Penicillin amidase (peptidase S45 family)

 

Penicillin acylase is a widely distributed enzyme among bacteria, yeast and fungi. It is used industrially in the production of 6-aminopenicillanic acid, the starting point for the synthesis of penicillins. In vivo it is thought to have a role in the use of aromatic compounds as carbon sources.

 

Reference Protein and Structure

Sequence
P06875 UniProt (3.5.1.11) IPR014395 (Sequence Homologues) (PDB Homologues)
Biological species
Escherichia coli (Bacteria) Uniprot
PDB
1pnl - PENICILLIN ACYLASE HAS A SINGLE-AMINO-ACID CATALYTIC CENTRE (2.5 Å) PDBe PDBsum 1pnl
Catalytic CATH Domains
3.60.20.10 CATHdb (see all for 1pnl)
Click To Show Structure

Enzyme Reaction (EC:3.5.1.11)

penicillinate anion
CHEBI:51356ChEBI
+
water
CHEBI:15377ChEBI
→
6-aminopenicillanic acid zwitterion
CHEBI:57869ChEBI
+
carboxylic acid anion
CHEBI:29067ChEBI
Alternative enzyme names: Alpha-acylamino-beta-lactam acylhydrolase, Ampicillin acylase, Benzylpenicillin acylase, Novozym 217, Penicillin acylase, Semacylase,

Enzyme Mechanism

Introduction

The penicillin binds in the active site near to the reactive N-terminal serine. The amino group enhances the serines nucleophilic character (via a bridging water) allowing it to attack the carbonyl carbon at the amide bond to be cleaved. The tetrahedral intermediate is stabilised by the side chain of asparagine 241 and the main chain nitrogen of alanine 69. The bond is then cleaved and the two reaction products are released.

Catalytic Residues Roles

UniProt PDB* (1pnl)
Asn530 (main-C) Asn241B (main-C) Stabilises the N-terminus of Ser 290 and increases its pKa so it more willingly accepts a proton from the hydroxyl of Ser 290. electrostatic stabiliser
Ser290 (N-term) Ser1B (N-term) Activates the serine side chain. proton acceptor, proton donor
Ser290 Ser1B Acts as a catalytic nucleophile. nucleofuge, nucleophile, proton acceptor, proton donor
Asn530 (main-N), Ala358 (main-N) Asn241B (main-N), Ala69B (main-N) Form the oxyanion hole, stabilising the reactive intermediates and transition states. 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, enzyme-substrate complex formation, intermediate formation, overall reactant used, rate-determining step, unimolecular elimination by the conjugate base, heterolysis, intermediate collapse, enzyme-substrate complex cleavage, native state of enzyme regenerated, overall product formed

References

  1. McVey CE et al. (2001), J Mol Biol, 313, 139-150. Crystal structures of penicillin acylase enzyme-substrate complexes: structural insights into the catalytic mechanism 1 1Edited by K. Nagai. DOI:10.1006/jmbi.2001.5043. PMID:11601852.
  2. Grigorenko BL et al. (2014), ACS Catal, 4, 2521-2529. Catalytic Cycle of Penicillin Acylase from Escherichia coli: QM/MM Modeling of Chemical Transformations in the Enzyme Active Site upon Penicillin G Hydrolysis. DOI:10.1021/cs5002898.
  3. Suresh CG et al. (1999), Nat Struct Biol, 6, 414-416. Penicillin V acylase crystal structure reveals new Ntn-hydrolase family members. DOI:10.1038/8213. PMID:10331865.
  4. Done SH et al. (1998), J Mol Biol, 284, 463-475. Ligand-induced conformational change in penicillin acylase. DOI:10.1006/jmbi.1998.2180. PMID:9813130.
  5. Duggleby HJ et al. (1995), Nature, 373, 264-268. Penicillin acylase has a single-amino-acid catalytic centre. DOI:10.1038/373264a0. PMID:7816145.

Step 1. Ser 290 N-terminus deprotonates its own hydroxyl group which activates it to nucleophilically attack the carbonyl group of the amide bond of penicillin.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Ala69B (main-N) electrostatic stabiliser
Asn241B (main-N) electrostatic stabiliser
Asn241B (main-C) electrostatic stabiliser
Ser1B proton donor, nucleophile
Ser1B (N-term) proton acceptor

Chemical Components

proton transfer, ingold: bimolecular nucleophilic addition, enzyme-substrate complex formation, intermediate formation, overall reactant used, rate-determining step

Step 2. The oxyanion initiates an elimination which results in the cleavage of the amide bond and the N-terminal product accepts a proton from the N-terminus of Ser290.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Ala69B (main-N) electrostatic stabiliser
Asn241B (main-N) electrostatic stabiliser
Asn241B (main-C) electrostatic stabiliser
Ser1B (N-term) proton donor

Chemical Components

ingold: unimolecular elimination by the conjugate base, proton transfer, heterolysis, intermediate collapse

Step 3. The N-terminus of Ser290 abstracts a proton from a water molecule which activates it to nucleophilically attack the carbon of the carbonyl group.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Ala69B (main-N) electrostatic stabiliser
Asn241B (main-N) electrostatic stabiliser
Asn241B (main-C) electrostatic stabiliser
Ser1B (N-term) proton acceptor

Chemical Components

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

Step 4. The oxyanion initiates an elimination which results in the cleavage acyl-enzyme bond which releases Ser290 which then accepts a proton from its N-terminus and thus returns the active site to its native state.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Ala69B (main-N) electrostatic stabiliser
Asn241B (main-N) electrostatic stabiliser
Asn241B (main-C) electrostatic stabiliser
Ser1B nucleofuge
Ser1B (N-term) proton donor
Ser1B proton acceptor

Chemical Components

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

Step 1. Ser 290 N-terminus deprotonates its own hydroxyl group which activates it to nucleophilically attack the carbonyl group of the amide bond of penicillin.

Step 2. The oxyanion initiates an elimination which results in the cleavage of the amide bond and the N-terminal product accepts a proton from the N-terminus of Ser290.

Step 3. The N-terminus of Ser290 abstracts a proton from a water molecule which activates it to nucleophilically attack the carbon of the carbonyl group.

Step 4. The oxyanion initiates an elimination which results in the cleavage acyl-enzyme bond which releases Ser290 which then accepts a proton from its N-terminus and thus returns the active site to its native state.

Products.

Contributors

Alex Gutteridge, Craig Porter, Gemma L. Holliday, Charity Hornby