Penicillin amidase (peptidase C59 family)

 

Penicillin V acylase (PVA), isolated from Bacillus sphaericus, catalyses the hydrolysis of penicillin V to 6-aminopenicillanic acid (6-APA) and phenoxyacetic acid. This reaction is commercially important because 6-APA is used in the synthesis of semi-synthetic penicillins. The enzyme undergoes post-translational autocatalytic self-cleavage to produce the mature enzyme. A tripeptide of Met-Leu-Gly is removed to expose the catalytic N-terminal cysteine residue. PVA belongs to the N-terminal nucleophile (Ntn) superfamily.

 

Reference Protein and Structure

Sequence
P12256 UniProt (3.5.1.11) IPR029055 (Sequence Homologues) (PDB Homologues)
Biological species
Lysinibacillus sphaericus (Bacteria) Uniprot
PDB
3pva - PENICILLIN V ACYLASE FROM B. SPHAERICUS (2.8 Å) PDBe PDBsum 3pva
Catalytic CATH Domains
3.60.60.10 CATHdb (see all for 3pva)
Click To Show Structure

Enzyme Reaction (EC:3.5.1.11)

phenoxymethylpenicillin
CHEBI:27446ChEBI
+
water
CHEBI:15377ChEBI
→
phenoxyacetic acid
CHEBI:8075ChEBI
+
6-aminopenicillanic acid
CHEBI:16705ChEBI
Alternative enzyme names: Alpha-acylamino-beta-lactam acylhydrolase, Ampicillin acylase, Benzylpenicillin acylase, Novozym 217, Penicillin acylase, Semacylase,

Enzyme Mechanism

Introduction

The alpha-amine of Cys1 deprotonates the thiol group of the residue. The resulting thiolate attacks the amide carbon of penicillin V, forming a tetrahedral intermediate that can be stabilised by the oxyanion hole (Asn175 and Tyr82). The intermediate collapses and eliminates 6-APA, which is protonated by the Cys1 amine, possibly via water. The alpha-amine then deprotonates water, which acts as the nucleophile for attack on the thiol ester carbon of the intermediate. The resulting tetrahedral intermediate, which is again stabilised by the oxyanion hole, collapses and eliminates the Cys1 thiolate, which is protonated by the alpha-amine.

Catalytic Residues Roles

UniProt PDB* (3pva)
Tyr85 (main-N) Tyr82A (main-N) Tyr82 forms part of the oxyanion hole and so can stabilise the tetrahedral intermediates produced during the reaction. hydrogen bond donor, electrostatic stabiliser
Arg231 Arg228A Arg228 is thought to lower the pKa of the alpha-amine of Cys1, thus aiding acid/base catalysis. hydrogen bond donor, repulsive charge-charge interaction, activator, increase acidity, increase basicity
Cys4 (N-term), Cys4 Cys1A (N-term), Cys1A The alpha-amine of Cys1 deprotonates the thiol sidechain. The thiolate is then the nucleophile for attack on the amide carbon of the substrate. The amine is responsible for protonating the amine leaving group, possibly via a water molecule. The amine deprotonates water for attack on the intermediate and the Cys1 thiolate is then eliminated. The amine group protonates the thiolate. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor
Asn178 Asn175A Asn175 forms part of the oxyanion hole and so can stabilise the tetrahedral intermediates produced during the reaction. hydrogen bond donor, 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, intermediate formation, overall reactant used, enzyme-substrate complex formation, unimolecular elimination by the conjugate base, intermediate collapse, overall product formed, hydrolysis, enzyme-substrate complex cleavage, native state of enzyme regenerated, intermediate terminated

