
10
1293-1302
2003
Escherichia coli
Jordan, S.W.; Cronan, J.E.
A new metabolic link. The acyl carrier protein of lipid synthesis donates lipoic acid to the pyruvate dehydrogenase complex in Escherichia coli
J. Biol. Chem.
272
17903-17906
1997
Escherichia coli, Neurospora crassa
Green, D.E.; Morris, T.W.; Green, J.; Cronan, J.E., Jr.; Guest, J.R.
Purification and properties of the lipoate protein ligase of Escherichia coli
Biochem. J.
309
853-862
1995
Escherichia coli
Brookfield, D.E.; Green, J.; Ali, S.T.; Machado, R.S.; Guest, J.R.
Evidence for two protein-lipoylation activities in Escherichia coli
FEBS Lett.
295
13-16
1991
Escherichia coli
Morris, T.W.; Reed, K.E.; Cronan, J.E., Jr.
Lipoic acid metabolism in Escherichia coli: the lplA and lipB genes define redundant pathways for ligation of lipoyl groups to apoprotein
J. Bacteriol.
177
1-10
1995
Escherichia coli
Morris, T.W.; Reed, K.E.; Cronan, J.E., Jr.
Identification of the gene encoding lipoate-protein ligase A of Escherichia coli. Molecular cloning and characterization of the lplA gene and gene product
J. Biol. Chem.
269
16091-16100
1994
Escherichia coli (P32099), Escherichia coli
Fujiwara, K.; Toma, S.; Okamura-Ikeda, K.; Motokawa, Y.; Nakagawa, A.; Taniguchi, H.
Crystal structure of lipoate-protein ligase A from Escherichia coli. Determination of the lipoic acid-binding site
J. Biol. Chem.
280
33645-33651
2005
Escherichia coli
Fujiwara, K.; Hosaka, H.; Nakagawa, A.; Motokawa, Y.
Lipoate-protein ligase A: Structure and function
Oxid. Stress Dis.
24
217-233
2008
Escherichia coli, Thermoplasma acidophilum
-
Puthenveetil, S.; Liu, D.S.; White, K.A.; Thompson, S.; Ting, A.Y.
Yeast display evolution of a kinetically efficient 13-amino acid substrate for lipoic acid ligase
J. Am. Chem. Soc.
131
16430-16438
2009
Escherichia coli
Hermes, F.A.; Cronan, J.E.
Scavenging of cytosolic octanoic acid by mutant LplA lipoate ligases allows growth of Escherichia coli strains lacking the LipB octanoyltransferase of lipoic acid synthesis
J. Bacteriol.
191
6796-6803
2009
Escherichia coli
Fujiwara, K.; Maita, N.; Hosaka, H.; Okamura-Ikeda, K.; Nakagawa, A.; Taniguchi, H.
Global conformational change associated with the two-step reaction catalyzed by Escherichia coli lipoate-protein ligase A
J. Biol. Chem.
285
9971-9980
2010
Escherichia coli (P32099), Escherichia coli
Hauke, S.; Best, M.; Schmidt, T.T.; Baalmann, M.; Krause, A.; Wombacher, R.
Two-step protein labeling utilizing lipoic acid ligase and Sonogashira cross-coupling
Bioconjug. Chem.
25
1632-1637
2014
Escherichia coli
Best, M.; Degen, A.; Baalmann, M.; Schmidt, T.T.; Wombacher, R.
Two-step protein labeling by using lipoic acid ligase with norbornene substrates and subsequent inverse-electron demand Diels-Alder reaction
ChemBioChem
16
1158-1162
2015
Escherichia coli
Florian, P.; Petrareanu, G.; Ruta, S.; Roseanu, A.
Optimization of recombinant lipoic acid ligase expression from bacterial cells
Rom. Biotechnol. Lett.
21
11539-11542
2016
Escherichia coli (P32099)
-
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