(S)-2-hydroxy-acid oxidase
Glycolate oxidase is a flavoenzyme, which contains a permanently bound FNN cofactor. It catalyses the oxidation of (S)-2-hydroxy-acid to 2-oxo acid. In green plants, glycolate oxidase is one of the key enzymes in photorespiration where it oxidizes glycolate to glyoxylate. Comparison of properties with the extensively studied L-lactate oxidase and the topography of the active site and the amino acids surrounding the prosthetic group FMN led to the proposal of a reaction mechanism.
Reference Protein and Structure
- Sequence
-
P05414
(1.1.3.15)
(Sequence Homologues)
(PDB Homologues)
- Biological species
-
Spinacia oleracea (spinach)
- PDB
-
1gox
- REFINED STRUCTURE OF SPINACH GLYCOLATE OXIDASE AT 2 ANGSTROMS RESOLUTION
(2.0 Å)
- Catalytic CATH Domains
-
3.20.20.70
(see all for 1gox)
- Cofactors
- Fmnh2(2-) (1)
Enzyme Reaction (EC:1.1.3.15)
Enzyme Mechanism
Introduction
The reaction proceeds via a ping-pong mechanism and the reduction of FMN is found to be rate-limiting. In the first reductive step a proton is abstracted from the substance C2 carbon atom producing a carbanion. This carbanion can subsequently attach to a N5 atom of FMN, via formation of a covalent adduct, two electrons are then transferred to the isoalloxazine ring. The reduced FMN is reoxidised by oxygen to form hydrogen peroxide.
Catalytic Residues Roles
| UniProt | PDB* (1gox) | ||
| Lys230 | Lys230(231)A | Lys230 interacts with atoms N1 and O2 of the isoalloxazine ring, which produces an inductive effect that could enhance the nucleophilicity of the electron acceptor N5 of the flavin ring | enhance reactivity, electrostatic stabiliser |
| Tyr129 | Tyr129(130)A | Tyr129 lowers the pKa of the N(3) of oxidized flavin. However, the main function of the hydroxyl group of Tyr129 is the stabilisation of the transition states formed during the course of the reaction. | modifies pKa, electrostatic stabiliser |
| Ser106, Thr155 | Ser106(107)A, Thr155(156)A | Ser106 and Thr155 are also important for stabilising the FMN cofactor. | electrostatic stabiliser |
| His254 | His254(255)A | Acts as a general acid/base. Abstracts a proton from the C2 atom of the substrate. | proton shuttle (general acid/base) |
Chemical Components
References
- Dellero Y et al. (2015), J Biol Chem, 290, 1689-1698. Experimental Evidence for a Hydride Transfer Mechanism in Plant Glycolate Oxidase Catalysis. DOI:10.1074/jbc.m114.618629. PMID:25416784.
- Stenberg K et al. (1997), Protein Sci, 6, 1009-1015. Three-dimensional structures of glycolate oxidase with bound active-site inhibitors. DOI:10.1002/pro.5560060506. PMID:9144771.
- Stenberg K et al. (1995), Eur J Biochem, 228, 408-416. Involvement of Tyr24 and Trp108 in substrate binding and substrate specificity of glycolate oxidase. PMID:7705356.
- Macheroux P et al. (1993), Eur J Biochem, 213, 1047-1054. Role of tyrosine 129 in the active site of spinach glycolate oxidase. DOI:10.1111/j.1432-1033.1993.tb17852.x. PMID:8504801.
- Lindqvist Y et al. (1989), J Biol Chem, 264, 3624-3628. The active site of spinach glycolate oxidase. PMID:2644287.
- Lindqvist Y (1989), J Mol Biol, 209, 151-166. Refined structure of spinach glycolate oxidase at 2 A resolution. PMID:2681790.
Catalytic Residues Roles
| Residue | Roles |
|---|---|
| His254(255)A | proton shuttle (general acid/base) |
| Tyr129(130)A | modifies pKa |
| Ser106(107)A | electrostatic stabiliser |
| Tyr129(130)A | electrostatic stabiliser |
| Thr155(156)A | electrostatic stabiliser |
| Lys230(231)A | enhance reactivity, electrostatic stabiliser |