N-succinylamino acid racemase

 

N-succinylamino acid racemase (NAAAR) catalyses the racemisation of N-succinyl-D/L-amino acids, as part of a pathway for the conversion of D- to L-amino acids. NAAARs act on a broad range of N-acylamino acids rather than amino acids. This function is of significant interest in industry where enantiopure amino acids are inportant as chiral building blocks for antibiotics, herbicides, and drugs. The experimentally characterised enzyme from Geobacillus kaustophilus efficiently catalyses the racemisation of hydrophobic, polar, and some basic N-succinyl-D/L-amino acids.

These enzymes are members of the enolase superfamily and highly similar to enzymes in the o-succinylbenzoate synthase family, and some may also function biologically as OSBSs. Although the reaction catalyzed by this family is similar to that catalyzed by the NSAR2 family (differing only in the preference of N-succinyl arginine/lysine for NSAR2 versus N-succinyl hydrophobic amino acids for NSAR), phylogenetic analysis suggests that these two families have independent evolutionary origins within the enolase superfamily. Not surprisingly, the amino acids responsible for substrate recognition appear to differ between the two families.

 

Reference Protein and Structure

Sequence
Q9RYA6 UniProt (4.2.1.113) IPR010197 (Sequence Homologues) (PDB Homologues)
Biological species
Deinococcus radiodurans R1 (Bacteria) Uniprot
PDB
1r0m - Structure of Deinococcus radiodurans N-acylamino acid racemase at 1.3 : insights into a flexible binding pocket and evolution of enzymatic activity (1.3 Å) PDBe PDBsum 1r0m
Catalytic CATH Domains
3.20.20.120 CATHdb (see all for 1r0m)
Cofactors
Magnesium(2+) (1)
Click To Show Structure

Enzyme Reaction (EC:5.1.1.10)

N-succinyl-L-amino acid residue
CHEBI:85535ChEBI
→
N-succinyl-D-amino acid
CHEBI:85536ChEBI
Alternative enzyme names: L-amino acid racemase,

Enzyme Mechanism

Introduction

The mechanism is proposed to proceed via a two-base proton abstraction. The alpha-proton of an N-acylamino acid is abstracted by a neutral Lys to produce the enolic intermediate stabilized by the divalent metal ion. A proton is donated by the other (positive) Lys to the intermediate to yield the racemised product.This second Lys can then can then reversibly act as the principle base as shown in the scheme.

Catalytic Residues Roles

UniProt PDB* (1r0m)
Lys170 Lys170A General acid/base. In the direction shown here, donates its proton to the substrate to give the product. proton donor
Asp195, Glu220, Asp245 Asp195A, Glu220A, Asp245A Bind the Mg(II) ion. metal ligand
Lys269 Lys269A Acts as a general acid/base. proton acceptor
Gly297 (main-N), Ser142, Lys168 Gly297A (main-N), Ser142A, Lys168A Stabilise the oxyanion intermediate through hydrogen bonding. transition state 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

assisted keto-enol tautomerisation, proton transfer

References

  1. Chiu WC et al. (2006), J Mol Biol, 359, 741-753. Structure–Stability–Activity Relationship in Covalently Cross-linked N-Carbamoyl d-Amino acid Amidohydrolase and N-Acylamino acid Racemase. DOI:10.1016/j.jmb.2006.03.063. PMID:16650857.
  2. Soriano-Maldonado P et al. (2015), Mol Biotechnol, 57, 454-465. Biochemical and Mutational Characterization of N-Succinyl-Amino Acid Racemase from Geobacillus stearothermophilus CECT49. DOI:10.1007/s12033-015-9839-4. PMID:25875730.
  3. Sakai A et al. (2006), Biochemistry, 45, 4455-4462. Evolution of enzymatic activities in the enolase superfamily: N-succinylamino acid racemase and a new pathway for the irreversible conversion of D- to L-amino acids. DOI:10.1021/bi060230b. PMID:16584181.
  4. Wang WC et al. (2004), J Mol Biol, 342, 155-169. Structural Basis for Catalytic Racemization and Substrate Specificity of an N-Acylamino Acid Racemase Homologue from Deinococcus radiodurans. DOI:10.1016/j.jmb.2004.07.023. PMID:15313614.
  5. Gulick AM et al. (2001), Biochemistry, 40, 15716-15724. Evolution of Enzymatic Activities in the Enolase Superfamily:  Crystal Structures of thel-Ala-d/l-Glu Epimerases fromEscherichia coliandBacillus subtilis†,‡. DOI:10.1021/bi011641p.

Step 1. Lys269 abstracts a proton from the substrate with concomitant double bond rearrangement.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Ser142A transition state stabiliser
Lys168A transition state stabiliser
Gly297A (main-N) transition state stabiliser
Glu220A metal ligand
Asp245A metal ligand
Asp195A metal ligand
Lys269A proton acceptor

Chemical Components

assisted keto-enol tautomerisation, proton transfer

Step 2. The oxyanion intermediate collapses, with concomitant double bond rearrangement and deprotonation of Lys170.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp245A metal ligand
Asp195A metal ligand
Gly297A (main-N) transition state stabiliser
Lys168A transition state stabiliser
Ser142A transition state stabiliser
Lys170A proton donor

Chemical Components

proton transfer, assisted keto-enol tautomerisation
Download: Image, Marvin File

Step 1. Lys269 abstracts a proton from the substrate with concomitant double bond rearrangement.

Step 2. The oxyanion intermediate collapses, with concomitant double bond rearrangement and deprotonation of Lys170.

Products.

Contributors

Gemma L. Holliday, Shoshana Brown