Diaminopimelate epimerase

 

Diaminopimelate epimerase catalyses the isomerisation of L,L-dimaminopimelate to meso-DAP in the biosynthetic pathway leading from aspartate to lysine. It is a member of the broader family of PLP-independent amino acid racemases. Diaminopimelic acid is an essential component of bacterial cell wall biosynthesis. Diaminopimelate epimerase utilises a pair of cysteine residues in catalysis, as seen in other non-PLP dependent alpha-amino acid racemases. However, its specificity for a substrate with two stereo-centres separates the kinetic from data that presented by functionally related enzymes [PMID:16723397, PMID:10194362].

 

Reference Protein and Structure

Sequence
P44859 UniProt (5.1.1.7) IPR001653 (Sequence Homologues) (PDB Homologues)
Biological species
Haemophilus influenzae Rd KW20 (Bacteria) Uniprot
PDB
1bwz - DIAMINOPIMELATE EPIMERASE FROM HEMOPHILUS INFLUENZAE (2.72 Å) PDBe PDBsum 1bwz
Catalytic CATH Domains
3.10.310.10 CATHdb (see all for 1bwz)
Click To Show Structure

Enzyme Reaction (EC:5.1.1.7)

(2S,6S)-2,6-diaminopimelic acid dizwitterion
CHEBI:57609ChEBI
→
meso-2,6-diaminopimelic acid dizwitterion
CHEBI:57791ChEBI

Enzyme Mechanism

Introduction

The Cys73 thiolate abstracts the substrate's alpha position proton, generating a carbanion transition state which rapidly abstracts a proton from Cys217. This residue is positioned on the alternative face of the molecule Cys73 and therefore, on reprotonation the stereochemistry at this centre is inverted. The enzyme is able to catalyse the interconversion of either S or R stereochemistry, creating an equilibrium between the diastereoisomer and the meso-product.

Catalytic Residues Roles

UniProt PDB* (1bwz)
Cys217, Cys73 Cys217A, Cys73A Act as a general acid/base. In the 2S,6S to 2R,6S direction Cys73 is negatively charged in the ground state, acting to abstract a proton from the substrate. Cys217 is neutral and donates a proton to the transition state to form the product. In the reverse reaction direction, the roles of the cysteine residues are reversed. hydrogen bond donor, proton donor
Gly220 (main-N), His159 Gly220A (main-N), His159A Activates and stabilises Cys217 hydrogen bond donor, electrostatic stabiliser
Glu208 Glu208A Stabilises and activates Cys73. activator, 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, isomerisation reaction (not covered by named reactions), overall reactant used, overall product formed, native state of enzyme is not regenerated, rate-determining step

References

  1. Koo CW et al. (1999), Biochemistry, 38, 4416-4422. Chemical Mechanism ofHaemophilus influenzaeDiaminopimelate Epimerase†. DOI:10.1021/bi982911f. PMID:10194362.
  2. Stenta M et al. (2009), J Chem Theory Comput, 5, 1915-1930. Catalytic Mechanism of Diaminopimelate Epimerase: A QM/MM Investigation. DOI:10.1021/ct900004x. PMID:26610016.
  3. Pillai B et al. (2006), Proc Natl Acad Sci U S A, 103, 8668-8673. Structural insights into stereochemical inversion by diaminopimelate epimerase: An antibacterial drug target. DOI:10.1073/pnas.0602537103. PMID:16723397.
  4. Diaper CM et al. (2005), Org Biomol Chem, 3, 4402-4411. The stereoselective synthesis of aziridine analogues of diaminopimelic acid (DAP) and their interaction with dap epimerase. DOI:10.1039/b513409a. PMID:16327902.
  5. Cirilli M et al. (1998), Biochemistry, 37, 16452-16458. Structural Symmetry:  The Three-Dimensional Structure ofHaemophilus InfluenzaeDiaminopimelate Epimerase†,‡. DOI:10.1021/bi982138o. PMID:9843410.

Step 1. The Cys73 thiolate abstracts the substrate's alpha position proton, generating a carbanion transition state which rapidly abstracts a proton from Cys217. This residue is positioned on the alternative face of the molecule Cys73 and therefore, on reprotonation the stereochemistry at this centre is inverted. The stereochemsitry of the bound substrate interconverts until it dissociates from the active site. The enzyme is therefore not regenerated, per say, before a second round of catalysis occurs [PMID:16723397, PMID:10194362].

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Gly220A (main-N) hydrogen bond donor, electrostatic stabiliser
Cys217A hydrogen bond donor
Cys73A hydrogen bond acceptor
His159A electrostatic stabiliser
Glu208A electrostatic stabiliser
His159A activator
Glu208A activator
Cys217A proton donor
Cys73A proton acceptor

Chemical Components

proton transfer, isomerisation reaction (not covered by named reactions), overall reactant used, overall product formed, native state of enzyme is not regenerated, rate-determining step
Download: Image, Marvin File

Step 1. The Cys73 thiolate abstracts the substrate's alpha position proton, generating a carbanion transition state which rapidly abstracts a proton from Cys217. This residue is positioned on the alternative face of the molecule Cys73 and therefore, on reprotonation the stereochemistry at this centre is inverted. The stereochemsitry of the bound substrate interconverts until it dissociates from the active site. The enzyme is therefore not regenerated, per say, before a second round of catalysis occurs [PMID:16723397, PMID:10194362].

Products.

Contributors

Sophie T. Williams, Nozomi Nagano, Craig Porter, Gemma L. Holliday, James Willey