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Literature summary extracted from

  • Silvaggi, N.R.; Boldt, G.E.; Hixon, M.S.; Kennedy, J.P.; Tzipori, S.; Janda, K.D.; Allen, K.N.
    Structures of Clostridium botulinum neurotoxin serotype A light chain complexed with small-molecule inhibitors highlight active-site flexibility (2007), Chem. Biol., 14, 533-542.
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
3.4.24.69 expression of His-tagged truncated enzyme, residues 1-424, in Escherichia coli strain BL21(DE3) Clostridium botulinum

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
3.4.24.69 purified recombinant His-tagged truncated enzyme, residues 1-424, in complex with inhibitors 4-chlorocinnamic hydroxamate, 2,4-dichlorocinnamic hydroxamate, and L-arginine hydroxamate, hanging drop vapor diffusion method, drops are formed of equal parts BoNT/A-LC(1-424) at 10 mg/ml in 20 mM HEPES, pH 7.5, 50 mM NaCl and well solution containing 10%-15% PEG-2000 monomethyl ether, 0.3M(NH4)2HPO4, 50 mM Tris, pH 8.5, co-crystallization with inhibitor by addition of 0.5 mM ligand to the protein solution, 1.3 days, larger crystals by microseeding, X-ray diffraction structure determination and analysis at 1.9-2.5 A resolution, modeling Clostridium botulinum

Inhibitors

EC Number Inhibitors Comment Organism Structure
3.4.24.69 2,4-dichlorocinnamic hydroxamate binding site and complex structure, overview Clostridium botulinum
3.4.24.69 4-chlorocinnamic hydroxamate binding site and complex structure, overview Clostridium botulinum
3.4.24.69 L-Arginine hydroxamate binding site and complex structure, overview Clostridium botulinum

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
3.4.24.69 Zn2+ binding site structure, a long a helix contains the HEXXH Zn(II)-binding motif, modeling, overview Clostridium botulinum

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
3.4.24.69 SNAP-25 + H2O Clostridium botulinum i.e. synaptosomal associated protein of 25 kDa ?
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
3.4.24.69 Clostridium botulinum
-
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
3.4.24.69 recombinant His-tagged truncated enzyme, residues 1-424, from Escherichia coli strain BL21(DE3) by metal ion affinity chromatography Clostridium botulinum

Reaction

EC Number Reaction Comment Organism Reaction ID
3.4.24.69 Limited hydrolysis of proteins of the neuroexocytosis apparatus, synaptobrevin (also known as neuronal vesicle-associated membrane protein, VAMP), synaptosome-associated protein of 25 kDa (SNAP25) or syntaxin. No detected action on small molecule substrates active site structure and structure-function relationship Clostridium botulinum
a

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
3.4.24.69 SNAP-25 + H2O i.e. synaptosomal associated protein of 25 kDa Clostridium botulinum ?
-
 ?
3.4.24.69 SNAP-25 + H2O i.e. synaptosomal associated protein of 25 kDa, all botulinus neurotoxin serotypes cleave the substrate at a unique peptide bond, BoNT/A cleaves SNAP-25 between residues Gln197 and Arg198. Phe194, Ile161, and Asp370 form the S1' subsite responsible for binding the P1' arginine side chain of SNAP-25, overview Clostridium botulinum ?
-
 ?

Synonyms

EC Number Synonyms Comment Organism
3.4.24.69 Clostridium botulinum neurotoxin serotype A light chain
-
 Clostridium botulinum

IC50 Value

EC Number IC50 Value IC50 Value Maximum Comment Organism Inhibitor Structure
3.4.24.69 0.0003
-
-
 Clostridium botulinum 2,4-dichlorocinnamic hydroxamate
3.4.24.69 0.015
-
-
 Clostridium botulinum 4-chlorocinnamic hydroxamate
3.4.24.69 0.06
-
-
 Clostridium botulinum L-Arginine hydroxamate