Alpha-amino-acid esterase

 

alpha-amino acid ester hydrolases (AEHs) catalyse the hydrolysis and synthesis of esters and amides with an alpha-amino group. They can be used to acylate a beta-lactam using an ester as acyl donor without cleaving the beta-lactam nuclei, so the enzyme is suitable for generating widely used antibiotics such as ampicillin, amoxicillin, and the cephalosporins, cephadroxil and cephalexin.

 

Reference Protein and Structure

Sequence
Q6YBS3 UniProt (3.1.1.43) IPR005674 (Sequence Homologues) (PDB Homologues)
Biological species
Xanthomonas citri (Bacteria) Uniprot
PDB
1mpx - ALPHA-AMINO ACID ESTER HYDROLASE LABELED WITH SELENOMETHIONINE (1.9 Å) PDBe PDBsum 1mpx
Catalytic CATH Domains
3.40.50.1820 CATHdb (see all for 1mpx)
Click To Show Structure

Enzyme Reaction (EC:3.1.1.43)

alpha-amino acid ester(1+)
CHEBI:83410ChEBI
+
water
CHEBI:15377ChEBI
→
alpha-amino acid zwitterion
CHEBI:78608ChEBI
+
hydron
CHEBI:15378ChEBI
+
alcohol
CHEBI:30879ChEBI
Alternative enzyme names: Alpha-amino acid ester hydrolase, Alpha-amino-acid ester hydrolase,

Enzyme Mechanism

Introduction

The catalytic mechanism for both hydrolysis and ester synthesis follows that of serine hydrolases. But during ester synthesis, instead of transferring the acyl group in the intermediate to a water molecule, the acyl group is transferred to a beta-lactam nuclei. His340 acts as a base to deprotonate Ser174 to allow its nucleophilic attack on the ester bond, forming an acylenzyme intermediate. His340 donates a proton to the leaving group. In hydrolysis, it activates a water molecule to restore the enzyme. Asp307 alters the pKa of the His340 to allow to act as an effective acid and base in the reaction. Backbone amide of Tyr175 and side chain of Tyr82 form the oxyanion hole to stabilise the transition state.

Catalytic Residues Roles

UniProt PDB* (1mpx)
Tyr82 Tyr82(60)A It forms the oxyanion hole to stabilise the transition state. electrostatic stabiliser
Asp307 Asp307(285)A It alters the pKa of the His 340 to allow to act as an effective acid and base in the reaction. increase basicity, electrostatic stabiliser
Tyr175 (main-N) Tyr175(153)A (main-N) Its backbone amide forms the oxyanion hole to stabilise the transition state. electrostatic stabiliser
Ser174 Ser174(152)A It acts as a nucleophile to attack the ester bond. covalently attached, nucleofuge, nucleophile, proton acceptor, proton donor
His340 His340(318)A It deprotonates Ser 174 to allow its nucleophilic attack on the ester or peptide bond. It donates a proton to the leaving group. In hydrolysis, it activates a water molecule to regenerate the enzyme. proton acceptor, proton donor
*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

overall reactant used, intermediate formation, proton transfer, bimolecular nucleophilic addition, overall product formed, unimolecular elimination by the conjugate base, native state of enzyme regenerated, intermediate terminated

References

  1. Barends TR et al. (2003), J Biol Chem, 278, 23076-23084. The Sequence and Crystal Structure of the  -Amino Acid Ester Hydrolase from Xanthomonas citri Define a New Family of  -Lactam Antibiotic Acylases. DOI:10.1074/jbc.m302246200. PMID:12684501.

Step 1. His340 acts as a general base activating the Ser174 hydroxyl group for nucleophilic attack on the carbonyl carbon. The third component of this catalytic triad- Asp307 acts to increase the basicity of the histidine. The oxyanion intermediate formed is stabilized by the amide group of Tyr175 and the side chain of Tyr82.

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Catalytic Residues Roles

Residue Roles
Tyr175(153)A (main-N) electrostatic stabiliser
Ser174(152)A covalently attached
Tyr82(60)A electrostatic stabiliser
Asp307(285)A electrostatic stabiliser, increase basicity
Ser174(152)A nucleophile, proton donor
His340(318)A proton acceptor

Chemical Components

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

Step 2. The tetrahedral intermediate collapses and an alcohol is eliminated.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Ser174(152)A covalently attached
Tyr82(60)A electrostatic stabiliser
Tyr175(153)A (main-N) electrostatic stabiliser
Asp307(285)A electrostatic stabiliser
His340(318)A proton donor

Chemical Components

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

Step 3. His340 activates water for nucleophilic attack and another oxyanion intermediate is formed.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Ser174(152)A covalently attached
Tyr82(60)A electrostatic stabiliser
Tyr175(153)A (main-N) electrostatic stabiliser
Asp307(285)A electrostatic stabiliser, increase basicity
His340(318)A proton acceptor

Chemical Components

ingold: bimolecular nucleophilic addition, proton transfer

Step 4. The tetrahedral intermediate collapses and Ser174 is eliminated.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Tyr82(60)A electrostatic stabiliser
Tyr175(153)A (main-N) electrostatic stabiliser
Asp307(285)A electrostatic stabiliser
Ser174(152)A nucleofuge, proton acceptor
His340(318)A proton donor

Chemical Components

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

Step 1. His340 acts as a general base activating the Ser174 hydroxyl group for nucleophilic attack on the carbonyl carbon. The third component of this catalytic triad- Asp307 acts to increase the basicity of the histidine. The oxyanion intermediate formed is stabilized by the amide group of Tyr175 and the side chain of Tyr82.

Step 2. The tetrahedral intermediate collapses and an alcohol is eliminated.

Step 3. His340 activates water for nucleophilic attack and another oxyanion intermediate is formed.

Step 4. The tetrahedral intermediate collapses and Ser174 is eliminated.

Products.

Contributors

Mei Leung, Gemma L. Holliday, James Willey