Cephalosporin-C deacetylase

 

Cephalosporin-C deacetylase is an esterase that removes acetyl groups from a number of O-acetylated small substrates, such as acetylated xylose, short xylooligosaccharides and cephalosporin C. It is known not to cleave amide bonds. It is used in the pharmaceutical industry for the chemoenzymatic deacetylation of cephalosporins and the synthesis of novel antibiotics. It is an alpha-beta hydrolase, a member of the carbohydrate esterase 7 family.

 

Reference Protein and Structure

Sequence
P94388 UniProt (3.1.1.41, 3.1.1.72) IPR008391 (Sequence Homologues) (PDB Homologues)
Biological species
Bacillus subtilis subsp. subtilis str. 168 (Bacteria) Uniprot
PDB
1l7a - structural Genomics, crystal structure of Cephalosporin C deacetylase (1.5 Å) PDBe PDBsum 1l7a
Catalytic CATH Domains
3.40.50.1820 CATHdb (see all for 1l7a)
Click To Show Structure

Enzyme Reaction (EC:3.1.1.41)

water
CHEBI:15377ChEBI
+
cephalosporin C(1-)
CHEBI:57511ChEBI
→
deacetylcephalosporin C(1-)
CHEBI:58366ChEBI
+
hydron
CHEBI:15378ChEBI
+
acetate
CHEBI:30089ChEBI
Alternative enzyme names: Cephalosporin C acetyl-esterase, Cephalosporin C acetyl-hydrolase, Cephalosporin C acetylase, Cephalosporin C acetylesterase, Cephalosporin C deacetylase, Cephalosporin acetylesterase,

Enzyme Mechanism

Introduction

Serine is activated by the histidine of the Ser-His-Asp catalytic triad. Serine then attacks the substrate in a nucleophilic addition reaction, eliminating the acetate group. Water then cleaves the enzyme-substrate bond to regenerate the active site and produce the final product.

Catalytic Residues Roles

UniProt PDB* (1l7a)
Ser181 Ser181A Forms part of the catalytic Ser-His-Asp triad. Functions as the nucleophile. covalently attached, nucleofuge, nucleophile, proton acceptor, proton donor, electrostatic stabiliser
Asp269 Asp269A Forms part of the catalytic Ser-His-Asp triad. Activates and stabilises the catalytic histidine. increase basicity, electrostatic stabiliser
His298 His298A Forms part of the catalytic Ser-His-Asp triad. Acts as a general acid/base, abstracting a proton from the catalytic serine. proton acceptor, proton donor
Gln182 (main-N), Tyr91 (main-N) Gln182A (main-N), Tyr91A (main-N) Forms part of the oxyanion hole. 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, overall reactant used, intermediate formation, bimolecular nucleophilic addition, unimolecular elimination by the conjugate base, overall product formed, intermediate terminated, native state of enzyme regenerated

References

  1. Vincent F et al. (2003), J Mol Biol, 330, 593-606. Multifunctional Xylooligosaccharide/Cephalosporin C Deacetylase Revealed by the Hexameric Structure of the Bacillus subtilis Enzyme at 1.9Å Resolution. DOI:10.1016/s0022-2836(03)00632-6. PMID:12842474.
  2. Dodson G et al. (1998), Trends Biochem Sci, 23, 347-352. Catalytic triads and their relatives. DOI:10.1016/s0968-0004(98)01254-7. PMID:9787641.

Step 1. His298 acts as a general base activating the Ser181 hydroxyl group for nucleophilic attack on the carbonyl carbon of the ester bond. The third component of this catalytic triad- Asp269 acts to increase the basicity of the histidine. The oxyanion intermediate formed is stabilized by the amide groups of Gln182 and Tyr91.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Gln182A (main-N) electrostatic stabiliser
Asp269A electrostatic stabiliser, increase basicity
Ser181A covalently attached
Tyr91A (main-N) electrostatic stabiliser
His298A proton acceptor
Ser181A proton donor, nucleophile

Chemical Components

proton transfer, overall reactant used, intermediate formation, ingold: bimolecular nucleophilic addition

Step 2. The tetrahedral intermediate collapses and deacetylcephalosporin is eliminated.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Tyr91A (main-N) electrostatic stabiliser
Gln182A (main-N) electrostatic stabiliser
Asp269A electrostatic stabiliser
Ser181A covalently attached
His298A proton donor

Chemical Components

ingold: unimolecular elimination by the conjugate base, proton transfer, overall product formed

Step 3. His298 activates water for nucleophilic attack and another oxyanion intermediate is formed.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Tyr91A (main-N) electrostatic stabiliser
Gln182A (main-N) electrostatic stabiliser
Asp269A electrostatic stabiliser, increase basicity
Ser181A covalently attached
His298A proton acceptor

Chemical Components

proton transfer, ingold: bimolecular nucleophilic addition

Step 4. The tetrahedral intermediate collapses and Ser181 is eliminated.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Tyr91A (main-N) electrostatic stabiliser
Ser181A electrostatic stabiliser
Asp269A electrostatic stabiliser
His298A proton donor
Ser181A nucleofuge, proton acceptor

Chemical Components

overall product formed, ingold: unimolecular elimination by the conjugate base, intermediate terminated, proton transfer, native state of enzyme regenerated
Download: Image, Marvin File

Step 1. His298 acts as a general base activating the Ser181 hydroxyl group for nucleophilic attack on the carbonyl carbon of the ester bond. The third component of this catalytic triad- Asp269 acts to increase the basicity of the histidine. The oxyanion intermediate formed is stabilized by the amide groups of Gln182 and Tyr91.

Step 2. The tetrahedral intermediate collapses and deacetylcephalosporin is eliminated.

Step 3. His298 activates water for nucleophilic attack and another oxyanion intermediate is formed.

Step 4. The tetrahedral intermediate collapses and Ser181 is eliminated.

Products.

Contributors

Alex Gutteridge, Craig Porter, Gemma L. Holliday, James Willey, Marko Babić