D-stereospecific aminopeptidase (peptidase S12 family)
Antibiotic resistance to the beta lactam family of antibiotics, which includes penicillin, is often conferred to bacteria by serine beta lactamases which can break down the inhibitor to harmless products. They are believed to have evolved from D amino peptidases, which hydrolyse the peptide bond between two D amino acids after the N terminal residue. DAP from Othrobacrum anthropi is one such peptidase, and is inhibited by beta lactams. Study of this protein will thus reveal details of how the antibiotic resistance conferring peptidases work. It is found to show significant active site homology with other penicillin binding proteins, including the serine beta lactamases, but very low sequence identity with either group, suggesting that it is part of a novel family which may have given rise to the resistance-conferring enzymes.
Reference Protein and Structure
- Sequence
-
Q9ZBA9
(3.4.11.19)
(Sequence Homologues)
(PDB Homologues)
- Biological species
-
Ochrobactrum anthropi (Bacteria)
- PDB
-
1ei5
- CRYSTAL STRUCTURE OF A D-AMINOPEPTIDASE FROM OCHROBACTRUM ANTHROPI
(1.9 Å)
- Catalytic CATH Domains
-
3.40.710.10
(see all for 1ei5)
Enzyme Mechanism
Introduction
The reaction proceeds via nucleophilic attack by Ser 62 on the peptide substrate, activated by proton abstraction by Lys 62. Lys 62's pKa is lowered sufficiently for it to carry out this role by contacts with Tyr 153 and Asn 155. This forms a tetrahedral intermediate, which collapses, assisted by protonation of the leaving group by Lys 65 to form an acyl enzyme intermediate. Hydrolysis of this intermediate by a water molecule, activated by deprotonation by Tyr 153 leads to the products of the peptidase reaction.
Catalytic Residues Roles
| UniProt | PDB* (1ei5) | ||
| Tyr153 | Tyr153A | Acts as general base for the deacylation step deprotonating a water molecule to activate it for hydrolysis of the acyl enzyme intermediate. Also acts to reduce the pKa of Lys 65 so that it remains unprotonated at physiological pH and can itself act as an acid-base. | proton shuttle (general acid/base), electrostatic stabiliser |
| Asn155 | Asn155A | Acts to reduce the pKa of Lys 65 so that it is deprotonated at physiological pH and can act as an acid-base. | electrostatic stabiliser |
| His287 | His287A | Activates Tyr 153 by reducing its pKa so that it is deprotonated at physiological pH and can act as an acid base for the deacylation step. | electrostatic stabiliser |
| Ser62 | Ser62A | Acts as a nucleophile to attack the peptide bond, forming an acyl enzyme intermediate which is hydrolysed to give the products. | covalently attached |
| Lys65 | Lys65A | Acts as the general base during the acylation step, removing a proton from Ser 62 to allow it to act as a nucleophile. Subsequently protonates the leaving group so that the tetrahedral intermediate collapses. | proton shuttle (general acid/base) |
Chemical Components
References
- Bompard-Gilles C et al. (2000), Structure, 8, 971-980. Crystal structure of a d-aminopeptidase from Ochrobactrum anthropi, a new member of the ‘penicillin-recognizing enzyme’ family. DOI:10.1016/s0969-2126(00)00188-x. PMID:10986464.
- Arima J et al. (2016), FEBS J, 283, 337-349. Crystal structure of D-stereospecific amidohydrolase from Streptomyces sp. 82F2 - insight into the structural factors for substrate specificity. DOI:10.1111/febs.13579. PMID:26513520.
Catalytic Residues Roles
| Residue | Roles |
|---|---|
| Asn155A | electrostatic stabiliser |
| His287A | electrostatic stabiliser |
| Ser62A | covalently attached |
| Lys65A | proton shuttle (general acid/base) |
| Tyr153A | electrostatic stabiliser, proton shuttle (general acid/base) |