Chloramphenicol O-acetyltransferase

 

Chloramphenicol acetyltransferase (CAT) [PMID:1867721] catalyses the acetyl-CoA dependent acetylation of chloramphenicol (Cm), an antibiotic which inhibits prokaryotic peptidyltransferase activity. Acetylation of Cm by CAT inactivates the antibiotic. A histidine residue, located in the C-terminal section of the enzyme, plays a central role in its catalytic mechanism. CAT enzymes are divided into three classes: CATI, CATII, and CATIII, with the enzymes of all three classes catalysing acetyl transfer to CAM to yield 3-O-acetyl-CAM. This entry represents the type III protein, although the mechanism is likely to be similar between the three classes.

 

Reference Protein and Structure

Sequence
P00484 UniProt (2.3.1.28) IPR001707 (Sequence Homologues) (PDB Homologues)
Biological species
Escherichia coli (Bacteria) Uniprot
PDB
3cla - REFINED CRYSTAL STRUCTURE OF TYPE III CHLORAMPHENICOL ACETYLTRANSFERASE AT 1.75 ANGSTROMS RESOLUTION (1.75 Å) PDBe PDBsum 3cla
Catalytic CATH Domains
3.30.559.10 CATHdb (see all for 3cla)
Cofactors
Water (1)
Click To Show Structure

Enzyme Reaction (EC:2.3.1.28)

acetyl-CoA(4-)
CHEBI:57288ChEBI
+
chloramphenicol
CHEBI:17698ChEBI
→
chloramphenicol 3-acetate
CHEBI:16730ChEBI
+
coenzyme A(4-)
CHEBI:57287ChEBI
Alternative enzyme names: CAT I, CAT II, CAT III, Chloramphenicol acetylase, Chloramphenicol acetyltransferase, Chloramphenicol transacetylase,

Enzyme Mechanism

Introduction

His195 abstracts the proton from the substrate hydroxyl group, which initiates a nucleophilic attack on the carbonyl carbon of the acyl CoA molecule. The oxyanion formed is stabilises by a hydrogen bonding network with a conserved water molecule and Thr174. The intermediate then collapses, forming the final products.

Catalytic Residues Roles

UniProt PDB* (3cla)
His189 His195(189)A His195 is appropriately positioned to act as a general base catalyst in the reaction, and the required tautomeric stabilisation is provided by an unusual interaction with a main-chain carbonyl oxygen [PMID: 2187098]. proton shuttle (general acid/base)
Arg13, Asp193 Arg18(13)A, Asp199(193)A Form part of the Arg-Asp-His catalytic triad that activates the histidine to act as a general acid/base. electrostatic stabiliser
Thr168 Thr174(168)A(AB) Helps stabilise the transition states formed during the course of the reaction. Thr174 bonds to water, which in turn forms a hydrogen bond to the transition state. activator, transition state 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. Shaw WV et al. (1991), Annu Rev Biophys Biophys Chem, 20, 363-386. Chloramphenicol Acetyltransferase. DOI:10.1146/annurev.bb.20.060191.002051. PMID:1867721.
  2. Beaman TW et al. (1998), Biochemistry, 37, 6689-6696. Structure of the hexapeptide xenobiotic acetyltransferase from Pseudomonas aeruginosa. DOI:10.1021/bi980106v. PMID:9578552.
  3. Lewendon A et al. (1994), Biochemistry, 33, 1944-1950. Replacement of catalytic histidine-195 of chloramphenicol acetyltransferase: Evidence for a general base role for glutamate. DOI:10.1021/bi00173a043. PMID:7906544.
  4. Lewendon A et al. (1993), J Biol Chem, 268, 20997-21001. Transition state stabilization by chloramphenicol acetyltransferase. Role of a water molecule bound to threonine 174. PMID:8407936.
  5. Leslie AG (1990), J Mol Biol, 213, 167-186. Refined crystal structure of type III chloramphenicol acetyltransferase at 1·75 Å resolution. DOI:10.1016/s0022-2836(05)80129-9. PMID:2187098.

Step 1.

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Catalytic Residues Roles

Residue Roles
His195(189)A proton shuttle (general acid/base)
Thr174(168)A(AB) activator, transition state stabiliser
Arg18(13)A electrostatic stabiliser
Asp199(193)A electrostatic stabiliser, modifies pKa

Chemical Components

Step 1.

Contributors

Craig Porter, Gemma L. Holliday