Mandelate racemase

 

Mandelate racemase enzymes catalyse the interconversion of the (R)- and (S)-enantiomers of mandelate, in the pathway for catabolism of mandelate. This pathway, that synthesises benzoate from, (R)-mandelate, is quite specialised, and, to date, only been identified in a small number of bacteria, including Pseudomonas putida.

 

Reference Protein and Structure

Sequence
P11444 UniProt (5.1.2.2) IPR015654 (Sequence Homologues) (PDB Homologues)
Biological species
Pseudomonas putida (Bacteria) Uniprot
PDB
1mns - ON THE ROLE OF LYSINE 166 IN THE MECHANISM OF MANDELATE RACEMASE FROM PSEUDOMONAS PUTIDA: MECHANISTIC AND CRYSTALLOGRAPHIC EVIDENCE FOR STEREOSPECIFIC ALKYLATION BY (R)-ALPHA-PHENYLGLYCIDATE (2.0 Å) PDBe PDBsum 1mns
Catalytic CATH Domains
3.20.20.120 CATHdb (see all for 1mns)
Cofactors
Magnesium(2+) (1) Metal MACiE
Click To Show Structure

Enzyme Reaction (EC:5.1.2.2)

(R)-mandelate
CHEBI:32382ChEBI
→
(S)-mandelate
CHEBI:17756ChEBI

Enzyme Mechanism

Introduction

The mechanism shown in the following steps refers to the direction shown in overall reaction not in the reverse direction. Lys166 is the (S)-specific acid/base catalyst and His297 is the (R)-specific acid/base catalyst, thus in the reverse reaction, Lys166 deprotonates the (S)-mandelate and the intermediate is reprotonated from His297.

Catalytic Residues Roles

UniProt PDB* (1mns)
Lys166 Lys166(164)A Acts as the general acid/base. Abstracts the proton from S-mandelate. Also helps stabilise the transition states formed. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor, electrostatic stabiliser
Lys164 Lys164(162)A Helps to stabilise the reactive intermediates. hydrogen bond donor, electrostatic stabiliser
Asp270 Asp270(268)A Alters the pKa of His297 so that it is neutral at physiological pH. increase basicity, hydrogen bond acceptor, electrostatic stabiliser
His297 His297(295)A Acts as the general acid/base. Abstracts the proton from (R)-mandelate. Also helps stabilise the transition states formed. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor, electrostatic stabiliser
Glu317 Glu317(315)A Acts as a general acid/base to stabilise the reactive intermediates. proton acceptor, hydrogen bond donor, electrostatic stabiliser, proton donor
Asn197 Asn197(195)A The interaction between Asn 197 and the substrate's alpha-hydroxyl function provides approximately 3.5 kcal/mol of transition-state stabilisation free energy to differentially stabilise the transition state relative to the ground state. electrostatic stabiliser
Glu247, Asp195, Glu221 Glu247(245)A, Asp195(193)A, Glu221(219)A Forms magnesium binding site. metal ligand
*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, overall reactant used, intermediate formation, proton transfer, intermediate terminated, overall product formed, inferred reaction step, native state of enzyme regenerated

