Literature summary for 5.1.2.2 extracted from

Nagar, M.; Lietzan, A.D.; St Maurice, M.; Bearne, S.L.
Potent inhibition of mandelate racemase by a fluorinated substrate-product analogue with a novel binding mode (2014), Biochemistry, 53, 1169-1178.

Crystallization (Commentary)

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

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

Protein Variants	Comment	Organism
C92S/C264S/K166C	site-directed mutagenesis	Pseudomonas putida

Inhibitors

Inhibitors	Comment	Organism
(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 [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

Metals/Ions	Comment	Organism	Structure
Mg2+	dependent on, active site bound	Pseudomonas putida

Organism

Organism	UniProt	Comment	Textmining
Pseudomonas putida	P11444	-	-

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
(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 [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
25	-	assay at	Pseudomonas putida

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7.5	-	assay at	Pseudomonas putida

Ki Value [mM]

Ki Value [mM]	Ki Value maximum [mM]	Inhibitor	Comment	Organism
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

General Information	Comment	Organism
additional information	the enzyme's 20s loop likely undergoes a significant conformational change upon binding (R)-mandelate	Pseudomonas putida