Bontoxilysin

 

Botulinum neurotoxin acts by inhibiting neurotransmitter release. It binds to peripheral neuronal synapses, is internalised and moves by retrograde transport up the axon into the spinal cord where it can move between postsynaptic and presynaptic neurons. It disrupts neurotransmitter release by acting as a zinc endopeptidase that cleaves the 76-Gln-|-Phe-77 bond of synaptobrevin-2(Sb2), one of three SNARE proteins involved in neuronal synaptic vesicle fusion.

There are in total 7 serotypes of botulinum neurotoxin. All seven serotypes possess an identical Zn2+-binding motif, HEXXH, but are unique either in substrate selection or in their cleavage site. Their catalytic domains share high degree of sequence homology and holotoxin structures of serotype A and serotype B have been reported to be structurally similar. Botulinum belongs to the same class of neurotoxin with tetanus neurotoxin and they share significant sequence homology. They possess similar structural and functional domains and have a similar mechanism of toxicity. However, Botulinum neurotoxins acts at the neuromuscular junction causing flaccid paralysis by inhibiting the release of acetylcholine into the synapse, while tetanus toxin acts at the central nervous system causing spastic paralysis. In addition botulinum neurotoxin is the protein also known as Botox which is widely used in the cosmetics industry for the reduction of facial wrinkles through relaxation of the muscles.

 

Reference Protein and Structure

Sequence
P10844 UniProt (3.4.24.69) IPR000395 (Sequence Homologues) (PDB Homologues)
Biological species
Clostridium botulinum (Bacteria) Uniprot
PDB
1epw - CRYSTAL STRUCTURE OF CLOSTRIDIUM NEUROTOXIN TYPE B (1.9 Å) PDBe PDBsum 1epw
Catalytic CATH Domains
3.90.1240.10 CATHdb (see all for 1epw)
Cofactors
Zinc(2+) (1)
Click To Show Structure

Enzyme Reaction (EC:3.4.24.69)

water
CHEBI:15377ChEBI
+
dipeptide zwitterion
CHEBI:90799ChEBI
→
L-alpha-amino acid zwitterion
CHEBI:59869ChEBI
Alternative enzyme names: BoNT, Botulinum neurotoxin,

Enzyme Mechanism

Introduction

Glu231 is abase which deprotonates water and in conjugation with polarization by Zn2+ activates water so that it can act as a nucleophile and attack the carbonyl of the peptide bond. Arg369 and Zn2+ stabilise the transition state by interacting with the negatively charged carbonyl oxygen atom of the tetrahedral intermediate. The collapse of the oxyanion results in the cleavage of the peptide bond and the kinetically favoured products of this reaction are the carboxyanion and the amine cation, thus in a final step the N-terminal amine deprotonates the C-terminal carboxyacid.

Catalytic Residues Roles

UniProt PDB* (1epw)
Glu231 Glu230A Acts as a general base on water, to create nucleophile from attack on the amide carbonyl. proton acceptor, proton donor
His230, His234, Glu268 His229A, His233A, Glu267A Form the Zinc binding site in the enzyme metal ligand
Arg370 Arg369A It stabilises the transition state by stabilising the negative charge developed on the substrate carbonyl oxygen atom. electrostatic stabiliser
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

proton transfer, bimolecular nucleophilic addition, coordination, coordination to a metal ion, intermediate formation, overall reactant used, rate-determining step, cofactor used, unimolecular elimination by the conjugate base, heterolysis, intermediate collapse, decoordination from a metal ion, native state of cofactor regenerated, native state of enzyme regenerated, overall product formed

References

  1. Swaminathan S et al. (2000), Nat Struct Biol, 7, 693-699. Structural analysis of the catalytic and binding sites of Clostridium botulinum neurotoxin B. PMID:10932256.
  2. Ahmed SA et al. (2008), Protein J, 27, 151-162. Identification of residues surrounding the active site of type A botulinum neurotoxin important for substrate recognition and catalytic activity. DOI:10.1007/s10930-007-9118-8. PMID:18213512.
  3. Swaminathan S et al. (2004), Mov Disord, 19 Suppl 8, S17-S22. Structure and enzymatic activity of botulinum neurotoxins. DOI:10.1002/mds.20005. PMID:15027050.
  4. Breidenbach MA et al. (2004), Nature, 432, 925-929. Substrate recognition strategy for botulinum neurotoxin serotype A. DOI:10.1038/nature03123. PMID:15592454.
  5. Rigoni M et al. (2001), Biochem Biophys Res Commun, 288, 1231-1237. Site-Directed Mutagenesis Identifies Active-Site Residues of the Light Chain of Botulinum Neurotoxin Type A. DOI:10.1006/bbrc.2001.5911. PMID:11700044.
  6. Hanson MA et al. (2000), Nat Struct Biol, 16, 795-795. Retraction: Cocrystal structure of synaptobrevin-II bound to botulinum neurotoxin type B at 2.0 Å resolution. DOI:10.1038/nsmb0709-795. PMID:10932255.
  7. Schiavo G et al. (1992), J Biol Chem, 267, 23479-23483. Botulinum neurotoxins are zinc proteins. PMID:1429690.

Step 1. Glu231 deprotonates water which then attacks the peptide bond in a nucleophilic addition. This results in the oxyanion coordinating to the Zinc. Arg369 stabilises the transition state by interacting with the negatively charged carbonyl of the tetrahedral intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Arg369A electrostatic stabiliser
His229A metal ligand
His233A metal ligand
Glu267A metal ligand
Glu230A proton acceptor

Chemical Components

proton transfer, ingold: bimolecular nucleophilic addition, coordination, coordination to a metal ion, intermediate formation, overall reactant used, rate-determining step, cofactor used

Step 2. The oxyanion initiates an elimination that results in the cleavage of the peptide bond. The N-terminal product accepts a proton from Glu231.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Arg369A electrostatic stabiliser
His229A metal ligand
His233A metal ligand
Glu267A metal ligand
Glu230A proton donor

Chemical Components

proton transfer, ingold: unimolecular elimination by the conjugate base, heterolysis, intermediate collapse, decoordination from a metal ion, native state of cofactor regenerated, native state of enzyme regenerated

Step 3. The N-terminal product deprotonates the C-terminal product to produce kinetically favourable products.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Arg369A electrostatic stabiliser
His229A metal ligand
His233A metal ligand
Glu267A metal ligand

Chemical Components

proton transfer, overall product formed
Download: Image, Marvin File

Step 1. Glu231 deprotonates water which then attacks the peptide bond in a nucleophilic addition. This results in the oxyanion coordinating to the Zinc. Arg369 stabilises the transition state by interacting with the negatively charged carbonyl of the tetrahedral intermediate.

Step 2. The oxyanion initiates an elimination that results in the cleavage of the peptide bond. The N-terminal product accepts a proton from Glu231.

Step 3. The N-terminal product deprotonates the C-terminal product to produce kinetically favourable products.

Products.

Contributors

Mei Leung, Gemma L. Holliday, Charity Hornby