NAD+---diphthamide ADP-ribosyltransferase

 

The ADP-ribosyltransferases are a class of enzymes that display activity in a variety of bacterial pathogens responsible for causing diseases in plants and animals, including those affecting mankind, such as diphtheria, cholera, and whooping cough.

 

Reference Protein and Structure

Sequences
P01555 UniProt (2.4.2.-)
P01556 UniProt IPR001144 (Sequence Homologues) (PDB Homologues)
Biological species
Vibrio cholerae O1 biovar El Tor str. N16961 (Bacteria) Uniprot
PDB
1xtc - CHOLERA TOXIN (2.4 Å) PDBe PDBsum 1xtc
Catalytic CATH Domains
3.90.210.10 CATHdb (see all for 1xtc)
Click To Show Structure

Enzyme Reaction (EC:2.4.2.36)

diphthamide residue
CHEBI:16692ChEBI
+
NAD(1-)
CHEBI:57540ChEBI
→
N-(ADP-D-ribosyl)diphthamide(1-) residue
CHEBI:82697ChEBI
+
hydron
CHEBI:15378ChEBI
+
nicotinamide
CHEBI:17154ChEBI
Alternative enzyme names: ADP-ribosyltransferase, Mono(ADPribosyl)transferase, Mono(ADP-ribosyl)transferase, NAD--diphthamide ADP-ribosyltransferase, NAD(+):peptide-diphthamide N-(ADP-D-ribosyl)transferase,

Enzyme Mechanism

Introduction

The mechanism for this enzyme is currently unclear, however an SN2-like mechanism has been proposed. Here, the attacking nucleophile may be the substrate arginine, diphthamide or asparagine, depending on the toxin involved, and may even be water in auto-hydrolysis. This nucleophile is then deprotonated by a conserved active site glutamate (Glu110?). The activated nucleophile then attacks the anomeric carbon of the ribose ring which, due to the conformation of the NAD, has been exposed to the solvent forming a pentacoordinate oxocarbenium transition state intermediate. This intermediate is partially stabilised by a catalytic glutamate (Glu112) forming a hydrogen bond with the O2 atom on the nicotinamide ribose. This makes the ring more electronegative, which stabilises the positively charged oxocarbenium ion before the N-glycosidic bond is cleaved, completing the transfer of the ADP-ribose+ moiety on to the substrate.

Catalytic Residues Roles

UniProt PDB* (1xtc)
Glu128 Glu110A Acts as a general acid/base. proton shuttle (general acid/base)
Arg25 Arg7A Binds the diphoshate moiety, helping to stabilise the reactive intermediates and transition states formed during the course of the reaction. electrostatic stabiliser
Ser79 Ser61A Forms a hydrogen bonding network with Arg7 in the absence of NAD. With NAD bound, thought to be involved in the stabilisation of the catalytic glutamate. electrostatic stabiliser
Glu130 Glu112A Binds the ribose ring of the NAD substrate, involved in stabilisation and activation of the substrate. 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

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. O'Neal CJ et al. (2004), Biochemistry, 43, 3772-3782. Crystal structures of an intrinsically active cholera toxin mutant yield insight into the toxin activation mechanism. DOI:10.1021/bi0360152. PMID:15049684.
  3. Zhang RG et al. (1995), J Mol Biol, 251, 563-573. The Three-dimensional Crystal Structure of Cholera Toxin. DOI:10.1006/jmbi.1995.0456. PMID:7658473.

Step 1.

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Catalytic Residues Roles

Residue Roles
Arg7A electrostatic stabiliser
Glu112A electrostatic stabiliser
Ser61A electrostatic stabiliser
Glu110A proton shuttle (general acid/base)

Chemical Components

Step 1.

Contributors

Craig Porter, Gemma L. Holliday