Mono-ADP-ribosyltransferase C3

 

Clostridium boltulinum C3 exoenzyme catalyses the transfer of the ADP-ribose moiety of NAD onto asparagine 41 of the small GTP-binding protein Rho. This ADP-ribosylation inactivates Rho, blocking Rho activity in regulating the cytoskeleton and leading to cytoskeleton depolymerisation. The protein synthesis inhibitors (inactivate the eukaryotic elongation factor 2 eEF2) diptheria toxin from Corynebacterium diphtheriae and exotoxin A (ETA) from Pseudomonas aeruginosa are also ADP-ribosyltransferases, contain a conserved Glu and thought to proceed through the same catalytic mechanism.

 

Reference Protein and Structure

Sequence
P15879 UniProt (2.4.2.-) IPR016678 (Sequence Homologues) (PDB Homologues)
Biological species
Clostridium botulinum D phage (Virus) Uniprot
PDB
1g24 - THE CRYSTAL STRUCTURE OF EXOENZYME C3 FROM CLOSTRIDIUM BOTULINUM (1.7 Å) PDBe PDBsum 1g24
Catalytic CATH Domains
3.90.176.10 CATHdb (see all for 1g24)
Click To Show Structure

Enzyme Reaction (EC:2.4.2.-)

asparagine residue
CHEBI:32664ChEBI
+
NAD(1-)
CHEBI:57540ChEBI
→
nicotinamide
CHEBI:17154ChEBI
+
hydron
CHEBI:15378ChEBI
+
[protein]-N-ADP-D-ribosyl-L-asparagine
CHEBI:137462ChEBI

Enzyme Mechanism

Introduction

The reaction requires loss of the nicotinamide moiety of NAD and attack on the C1' of ribose by Asn 41 (thought to be activated by Glu214) of Rho in an Sn1 like mechanism. The oxocarbenium ion intermediate is thought to be stabilised by Glu 214, which forms a hydrogen bond to the ribose 2' OH group and also Ser174. Out of the two mechanism proposals, this is more favoured due to mutagenesis experiments of Glu214 uncoupling the glycohydrolytic and transferase reactions. It is thought Exotoxin A and the Diphtheria toxin share the same catalytic mechanism as the C3 exoenzyme.

Catalytic Residues Roles

UniProt PDB* (1g24)
Ser174 Ser174(134)A Stabilises the positive charge in the oxocarbenium-like transition state. Apart of the conserved STS motif. electrostatic stabiliser, polar interaction
Glu214 Glu214(174)A Stabilises positive charge in the oxocarbenium-like intermediate by forming a hydrogen bond to the 2' OH group of the ribose moiety. proton acceptor, proton donor, electrostatic stabiliser, polar interaction
*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

heterolysis, intermediate formation, elimination (not covered by the Ingold mechanisms), overall product formed, bimolecular nucleophilic addition, overall reactant used, proton transfer, intermediate terminated, inferred reaction step, native state of enzyme regenerated

References

  1. Holbourn KP et al. (2006), FEBS J, 273, 4579-4593. A family of killer toxins. Exploring the mechanism of ADP-ribosylating toxins. DOI:10.1111/j.1742-4658.2006.05442.x. PMID:16956368.
  2. Vogelsgesang M et al. (2006), Biochemistry, 45, 1017-1025. Exchange of glutamine-217 to glutamate of Clostridium limosum exoenzyme C3 turns the asparagine-specific ADP-ribosyltransferase into an arginine-modifying enzyme. DOI:10.1021/bi052253g. PMID:16411778.
  3. Jørgensen R et al. (2005), Nature, 436, 979-984. Exotoxin A–eEF2 complex structure indicates ADP ribosylation by ribosome mimicry. DOI:10.1038/nature03871. PMID:16107839.
  4. Yates SP et al. (2004), Biochem J, 379, 563-572. Elucidation of eukaryotic elongation factor-2 contact sites within the catalytic domain of Pseudomonas aeruginosa exotoxin A. DOI:10.1042/bj20031731. PMID:14733615.
  5. Han S et al. (2001), J Mol Biol, 305, 95-107. Crystal structure and novel recognition motif of Rho ADP-ribosylating C3 exoenzyme from Clostridium botulinum: structural insights for recognition specificity and catalysis. DOI:10.1006/jmbi.2000.4292. PMID:11114250.
  6. Bell CE et al. (1997), Biochemistry, 36, 481-488. Crystal Structure of Nucleotide-Free Diphtheria Toxin†,‡. DOI:10.1021/bi962214s. PMID:9012663.
  7. Wilson BA et al. (1990), Biochemistry, 29, 8643-8651. Active-site mutations of diphtheria toxin: effects of replacing glutamic acid-148 with aspartic acid, glutamine, or serine. DOI:10.1021/bi00489a021. PMID:1980208.

Step 1. Glycosidic bond cleavage resulting in an oxocarbenium intermediate, stabilised by Glu214 and Ser174.

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Catalytic Residues Roles

Residue Roles
Glu214(174)A electrostatic stabiliser
Ser174(134)A electrostatic stabiliser, polar interaction
Glu214(174)A polar interaction

Chemical Components

heterolysis, intermediate formation, elimination (not covered by the Ingold mechanisms), overall product formed

Step 2. Nucleophilic attack by Asn41 on the Rho protein. Glu214 is proposed to deprotonate the arginine to increase it's nucleophilicity.

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Catalytic Residues Roles

Residue Roles
Ser174(134)A electrostatic stabiliser
Glu214(174)A electrostatic stabiliser
Ser174(134)A polar interaction
Glu214(174)A polar interaction, proton acceptor

Chemical Components

ingold: bimolecular nucleophilic addition, overall reactant used, proton transfer, overall product formed, intermediate terminated

Step 3. Glu214 is deprotonated by a water molecule.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu214(174)A proton donor

Chemical Components

inferred reaction step, proton transfer, native state of enzyme regenerated
Download: Image, Marvin File

Step 1. Glycosidic bond cleavage resulting in an oxocarbenium intermediate, stabilised by Glu214 and Ser174.

Step 2. Nucleophilic attack by Asn41 on the Rho protein. Glu214 is proposed to deprotonate the arginine to increase it's nucleophilicity.

Step 3. Glu214 is deprotonated by a water molecule.

Products.

Introduction

In an Sn2 manner, Asn41 is deprotonated and acts as a nucleophile, attacking C1 of the ribose sugar to generate an oxocarbenium ion like transition state. Glu214 hydrogen bonding to the nicotinamide ribose 2' OH increases the ring's electronegativity, stabilising the oxocarbenium ion thus facilitating glycosidic bond cleavage. The phosphates of NAD itself potentially increase the electrophilicity of C1 due to withdrawing electrons from the nicotinamide ring.

Catalytic Residues Roles

UniProt PDB* (1g24)
Glu214 Glu214(174)A Stabilises positive charge in the oxocarbenium-like transition state by forming a hydrogen bond to the 2' OH group of the ribose moiety. proton acceptor, proton donor, electrostatic stabiliser, polar interaction
*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

bimolecular nucleophilic substitution, overall reactant used, overall product formed, inferred reaction step, proton transfer, native state of enzyme regenerated

References

  1. Han S et al. (2001), J Mol Biol, 305, 95-107. Crystal structure and novel recognition motif of Rho ADP-ribosylating C3 exoenzyme from Clostridium botulinum: structural insights for recognition specificity and catalysis. DOI:10.1006/jmbi.2000.4292. PMID:11114250.
  2. Holbourn KP et al. (2006), FEBS J, 273, 4579-4593. A family of killer toxins. Exploring the mechanism of ADP-ribosylating toxins. DOI:10.1111/j.1742-4658.2006.05442.x. PMID:16956368.

Step 1. Glu214 is proposed to deprotonate Asn41 so that Asn41 nucleophilically attacks the C1 on the ribose ring.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu214(174)A electrostatic stabiliser
Glu214(174)A polar interaction, proton acceptor

Chemical Components

ingold: bimolecular nucleophilic substitution, overall reactant used, overall product formed

Step 2. Glu214 is deprotonated by a water molecule.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu214(174)A polar interaction, proton donor

Chemical Components

inferred reaction step, proton transfer, native state of enzyme regenerated
Download: Image, Marvin File

Step 1. Glu214 is proposed to deprotonate Asn41 so that Asn41 nucleophilically attacks the C1 on the ribose ring.

Step 2. Glu214 is deprotonated by a water molecule.

Products.

Contributors

Steven Smith, Gemma L. Holliday, Morwenna Hall, Ellie Wright, Jonathan T. W. Ng