Glutamate racemase

 

Glutamate racemase is responsible for the synthesis of D-glutamate, an essential building block of peptidoglycan, found in bacterial cell walls where it provides structural integrity. Due to its uniqueness to bacteria, peptidoglycan, and enzymes involved in its biosynthesis, are targets for designing new antibacterial drugs. Peptidoglycan is formed from a repeating unit of a disaccharide, N-acetylglucosamine and N-acetylmuramic acid to which a small group of amino acids (L-alanine, D-alaline, D-glutamate and either lysine or diaminopimelic acid) are covalently attached. The presence of D-amino acids protects the cell wall from proteases which can only recognise the L-isomer.

 

Reference Protein and Structure

Sequence
P56868 UniProt (5.1.1.3) IPR004391 (Sequence Homologues) (PDB Homologues)
Biological species
Aquifex pyrophilus (Bacteria) Uniprot
PDB
1b73 - GLUTAMATE RACEMASE FROM AQUIFEX PYROPHILUS (2.3 Å) PDBe PDBsum 1b73
Catalytic CATH Domains
3.40.50.1860 CATHdb (see all for 1b73)
Click To Show Structure

Enzyme Reaction (EC:5.1.1.3)

L-glutamate(1-)
CHEBI:29985ChEBI
D-glutamate(1-)
CHEBI:29986ChEBI

Enzyme Mechanism

Introduction

A two base mechanism is proposed where Cys70 and Cys178 are responsible for the deprotonation and protonation of the C-alpha atom of D-Glucose, respectively. Cys70 deprotonates the C-alpha to form an enolate intermediate before Cys178 reprotonates on the opposite face to produce L-Glucose. The carboxylates of Asp7 and Glu147 are also important in catalysis by assisting activation of the acid/base thiols.

Catalytic Residues Roles

UniProt PDB*
Asp7 Asp7A Acts as the general acid/base for Cys70 activation. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, activator, electrostatic stabiliser
Ser8 Ser8A Activates Asp7 increase basicity, hydrogen bond donor, electrostatic stabiliser
Cys178 Cys178A The catalytic general acid/base that re-protonates the substrate to produce the D-product. In the reverse reaction it deprotonates the D-substrate. activator, hydrogen bond acceptor, proton acceptor, proton donor
Cys70 Cys70A The catalytic general acid/base that deprotonates the L-substrate. In the reverse reaction, Cys70 re-protonates the substrate to produce the L-product. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor, activator, electrostatic stabiliser
His180 His180A Activates Glu147 hydrogen bond donor, electrostatic stabiliser
Glu147 Glu147A(A-2) Re-protonates Cys178 to return the active site to a favourable state. In the reverse reaction it accepts a proton from Cys178 to initiate the reaction. hydrogen bond acceptor, hydrogen bond donor, proton donor, activator, electrostatic stabiliser, increase basicity
*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, assisted keto-enol tautomerisation, atom stereo change, intermediate formation, native state of enzyme regenerated

References

  1. Hwang KY et al. (1999), Nat Struct Biol, 6, 422-426. Structure and mechanism of glutamate racemase from Aquifex pyrophilus. DOI:10.1038/8223. PMID:10331867.
  2. Mixcoha E et al. (2012), J Phys Chem B, 116, 12406-12414. Theoretical Analysis of the Catalytic Mechanism ofHelicobacter pyloriGlutamate Racemase. DOI:10.1021/jp3054982. PMID:22984984.
  3. Puig E et al. (2009), J Am Chem Soc, 131, 3509-3521. How the Substrated-Glutamate Drives the Catalytic Action ofBacillus subtilisGlutamate Racemase. DOI:10.1021/ja806012h. PMID:19227983.
  4. Sengupta S et al. (2008), Microbiology, 154, 2796-2803. Moonlighting function of glutamate racemase from Mycobacterium tuberculosis: racemization and DNA gyrase inhibition are two independent activities of the enzyme. DOI:10.1099/mic.0.2008/020933-0. PMID:18757813.
  5. Puig E et al. (2007), J Phys Chem B, 111, 2385-2397. New Insights into the Reaction Mechanism Catalyzed by the Glutamate Racemase Enzyme:  pH Titration Curves and Classical Molecular Dynamics Simulations. DOI:10.1021/jp066350a. PMID:17286428.
  6. Puig E et al. (2006), J Phys Chem A, 110, 717-725. On the Ionization State of the Substrate in the Active Site of Glutamate Racemase. A QM/MM Study about the Importance of Being Zwitterionic†. DOI:10.1021/jp054555y. PMID:16405345.
  7. Ashiuchi M et al. (2002), J Biol Chem, 277, 39070-39073. Glutamate Racemase Is an Endogenous DNA Gyrase Inhibitor. DOI:10.1074/jbc.c200253200. PMID:12213801.
  8. Glavas S et al. (2001), Biochemistry, 40, 6199-6204. Active Site Residues of Glutamate Racemase†. DOI:10.1021/bi002703z. PMID:11371180.
  9. Glavas S et al. (1999), Biochemistry, 38, 4106-4113. Catalytic Acid/Base Residues of Glutamate Racemase†. DOI:10.1021/bi982663n. PMID:10194325.

Catalytic Residues Roles

Residue Roles
Glu147A(A-2) hydrogen bond acceptor
Asp7A activator, hydrogen bond acceptor
His180A hydrogen bond donor
Cys70A activator, hydrogen bond donor
Ser8A hydrogen bond donor, electrostatic stabiliser, increase basicity
Cys70A proton donor
Asp7A proton acceptor

Chemical Components

proton transfer

Catalytic Residues Roles

Residue Roles
Glu147A(A-2) hydrogen bond acceptor
Asp7A electrostatic stabiliser, hydrogen bond donor
His180A hydrogen bond donor
Cys70A activator, hydrogen bond acceptor
Ser8A hydrogen bond donor
Cys70A proton acceptor

Chemical Components

assisted keto-enol tautomerisation, proton transfer, atom stereo change, intermediate formation

Catalytic Residues Roles

Residue Roles
Cys178A activator, hydrogen bond acceptor
Glu147A(A-2) electrostatic stabiliser, hydrogen bond donor, increase basicity
Asp7A electrostatic stabiliser, hydrogen bond donor
His180A hydrogen bond donor
Cys70A electrostatic stabiliser, hydrogen bond donor
Ser8A hydrogen bond donor
Cys178A proton donor

Chemical Components

assisted keto-enol tautomerisation, proton transfer, atom stereo change

Catalytic Residues Roles

Residue Roles
Cys178A activator, hydrogen bond acceptor
Glu147A(A-2) activator, hydrogen bond donor, hydrogen bond acceptor
Asp7A hydrogen bond acceptor
His180A hydrogen bond donor, electrostatic stabiliser
Ser8A hydrogen bond donor
Glu147A(A-2) proton donor
Cys178A proton acceptor

Chemical Components

proton transfer, native state of enzyme regenerated

Contributors

Gail J. Bartlett, Daniel E. Almonacid, Sophie T. Williams, Gemma L. Holliday, Alex Gutteridge, Craig Porter, Katherine Ferris