Cloned (Comment) | Organism |
---|---|
overexpression in Escherichia coli strain BL21(DE3) | Oryza sativa |
Crystallization (Comment) | Organism |
---|---|
enzyme KARI in complex with Mg2+ or with Mg2+ and NADPH, hanging-drop method by vaporphase diffusion at 18°C, 0.003 ml of protein solution containing 6 mg/ml enzyme, 50 mM Hepes, pH 7.5, 5 mM NADPH, and 5 mM MgCl2, is mixed with 0.001 ml of reservoir solution containing 0.2 M magnesium chloride hexahydrate, 0.1 M Tris-HCl, pH 8.5, and 15% w/v PEG 4000, a few days to 3 months, X-ray diffraction structure determination and analysis at 1.55 A and 2.80 A resolution, respectively, molecular replacement | Oryza sativa |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
ethylene glycol | exhibits competitive and uncompetitive inhibition | Oryza sativa |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Mg2+ | required for the alkyl migration reaction, Km is 0.0072 mM, binding structure, overview | Oryza sativa | |
additional information | the enzyme catalyzes a two-step reaction: an alkyl migration that requires Mg2+, and a reduction reaction involving NADPH. For the reduction reaction, a divalent metal ion is also required, with any of Mg2+, Mn2+, Co2+, or Ni2+ utilized in this role | Oryza sativa |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
(R)-2,3-dihydroxy-3-methylbutanoate + NADP+ | Oryza sativa | - |
(S)-2-hydroxy-2-methyl-3-oxobutanoate + NADPH + H+ | - |
r |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Oryza sativa | Q65XK0 | - |
- |
Reaction | Comment | Organism | Reaction ID |
---|---|---|---|
(2R)-2,3-dihydroxy-3-methylbutanoate + NADP+ = (2S)-2-hydroxy-2-methyl-3-oxobutanoate + NADPH + H+ | active site structure and catalytic mechanism, mechanism of induced fit, overview | Oryza sativa |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
(R)-2,3-dihydroxy-3-methylbutanoate + NADP+ | - |
Oryza sativa | (S)-2-hydroxy-2-methyl-3-oxobutanoate + NADPH + H+ | - |
r | |
(R)-2,3-dihydroxy-3-methylbutanoate + NADP+ | conformational changes in a plant ketol-acid reductoisomerase upon Mg2+ and NADPH binding, overview | Oryza sativa | (S)-2-hydroxy-2-methyl-3-oxobutanoate + NADPH + H+ | - |
r | |
2-aceto-2-hydroxybutyrate + NADP+ | - |
Oryza sativa | (2R,3R)-2,3-dihydroxy-3-methylvalerate + NADPH + H+ | - |
r | |
2-acetolactate + NADP+ | - |
Oryza sativa | (2R)-2,3-dihydroxy-3-isovalerate + NADPH + H+ | - |
r | |
additional information | KARI catalyzes two reactions: alkyl migration and reduction. Structure-function relationship, effects of ligand binding, MG2+ and NADPH, on the enzyme conformation, overview | Oryza sativa | ? | - |
? |
Subunits | Comment | Organism |
---|---|---|
dimer | structure-function relationship, effects of ligand, Mg2+ and NADPH, binding on the enzyme conformation, overview | Oryza sativa |
Synonyms | Comment | Organism |
---|---|---|
KARI | - |
Oryza sativa |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
30 | - |
assay at | Oryza sativa |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
8 | - |
assay at | Oryza sativa |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
NADP+ | - |
Oryza sativa | |
NADPH | required, the majority of the NADP+ molecule binds to the N-domain of KARI with polar contacts with 11 residues, including W133, G134, S135, Q136, R162, S165, S167, S201, D202, Q205, and G254. There are also contacts with the C-domain, although these are much fewer and involve only the phosphoryl oxygen atoms and the oxygen and nitrogen atoms of the nicotinamide group in NADPH, forming contacts with C517, S518, and T519, and with R589 in the polypeptide | Oryza sativa |
Ki Value [mM] | Ki Value maximum [mM] | Inhibitor | Comment | Organism | Structure |
---|---|---|---|---|---|
0.0025 | - |
ethylene glycol | pH 8.0, 30°C, recombinant enzyme, competitive mode | Oryza sativa | |
0.0032 | - |
ethylene glycol | pH 8.0, 30°C, recombinant enzyme, uncompetitive mode | Oryza sativa |
General Information | Comment | Organism |
---|---|---|
metabolism | the enzyme is involved in the branched-chain amino acid biosynthesis pathway | Oryza sativa |