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Literature summary extracted from

  • 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 (2015), ChemBioChem, 16, 1158-1162 .
    View publication on PubMed

Application

EC Number Application Comment Organism
6.3.1.20 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 Escherichia coli

Cloned(Commentary)

EC Number Cloned (Comment) Organism
6.3.1.20 recombinant expression of wild-type enzyme and enzyme mutant LplAW37V Escherichia coli

Protein Variants

EC Number Protein Variants Comment Organism
6.3.1.20 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 Escherichia coli
6.3.1.20 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 Escherichia coli

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
6.3.1.20 Mg2+ required Escherichia coli

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
6.3.1.20 ATP + (R)-lipoate + a [lipoyl-carrier protein]-L-lysine Escherichia coli
-
 a [lipoyl-carrier protein]-N6-(lipoyl)lysine + AMP + diphosphate
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
6.3.1.20 Escherichia coli
-
-
-

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
6.3.1.20 ATP + (R)-lipoate + a [lipoyl-carrier protein]-L-lysine
-
 Escherichia coli a [lipoyl-carrier protein]-N6-(lipoyl)lysine + AMP + diphosphate
-
 ?
6.3.1.20 ATP + 5-[[(1R,2R,4R)-bicyclo[2.2.1]hept-5-ene-2-carbonyl]amino]pentanoic acid + a [lipoyl-carrier protein]-L-lysine
-
 Escherichia coli ?
-
 ?
6.3.1.20 ATP + 5-[[(1R,2S,4R)-bicyclo[2.2.1]hept-5-ene-2-carbonyl]amino]pentanoic acid + a [lipoyl-carrier protein]-L-lysine
-
 Escherichia coli ?
-
 ?
6.3.1.20 ATP + 6-([[(1R,4R)-bicyclo[2.2.1]hept-5-en-2-yl]methyl]amino)hexanoic acid + a [lipoyl-carrier protein]-L-lysine
-
 Escherichia coli ?
-
 ?
6.3.1.20 ATP + 6-[[(1R,2R,4R)-bicyclo[2.2.1]hept-5-ene-2-carbonyl]amino]hexanoic acid + a [lipoyl-carrier protein]-L-lysine
-
 Escherichia coli ?
-
 ?
6.3.1.20 ATP + 6-[[(1S,2R,4S)-bicyclo[2.2.1]hept-5-ene-2-carbonyl]amino]hexanoic acid + a [lipoyl-carrier protein]-L-lysine
-
 Escherichia coli ?
-
 ?
6.3.1.20 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 Escherichia coli ?
-
 ?
6.3.1.20 ATP + 8-[[(1R,2S,4R)-bicyclo[2.2.1]hept-5-ene-2-carbonyl]amino]octanoic acid + a [lipoyl-carrier protein]-L-lysine
-
 Escherichia coli ?
-
 ?
6.3.1.20 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 Escherichia coli ?
-
 ?

Synonyms

EC Number Synonyms Comment Organism
6.3.1.20 lipoic acid ligase
-
 Escherichia coli

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
6.3.1.20 30
-
 assay at Escherichia coli

Cofactor

EC Number Cofactor Comment Organism Structure
6.3.1.20 ATP
-
 Escherichia coli

General Information

EC Number General Information Comment Organism
6.3.1.20 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 Escherichia coli