N-carbamoyl-D-amino-acid hydrolase

 

N-carbomyl-D-amino-acid amidohydrolase catalyses the hydrolysis of N-carbamoyl-D-amino acids to the corresponding D-amino acids which are useful intermediates in the preparation of beta-lactam antibiotics. Industrial production of beta-lactam antibiotics is now being developed using this enzyme.

 

Reference Protein and Structure

Sequence
Q44185 UniProt (3.5.1.77) IPR003010 (Sequence Homologues) (PDB Homologues)
Biological species
Agrobacterium tumefaciens (Bacteria) Uniprot
PDB
1fo6 - CRYSTAL STRUCTURE ANALYSIS OF N-CARBAMoYL-D-AMINO-ACID AMIDOHYDROLASE (1.95 Å) PDBe PDBsum 1fo6
Catalytic CATH Domains
3.60.110.10 CATHdb (see all for 1fo6)
Click To Show Structure

Enzyme Reaction (EC:3.5.1.77)

water
CHEBI:15377ChEBI
+
N-carbamoyl-D-alpha-amino acid anion
CHEBI:85602ChEBI
+
hydron
CHEBI:15378ChEBI
→
ammonium
CHEBI:28938ChEBI
+
carbon dioxide
CHEBI:16526ChEBI
+
D-alpha-amino acid zwitterion
CHEBI:59871ChEBI
Alternative enzyme names: D-N-carbamoylase, N-carbamoylase, N-carbamoyl-D-amino acid hydrolase,

Enzyme Mechanism

Introduction

N-carbomyl-D-amino-acid amidohydrolase catalyses the hydrolysis of N-carbamoyl-D-amino acids to the corresponding D-amino acids with the release of carbon dioxide and ammonia. The catalytic mechanism of N-carbomyl-D-amino-acid amidohydrolase begins with an acylation reaction. The sulphur atom of Cys 172 makes a nucleophilic attack on the C atom of the substrate carbonyl group, with Glu 47 acting as a general base in removing the proton from the sulphur atom. Lys 127 can then stabilise the tetrahedral transition state, and cleavage of the C-N bond occurs by the action of Glu 47 as a general acid catalyst to release ammonia. Deacylation of the acyl-enzyme intermediate occurs by Glu 47 acting as a general base to activate a water molecule for nucleophilic attack of the carbonyl carbon. The transition state is again stabilised by Lys 127 and Glu 47 again donates a proton, this time to Cys 172, which releases an acid component that decomposes further to release carbon dioxide leaving the D-amino acid product.

Catalytic Residues Roles

UniProt PDB* (1fo6)
Glu47 Glu47A It deprotonates Cys 171 to allow its nucleophilic attack on the substrate carbonyl carbon. It protonates the leaving group. It activates a water molecule to restore the enzyme from the acylenzyme intermediate. proton acceptor, proton donor
Lys127 Lys127A It forms an oxyanion hole to stabilise the negatively charged transition state. electrostatic stabiliser
Glu146 Glu146A Stabilises Lys 127 through hydrogen bonding. electrostatic stabiliser
Cys172 Cys172A It's thiol group acts as a nucleophile to attack the carbonyl carbon of the substrate to form an acylenzyme intermediate. nucleofuge, nucleophile, proton acceptor, proton donor
Asn110, Asn197 Asn110A, Asn197A Stabilises Glu 47 through hydrogen bonding which lowers the pKa of Glu 47 so it more willingly accepts a proton. 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, intermediate collapse, enzyme-substrate complex cleavage, native state of enzyme regenerated, heterolysis, inferred reaction step, overall product formed

References

  1. Wang WC et al. (2001), J Mol Biol, 306, 251-261. Crystal structure and site-directed mutagenesis studies of N-carbamoyl-d-amino-acid amidohydrolase from Agrobacterium radiobacter reveals a homotetramer and insight into a catalytic cleft11Edited by R. Huber. DOI:10.1006/jmbi.2000.4380. PMID:11237598.
  2. Han W et al. (2009), Chem Phys Lett, 472, 107-112. The substrate specificity and the catalytic mechanism of N-carbamyl- d -amino acid amidohydrolase: A theoretical investigation. DOI:10.1016/j.cplett.2009.01.086.
  3. Chen CY et al. (2003), J Biol Chem, 278, 26194-26201. Structural Basis for Catalysis and Substrate Specificity of Agrobacterium radiobacter N-Carbamoyl-D-amino Acid Amidohydrolase. DOI:10.1074/jbc.m302384200. PMID:12709423.
  4. Grifantini R et al. (1996), J Biol Chem, 271, 9326-9331. Topological Mapping of the Cysteine Residues of N-Carbamyl-D-amino-acid Amidohydrolase and Their Role in Enzymatic Activity. DOI:10.1074/jbc.271.16.9326. PMID:8621596.

Step 1. Glu47 deprotonates Cys172 which activates it nucleophilically attack the carbon of the carbonyl group.

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Catalytic Residues Roles

Residue Roles
Asn197A electrostatic stabiliser
Lys127A electrostatic stabiliser
Asn110A electrostatic stabiliser
Glu146A electrostatic stabiliser
Glu47A proton acceptor
Cys172A nucleophile, proton donor

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 amino group which is protonated by Glu47 to produce ammonia.

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Catalytic Residues Roles

Residue Roles
Asn110A electrostatic stabiliser
Lys127A electrostatic stabiliser
Glu146A electrostatic stabiliser
Asn197A electrostatic stabiliser
Glu47A proton donor

Chemical Components

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

Step 3. Glu47 deprotonates a water molecule which activates it to nucleophilically attack the carbon of the carbonyl group.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asn110A electrostatic stabiliser
Lys127A electrostatic stabiliser
Glu146A electrostatic stabiliser
Asn197A electrostatic stabiliser
Glu47A proton acceptor

Chemical Components

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

Step 4. The oxyanion once again initiates an elimination which results in the cleavage of the acyl-enzyme and Cys172 is protonated by Glu47 which returns the active site to its native state.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asn110A electrostatic stabiliser
Lys127A electrostatic stabiliser
Glu146A electrostatic stabiliser
Asn197A electrostatic stabiliser
Cys172A nucleofuge
Glu47A proton donor
Cys172A proton acceptor

Chemical Components

proton transfer, ingold: unimolecular elimination by the conjugate base, enzyme-substrate complex cleavage, intermediate collapse, intermediate formation, native state of enzyme regenerated

Step 5. The released intermediate further decomposes to release CO2 and the D-amino acid product, which is protonated by the surrounding solvent.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asn110A electrostatic stabiliser
Lys127A electrostatic stabiliser
Glu146A electrostatic stabiliser
Asn197A electrostatic stabiliser

Chemical Components

proton transfer, heterolysis, inferred reaction step, overall product formed
Download: Image, Marvin File

Step 1. Glu47 deprotonates Cys172 which activates it nucleophilically attack the carbon of the carbonyl group.

Step 2. The oxyanion initiates an elimination which results in the cleavage of the amino group which is protonated by Glu47 to produce ammonia.

Step 3. Glu47 deprotonates a water molecule which activates it to nucleophilically attack the carbon of the carbonyl group.

Step 4. The oxyanion once again initiates an elimination which results in the cleavage of the acyl-enzyme and Cys172 is protonated by Glu47 which returns the active site to its native state.

Step 5. The released intermediate further decomposes to release CO2 and the D-amino acid product, which is protonated by the surrounding solvent.

Products.

Contributors

Gary McDowell, Gemma L. Holliday, Mei Leung, Charity Hornby