Crystallization (Comment) | Organism |
---|---|
wild-type and C92S/C264S/K166C mutant enzymes in complex with inhibitors benzilate, 3,3,3-trifluoro-2-hydroxy-2-(trifluoromethyl)-propanoate and tratronate, hanging drop vapor diffusion method, mixing of 0.002 ml of each protein and reservoir solution, the protein solution contains for 3,3,3-trifluoro-2-hydroxy-2-(trifluoromethyl)-propanoate-enzyme crystals 3.3 mM MgCl2, 1 mM inhibitor, and HEPES, 50 mM, pH 7.5, and for the reservoir solution 20% w/v, 120 mM glycine, 70 mM KNO3, and 0.1 M Tris-HCl, pH 8.0, for tartronate-enzyme crystals , 6.0 mg/ml protein, 3.3 mM MgCl2, 20 mM sodium tartronate, and 50 mM HEPES, pH 7.5, and 14% w/v PEG 1500 , 100 mM glycine, and 0.1 M triethanolamin, pH 8.5, as reservoir solution, and for mutant enzyme-benzilate crystals, mutant protein in 3.3 mM MgCl2, 20 mM benzilate, and 50 mM HEPES, pH 7.5, reservoir solution containing 15% w/v PEG 1500, 150 mM glycine, 50 mM NaCl, and 0.1 M HEPES, pH 7.5, equilibration against 0.5 ml reservoir solution, 21°C, and 50% humidity, X-ray diffraction structure determination and analysis at 1.68-1.89 A resolution | Pseudomonas putida |
Protein Variants | Comment | Organism |
---|---|---|
C92S/C264S/K166C | site-directed mutagenesis | Pseudomonas putida |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
(R)-lactate | - |
Pseudomonas putida | |
(R,S)-1-hydroxyethylphosphonate | transition state analogue inhibitor | Pseudomonas putida | |
(R,S)-2,2,2-trifluoro-1-hydroxyethylphosphonate | transition state analogue inhibitor | Pseudomonas putida | |
(R,S)-alpha-hydroxybenzylphosphonate | transition state analogue inhibitor | Pseudomonas putida | |
(S)-lactate | - |
Pseudomonas putida | |
3,3,3-trifluoro-2-hydroxy-2-(trifluoromethyl)-propanoate | a substrate-product analogue and a potent competitive inhibitor with both (R)-mandelate and (R)-trifluorolactate. The inhibitor exhibits a different binding mode with the two trifluoromethyl groups closely packed against the 20s loop and the carboxylate bridging the two active site Broensted acid-base catalysts Lys166 and His297 | Pseudomonas putida | |
alpha-hydroxyisobutyrate | substrate-product analogue inhibitor | Pseudomonas putida | |
benzilate | a competitive, substrate-product analogue inhibitor | Pseudomonas putida | |
Tartronate | competitive | Pseudomonas putida |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
1 | - |
(S)-Mandelate | pH 7.5, 25°C, recombinant wild-type enzyme | Pseudomonas putida | |
1.2 | - |
(R)-mandelate | pH 7.5, 25°C, recombinant wild-type enzyme | Pseudomonas putida |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Mg2+ | dependent on, active site bound | Pseudomonas putida |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Pseudomonas putida | P11444 | - |
- |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
(R)-lactate | - |
Pseudomonas putida | (S)-lactate | - |
? | |
(R)-mandelate | - |
Pseudomonas putida | (S)-mandelate | - |
? | |
(S)-lactate | - |
Pseudomonas putida | (R)-lactate | - |
? | |
(S)-mandelate | - |
Pseudomonas putida | (R)-mandelate | - |
? | |
(S)-trifluorolactate | - |
Pseudomonas putida | (R)-trifluorolactate | - |
? | |
additional information | mandelate racemase catalyzes the Mg2+-dependent 1,1-proton transfer that interconverts the enantiomers of mandelate | Pseudomonas putida | ? | - |
? |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
25 | - |
assay at | Pseudomonas putida |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.5 | - |
assay at | Pseudomonas putida |
Ki Value [mM] | Ki Value maximum [mM] | Inhibitor | Comment | Organism | Structure |
---|---|---|---|---|---|
0.0047 | - |
(R,S)-alpha-hydroxybenzylphosphonate | pH 7.5, 25°C, recombinant wild-type enzyme | Pseudomonas putida | |
0.027 | - |
3,3,3-trifluoro-2-hydroxy-2-(trifluoromethyl)-propanoate | pH 7.5, 25°C, recombinant wild-type enzyme | Pseudomonas putida | |
0.67 | - |
benzilate | pH 7.5, 25°C, recombinant wild-type enzyme | Pseudomonas putida | |
0.67 | - |
(R,S)-2,2,2-trifluoro-1-hydroxyethylphosphonate | pH 7.5, 25°C, recombinant wild-type enzyme | Pseudomonas putida | |
1.8 | - |
Tartronate | pH 7.5, 25°C, recombinant wild-type enzyme | Pseudomonas putida | |
5.5 | - |
alpha-hydroxyisobutyrate | pH 7.5, 25°C, recombinant wild-type enzyme | Pseudomonas putida | |
26.1 | - |
(S)-lactate | pH 7.5, 25°C, recombinant wild-type enzyme | Pseudomonas putida | |
32 | - |
(R)-lactate | pH 7.5, 25°C, recombinant wild-type enzyme | Pseudomonas putida | |
40.2 | - |
(R,S)-1-hydroxyethylphosphonate | pH 7.5, 25°C, recombinant wild-type enzyme | Pseudomonas putida |
General Information | Comment | Organism |
---|---|---|
additional information | the enzyme's 20s loop likely undergoes a significant conformational change upon binding (R)-mandelate | Pseudomonas putida |