Ketol-acid reductoisomerase
Ketol-acid reductoisomerase (KARI) is a Mg(2+) -dependent enzyme in the branched-chain amino acid biosynthesis pathway. KARI catalyses two steps in the biosynthesis of branched-chain amino acids. The reaction involves an Mg(II) dependent alkyl migration followed by an NADPH-dependent reduction of the 2-keto group.
Reference Protein and Structure
- Sequence
-
Q01292
(1.1.1.86)
(Sequence Homologues)
(PDB Homologues)
- Biological species
-
Spinacia oleracea (spinach)
- PDB
-
1yve
- ACETOHYDROXY ACID ISOMEROREDUCTASE COMPLEXED WITH NADPH, MAGNESIUM AND INHIBITOR IPOHA (N-HYDROXY-N-ISOPROPYLOXAMATE)
(1.65 Å)
- Catalytic CATH Domains
-
1.10.1040.10
3.40.50.720
(see all for 1yve)
- Cofactors
- Magnesium(2+) (2), Water (1)
Enzyme Reaction (EC:1.1.1.86)
+
→
+
+
Alternative enzyme names: 2-hydroxy-3-keto acid reductoisomerase, Alpha-keto-beta-hydroxylacyl reductoisomerase, Acetohydroxy acid isomeroreductase, Acetohydroxy acid reductoisomerase, Acetolactate reductoisomerase, Dihydroxyisovalerate (isomerizing) dehydrogenase, Dihydroxyisovalerate dehydrogenase (isomerizing), Isomeroreductase, Ketol acid reductoisomerase, Reductoisomerase, Ketol-acid reductoisomerase, (R)-2,3-dihydroxy-3-methylbutanoate:NADP(+) oxidoreductase (isomerizing),
Enzyme Mechanism
Introduction
The reaction is initiated by a proton abstraction (by the hydroxide ion bridging the two metal ions), followed by a concerted isomerisation and hydride transfer. The two protons needed to protonate the product are proposed to be donated from the bridging water ligand and a conserved lysine
Catalytic Residues Roles
| UniProt | PDB* (1yve) | ||
| Asp315 | Asp315(244)K(C) | Acts as a bridging ligand between the two divalent metal ions. | metal ligand |
| Glu319 | Glu319(248)K(C) | Forms part of the metal 1 binding site. | metal ligand |
| Lys252 | Lys252(181)K(C) | Proposed to act as the general acid/base in the reaction. | proton acceptor, proton donor |
*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, intramolecular rearrangement, overall reactant used, hydride transfer, native state of cofactor regenerated, overall product formed, inferred reaction step, native state of enzyme regeneratedReferences
- Tadrowski S et al. (2016), Chemistry, 22, 7427-7436. Metal Ions Play an Essential Catalytic Role in the Mechanism of Ketol-Acid Reductoisomerase. DOI:10.1002/chem.201600620. PMID:27136273.
- Leung EW et al. (2009), J Mol Biol, 389, 167-182. Conformational changes in a plant ketol-acid reductoisomerase upon Mg(2+) and NADPH binding as revealed by two crystal structures. DOI:10.1016/j.jmb.2009.04.012. PMID:19362563.
- Tyagi R et al. (2005), FEBS J, 272, 593-602. Probing the mechanism of the bifunctional enzyme ketol-acid reductoisomerase by site-directed mutagenesis of the active site. DOI:10.1111/j.1742-4658.2004.04506.x. PMID:15654896.
- Dumas R et al. (2001), Acc Chem Res, 34, 399-408. Enzymology, Structure, and Dynamics of Acetohydroxy Acid Isomeroreductase. DOI:10.1021/ar000082w. PMID:11352718.
- Thomazeau K et al. (2000), Acta Crystallogr D Biol Crystallogr, 56, 389-397. Structure of spinach acetohydroxyacid isomeroreductase complexed with its reaction product dihydroxymethylvalerate, manganese and (phospho)-ADP-ribose. PMID:10739911.
- Halgand F et al. (1999), Biochemistry, 38, 6025-6034. Characterization of the conformational changes of acetohydroxy acid isomeroreductase induced by the binding of Mg2+ ions, NADPH, and a competitive inhibitor. DOI:10.1021/bi982412e. PMID:10320328.
- Biou V et al. (1997), EMBO J, 16, 3405-3415. The crystal structure of plant acetohydroxy acid isomeroreductase complexed with NADPH, two magnesium ions and a herbicidal transition state analog determined at 1.65 A resolution. DOI:10.1093/emboj/16.12.3405. PMID:9218783.
- Dumas R et al. (1995), Biochemistry, 34, 6026-6036. Evidence for two catalytically different magnesium-binding sites in acetohydroxy acid isomeroreductase by site-directed mutagenesis. PMID:7742305.
- Mrachko GT et al. (1992), Arch Biochem Biophys, 294, 446-453. The pH dependence of the kinetic parameters of ketol acid reductoisomerase indicates a proton shuttle mechanism for alkyl migration. PMID:1567200.
- Aulabaugh A et al. (1990), Biochemistry, 29, 2824-2830. Oxalyl hydroxamates as reaction-intermediate analogues for ketol-acid reductoisomerase. PMID:2189496.
Step 1. A magnesium activated water molecule abstracts the proton from the alcohol, initiating a 1,2-alkyl shift with concomitant deprotonation of the conserved Lys252 residue.
Download: Image, Marvin FileCatalytic Residues Roles
| Residue | Roles |
|---|---|
| Asp315(244)K(C) | metal ligand |
| Glu319(248)K(C) | metal ligand |
| Lys252(181)K(C) | proton donor |
Chemical Components
proton transfer, intramolecular rearrangement, overall reactant used
Step 2. NADPH eliminates a hydride ion, that is added to the newly formed carbonyl group, which in turn abstracts the proton from the magnesium bound water molecule, regenerating the bound hydroxide ion.
Download: Image, Marvin FileCatalytic Residues Roles
| Residue | Roles |
|---|---|
| Asp315(244)K(C) | metal ligand |
| Glu319(248)K(C) | metal ligand |
Chemical Components
hydride transfer, proton transfer, native state of cofactor regenerated, overall product formed, overall reactant used
Step 3. In an inferred return step, water donates a proton to the lysine to regenerate the enzyme's active site.
Download: Image, Marvin FileCatalytic Residues Roles
| Residue | Roles |
|---|---|
| Asp315(244)K(C) | metal ligand |
| Glu319(248)K(C) | metal ligand |
| Lys252(181)K(C) | proton acceptor |
Download: