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Literature summary extracted from

  • Tron, C.M.; McNae, I.W.; Nutley, M.; Clarke, D.J.; Cooper, A.; Walkinshaw, M.D.; Baxter, R.L.; Campopiano, D.J.
    Structural and functional studies of the biotin protein ligase from Aquifex aeolicus reveal a critical role for a conserved residue in target specificity (2009), J. Mol. Biol., 387, 129-146.
    View publication on PubMed

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
6.3.4.15 purified recombinant wild-type and mutant R40G enzymes free and in complex with ligands ATP and/or biotin, hanging drop vapour diffusion method, 0.002 ml of protein solution with 5 or 6 mg/ml wild-type or mutant protein, respectively, is mixed with 0.001 ml of reservoir solution containing 0.1M Mes, 0.2 M ammonium sulfate, 15% w/v PEG 5000 monoethyl ether, pH 6.5, with or without 2 mM D-biotin and 5 mM ATP, 17°C, a few hours for the free enzyme, 4-14 days for the complexed enzyme, X-ray diffraction structure determination and analysis at 2.3-2.55 A resolution, molecular modeling Aquifex aeolicus

Protein Variants

EC Number Protein Variants Comment Organism
6.3.4.15 R40G site-directed mutagenesis, the structure of the mutant enzyme in both the ligand-free and biotin-bound forms reveals that the mutated loop has collapsed, thus hindering ATP binding. The mutant is catalytically active but shows poor substrate specificity. The affinity for biotin is reduced 3.5fold by the R40G mutation, but binding of ATP to mutant R40G is very weak in the absence or in the presence of biotin. Comparison to the equivalent mutant of Escherichia coli BirA, overview Aquifex aeolicus

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
6.3.4.15 additional information
-
 additional information isothermal titration calorimetry measurements of wild-type an dR40G mutant BPL with biotin and ATP, ATP and biotin binding are enthalpydriven with a favourable entropy change, overview Aquifex aeolicus

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
6.3.4.15 Mg2+ the final step of the mechanism, the formation of biotinyl-5'-AMP, is Mg2+-dependent Aquifex aeolicus

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
6.3.4.15 ATP + biotin + apo-[acetyl-CoA carboxylase] Aquifex aeolicus
-
 AMP + diphosphate + acetyl-CoA carboxylase
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
6.3.4.15 Aquifex aeolicus O66837
-
-

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
6.3.4.15 ATP + biotin + apo-[acetyl-CoA carboxylase]
-
 Aquifex aeolicus AMP + diphosphate + acetyl-CoA carboxylase
-
 ?
6.3.4.15 ATP + biotin + apo-[acetyl-CoA carboxylase] biotin binding structure with importance of arginine 40 in the glycine-rich motif in the specificity of the biotinylation reaction. The Arg40 residue from the conserved GXGRXG motif interacts with the carboxyl group of biotin and stabilizes the alpha- and beta-phosphates of the nucleotide, overview. Presence of biotin is not required for ATP binding to the wild-type enzyme in the absence of Mg2+, the binding of biotin and ATP occurs via a random but cooperative process Aquifex aeolicus AMP + diphosphate + acetyl-CoA carboxylase
-
 ?

Subunits

EC Number Subunits Comment Organism
6.3.4.15 More ligand-free structure, and biotin-ATP intermediate complex of the enzyme, comparison of wild-type and mutant structures, overview Aquifex aeolicus

Synonyms

EC Number Synonyms Comment Organism
6.3.4.15 biotin protein ligase
-
 Aquifex aeolicus
6.3.4.15 BPL
-
 Aquifex aeolicus

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
6.3.4.15 65
-
 binding assays at Aquifex aeolicus

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
6.3.4.15 7.5
-
 assay at Aquifex aeolicus

Cofactor

EC Number Cofactor Comment Organism Structure
6.3.4.15 ATP binding structure, the solvent-exposed beta- and gamma-phosphates of ATP are located in the intersubunit cavity formed by the N- and C-terminal domains, the Arg40 residue from the conserved GXGRXG motif interacts with the carboxyl group of biotin and stabilizes the alpha- and beta-phosphates of the nucleotide, overview Aquifex aeolicus