N-carbamoyl-D-amino-acid hydrolase

 

N-carbomyl-D-amino acid amindohydrolase (DCase) catalyses the hydrolysis of N-carbomyl-D-amino acids to the corresponding D-amino acids, which are useful in preparation of physiologically active peptides and beta-lactam antibiotics.

 

Reference Protein and Structure

Sequence
P60327 UniProt (3.5.1.77) IPR003010 (Sequence Homologues) (PDB Homologues)
Biological species
Agrobacterium sp. KNK712 (Bacteria) Uniprot
PDB
1uf7 - Crystal structure of C171A/V236A Mutant of N-carbamyl-D-amino acid amidohydrolase complexed with N-carbamyl-D-valine (1.9 Å) PDBe PDBsum 1uf7
Catalytic CATH Domains
3.60.110.10 CATHdb (see all for 1uf7)
Click To Show Structure

Enzyme Reaction (EC:3.5.1.77)

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

Enzyme Mechanism

Introduction

The proposed mechanism suggests that Glu46 acts as a base to deprotonate the thiol group of Cys171 to promote its nucleophilic attack on the carbonyl carbon of the substrate to form an acylenzyme intermediate and ammonia. Glu46 then protonates the leaving group and activates a water molecule to restore the enzyme from the acylenzyme intermediate. The released N-carboxy-amino acid spontaneously collapses and converts to the corresponding D-amino acid and carbon dioxide. The negatively charged transition state in the reaction is stabilised by an oxyanion hole formed by Lys126.

Catalytic Residues Roles

UniProt PDB* (1uf7)
Glu47 Glu46A 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 shuttle (general acid/base)
Lys127 Lys126A It forms an oxyanion hole to stabilise the negatively charged transition state. electrostatic stabiliser
Cys172 Ala171A Its thiol group acts as a nucleophile to attack the carbonyl carbon of the substrate to form an acylenzyme intermediate. covalent catalysis, proton shuttle (general acid/base)
*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

References

  1. Nakai T et al. (2000), Structure, 8, 729-738. Crystal structure of N-carbamyl-d-amino acid amidohydrolase with a novel catalytic framework common to amidohydrolases. DOI:10.1016/s0969-2126(00)00160-x. PMID:10903946.
  2. 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.

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Catalytic Residues Roles

Residue Roles
Lys126A electrostatic stabiliser
Glu46A proton shuttle (general acid/base)
Ala171A covalent catalysis, proton shuttle (general acid/base)

Chemical Components

Step 1.

Contributors

Mei Leung, Gemma L. Holliday