References

  1. Nagar M et al. (2015), Biochemistry, 54, 2747-2757. Inactivation of Mandelate Racemase by 3-Hydroxypyruvate Reveals a Potential Mechanistic Link between Enzyme Superfamilies. DOI:10.1021/acs.biochem.5b00221. PMID:25844917.
  2. Nagar M et al. (2015), Biochemistry, 54, 6743-6752. An Additional Role for the Brønsted Acid–Base Catalysts of Mandelate Racemase in Transition State Stabilization. DOI:10.1021/acs.biochem.5b00982. PMID:26480244.
  3. Lietzan AD et al. (2012), Biochemistry, 51, 1160-1170. Structure of Mandelate Racemase with Bound Intermediate Analogues Benzohydroxamate and Cupferron. DOI:10.1021/bi2018514. PMID:22264153.
  4. Nagar M et al. (2011), Biochemistry, 50, 8846-8852. Redefining the Minimal Substrate Tolerance of Mandelate Racemase. Racemization of Trifluorolactate. DOI:10.1021/bi201188j. PMID:21894901.
  5. Bourque JR et al. (2008), Biochemistry, 47, 566-578. Mutational Analysis of the Active Site Flap (20s Loop) of Mandelate Racemase†. DOI:10.1021/bi7015525. PMID:18092808.
  6. Prat-Resina X et al. (2005), J Phys Chem B, 109, 21089-21101. Reaction Mechanism of the Mandelate Anion Racemization Catalyzed by Mandelate Racemase Enzyme:  A QM/MM Molecular Dynamics Free Energy Study. DOI:10.1021/jp052239d. PMID:16853732.
  7. St Maurice M et al. (2004), Biochemistry, 43, 2524-2532. Hydrophobic Nature of the Active Site of Mandelate Racemase†. DOI:10.1021/bi036207x. PMID:14992589.
  8. Garcia-Viloca M et al. (2001), J Am Chem Soc, 123, 709-721. A QM/MM Study of the Racemization of Vinylglycolate Catalyzed by Mandelate Racemase Enzyme. DOI:10.1021/ja002879o. PMID:11456585.
  9. St. Maurice M et al. (2000), Biochemistry, 39, 13324-13335. Reaction Intermediate Analogues for Mandelate Racemase:  Interaction between Asn 197 and the α-Hydroxyl of the Substrate Promotes Catalysis†. DOI:10.1021/bi001144t.
  10. Schafer SL et al. (1996), Biochemistry, 35, 5662-5669. Mechanism of the Reaction Catalyzed by Mandelate Racemase:  Structure and Mechanistic Properties of the D270N Mutant†,‡. DOI:10.1021/bi960174m. PMID:8639525.
  11. Babbitt PC et al. (1996), Biochemistry, 35, 16489-16501. The enolase superfamily: a general strategy for enzyme-catalyzed abstraction of the alpha-protons of carboxylic acids. DOI:10.1021/bi9616413. PMID:8987982.
  12. Mitra B et al. (1995), Biochemistry, 34, 2777-2787. Mechanism of the Reaction Catalyzed by Mandelate Racemase: Importance of Electrophilic Catalysis by Glutamic Acid 317. DOI:10.1021/bi00009a006. PMID:7893689.
  13. Kallarakal AT et al. (1995), Biochemistry, 34, 2788-2797. Mechanism of the Reaction Catalyzed by Mandelate Racemase: Structure and Mechanistic Properties of the K166R Mutant. DOI:10.1021/bi00009a007. PMID:7893690.
  14. Landro JA et al. (1994), Biochemistry, 33, 635-643. The role of lysine 166 in the mechanism of mandelate racemase from Pseudomonas putida: mechanistic and crystallographic evidence for stereospecific alkylation by (R)-alpha-phenylglycidate. PMID:8292591.
  15. Gerlt JA et al. (1993), J Am Chem Soc, 115, 11552-11568. An explanation for rapid enzyme-catalyzed proton abstraction from carbon acids: importance of late transition states in concerted mechanisms. DOI:10.1021/ja00077a062.
  16. Landro JA et al. (1991), Biochemistry, 30, 9274-9281. Mechanism of the reaction catalyzed by mandelate racemase. 3. Asymmetry in reactions catalyzed by the H297N mutant. PMID:1909893.
  17. Neidhart DJ et al. (1991), Biochemistry, 30, 9264-9273. Mechanism of the reaction catalyzed by mandelate racemase. 2. Crystal structure of mandelate racemase at 2.5-.ANG. resolution: identification of the active site and possible catalytic residues. DOI:10.1021/bi00102a019. PMID:1892834.

Step 1. His297 deprotonates the (R)-mandelate substrate, which results in a keto-enol tautomerisation.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys166(164)A hydrogen bond acceptor
Glu317(315)A hydrogen bond donor, electrostatic stabiliser
Lys164(162)A hydrogen bond donor, electrostatic stabiliser
His297(295)A hydrogen bond donor
Asp270(268)A hydrogen bond acceptor, electrostatic stabiliser
Lys166(164)A electrostatic stabiliser
Asn197(195)A electrostatic stabiliser
His297(295)A electrostatic stabiliser
Glu221(219)A metal ligand
Glu247(245)A metal ligand
Asp195(193)A metal ligand
His297(295)A proton acceptor
Glu317(315)A proton donor

Chemical Components

assisted keto-enol tautomerisation, overall reactant used, intermediate formation, proton transfer

Step 2. The intermediate undergoes another keto-enol tautomerisation which reprotonates the intermediate from Lys166.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys166(164)A hydrogen bond donor
Glu317(315)A electrostatic stabiliser
Lys164(162)A hydrogen bond donor, electrostatic stabiliser
His297(295)A hydrogen bond donor
Asp270(268)A hydrogen bond acceptor, electrostatic stabiliser
Asn197(195)A electrostatic stabiliser
Lys166(164)A electrostatic stabiliser
His297(295)A electrostatic stabiliser
Glu221(219)A metal ligand
Glu247(245)A metal ligand
Asp195(193)A metal ligand
Glu317(315)A proton acceptor
Lys166(164)A proton donor

Chemical Components

assisted keto-enol tautomerisation, intermediate terminated, overall product formed, proton transfer

Step 3. Lys166 deprotonates water, which deprotonates His297 in an inferred return step.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys166(164)A hydrogen bond donor
His297(295)A hydrogen bond acceptor
Asp270(268)A increase basicity, hydrogen bond acceptor
Glu221(219)A metal ligand
Glu247(245)A metal ligand
Asp195(193)A metal ligand
Lys166(164)A proton acceptor
His297(295)A proton donor

Chemical Components

inferred reaction step, native state of enzyme regenerated, proton transfer
Download: Image, Marvin File

Step 1. His297 deprotonates the (R)-mandelate substrate, which results in a keto-enol tautomerisation.

Step 2. The intermediate undergoes another keto-enol tautomerisation which reprotonates the intermediate from Lys166.

Step 3. Lys166 deprotonates water, which deprotonates His297 in an inferred return step.

Products.

Contributors

Gemma L. Holliday, Daniel E. Almonacid, Anna Waters, Craig Porter