References

  1. 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.
  2. Avinash VS et al. (2016), Crit Rev Biotechnol, 36, 303-316. Penicillin acylases revisited: importance beyond their industrial utility. DOI:10.3109/07388551.2014.960359. PMID:25430891.
  3. Rathinaswamy P et al. (2005), Acta Crystallogr Sect F Struct Biol Cryst Commun, 61, 680-683. Cloning, purification, crystallization and preliminary structural studies of penicillin V acylase fromBacillus subtilis. DOI:10.1107/s1744309105017987. PMID:16511127.
  4. 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.
  5. Morillas M et al. (1999), Biochem J, 338, 235-239. The kinetics of acylation and deacylation of penicillin acylase from Escherichia coli ATCC 11105: evidence for lowered pKa values of groups near the catalytic centre. DOI:10.1042/bj3380235. PMID:9931321.
  6. Duggleby HJ et al. (1995), Nature, 373, 264-268. Penicillin acylase has a single-amino-acid catalytic centre. DOI:10.1038/373264a0. PMID:7816145.
  7. Brannigan JA et al. (1995), Nature, 378, 416-419. A protein catalytic framework with an N-terminal nucleophile is capable of self-activation. DOI:10.1038/378416a0. PMID:7477383.

Step 1. The alpha-amine of Cys1 deprotonates the thiol group. The resulting thiolate is the nucleophile for attack on the carbonyl carbon. The tetrahedral intermediate is stabilised by the oxyanion hole.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asn175A hydrogen bond donor, electrostatic stabiliser
Cys1A (N-term) hydrogen bond acceptor
Arg228A repulsive charge-charge interaction, hydrogen bond donor, increase basicity
Tyr82A (main-N) hydrogen bond donor, electrostatic stabiliser
Cys1A (N-term) proton acceptor
Cys1A nucleophile, proton donor

Chemical Components

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

Step 2. The tetrahedral intermediate collapses and eliminates 6-aminopenicillanate, with proton transfer from the alpha amine to the leaving group. Proton transfer between the alpha-amine and the amine leaving group may be mediated by water.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Tyr82A (main-N) hydrogen bond donor
Asn175A hydrogen bond donor
Cys1A (N-term) hydrogen bond donor
Arg228A activator, repulsive charge-charge interaction, hydrogen bond donor
Cys1A covalently attached
Cys1A (N-term) proton donor

Chemical Components

ingold: unimolecular elimination by the conjugate base, intermediate collapse, intermediate formation, overall product formed

Step 3. The alpha amine deprotonates water, which then acts as the nucleophile for attack on the carbonyl carbon. The tetrahedral intermediate is stabilised by the oxyanion hole.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Cys1A covalently attached
Tyr82A (main-N) hydrogen bond donor, electrostatic stabiliser
Asn175A hydrogen bond donor, electrostatic stabiliser
Cys1A (N-term) hydrogen bond acceptor
Arg228A repulsive charge-charge interaction, hydrogen bond donor, increase basicity
Cys1A (N-term) proton acceptor

Chemical Components

proton transfer, ingold: bimolecular nucleophilic addition, hydrolysis

Step 4. The tetrahedral intermediate collapses and eliminates Cys1 phenoxyacetate. The Cys1 thiolate is protonated by the alpha amine.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Tyr82A (main-N) hydrogen bond donor, electrostatic stabiliser
Asn175A hydrogen bond donor, electrostatic stabiliser
Cys1A (N-term) hydrogen bond donor
Arg228A increase acidity, repulsive charge-charge interaction, hydrogen bond donor
Cys1A nucleofuge
Cys1A (N-term) proton donor
Cys1A proton acceptor

Chemical Components

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

Step 1. The alpha-amine of Cys1 deprotonates the thiol group. The resulting thiolate is the nucleophile for attack on the carbonyl carbon. The tetrahedral intermediate is stabilised by the oxyanion hole.

Step 2. The tetrahedral intermediate collapses and eliminates 6-aminopenicillanate, with proton transfer from the alpha amine to the leaving group. Proton transfer between the alpha-amine and the amine leaving group may be mediated by water.

Step 3. The alpha amine deprotonates water, which then acts as the nucleophile for attack on the carbonyl carbon. The tetrahedral intermediate is stabilised by the oxyanion hole.

Step 4. The tetrahedral intermediate collapses and eliminates Cys1 phenoxyacetate. The Cys1 thiolate is protonated by the alpha amine.

Products.

Contributors

Judith A. Reeks, Gemma L. Holliday, Charity Hornby