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
(5.1.2.2)
(Sequence Homologues) (PDB Homologues)
- Biological species
-
Pseudomonas putida (Bacteria)
- 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)
- Catalytic CATH Domains
-
3.20.20.120
(see all for 1mns)
- Cofactors
- Magnesium(2+) (1) Metal MACiE
Enzyme Reaction (EC:5.1.2.2)
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 |
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 regeneratedReferences
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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 FileCatalytic 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 transferStep 2. The intermediate undergoes another keto-enol tautomerisation which reprotonates the intermediate from Lys166.
Download: Image, Marvin FileCatalytic 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 transferStep 3. Lys166 deprotonates water, which deprotonates His297 in an inferred return step.
Download: Image, Marvin FileCatalytic 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 |