Literature summary for 4.1.3.17 extracted from

Mazurkewich, S.; Seah, S.Y.
Investigation into the mode of phosphate activation in the 4-hydroxy-4-methyl-2-oxoglutarate/4-carboxy-4-hydroxy-2-oxoadipate aldolase from Pseudomonas putida F1 (2016), PLoS ONE, 11, e0164556 .

Activating Compound

Activating Compound	Comment	Organism	Structure
additional information	neither 2 mM Na2SO4 nor Na2MoO4 activate the enzyme	Pseudomonas putida
phosphate	the enzyme is uncompetitively activated in the presence of phosphate	Pseudomonas putida

Protein Variants

Protein Variants	Comment	Organism
G144V	the variant has a 10fold increase in KM and a 73fold decrease in kcat for the 4-hydroxy-4-methyl-2-oxoglutarate cleavage reaction and the kcat increases 2-fold in the presence of phosphate	Pseudomonas putida
H75A	the variant has a 2fold increase in KM and a 1.4fold increase in kcat for the 4-hydroxy-4-methyl-2-oxoglutarate catalyzed reaction and does not have a significant effect on the pyruvate methyl proton exchange rate compared to the wild type enzyme	Pseudomonas putida
K147A	the mutation has significant effects on the steady state kinetics of the aldolase 4-hydroxy-4-methyl-2-oxoglutarate reaction with decreasing the kcat by 1114fold compared to the wild type enzyme	Pseudomonas putida
N71A	the mutation has significant effects on the steady state kinetics of the aldolase 4-hydroxy-4-methyl-2-oxoglutarate reaction with decreasing the kcat by 1114fold compared to the wild type enzyme	Pseudomonas putida
R123K	the mutation reduces the kcat of the 4-hydroxy-4-methyl-2-oxoglutarate catalyzed reaction by 181fold and reduces the pyruvate methyl proton exchange rate by 20fold compared to the wild type enzyme	Pseudomonas putida
R195A	the substitution moderately effects the 4-hydroxy-4-methyl-2-oxoglutarate cleavage reaction with a 7fold increase in KM and a 2fold decrease in kcat. The activation of kcat and the pyruvate methyl proton exchange rate by Pi is each reduced by 5fold	Pseudomonas putida
R40A	the variant has a 2fold decrease in kcat and is still activated to a similar extent as the wild type enzyme in the 4-hydroxy-4-methyl-2-oxoglutarate aldolase cleavage reaction	Pseudomonas putida
T145A	the variant is activated in the presence of phosphate resulting in a 12fold increase in kcat for the 4-hydroxy-4-methyl-2-oxoglutarate catalyzed reaction and a 24fold increase in the pyruvate methyl proton exchange rate compared to the wild type enzyme	Pseudomonas putida

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
0.015	-	4-carboxy-4-hydroxy-2-oxoadipate	wild type enzyme, in the absence of phosphate, at pH 8.0 and 25°C	Pseudomonas putida
0.104	-	4-carboxy-4-hydroxy-2-oxoadipate	wild type enzyme, in the presence of 2 mM phosphate, at pH 8.0 and 25°C	Pseudomonas putida
0.188	-	4-hydroxy-4-methyl-2-oxoglutarate	wild type enzyme, in the absence of phosphate, at pH 8.0 and 25°C	Pseudomonas putida
0.298	-	oxaloacetate	wild type enzyme, in the absence of phosphate, at pH 8.0 and 25°C	Pseudomonas putida
0.304	-	4-hydroxy-4-methyl-2-oxoglutarate	mutant enzyme R40A, in the absence of phosphate, at pH 8.0 and 25°C	Pseudomonas putida
0.389	-	4-hydroxy-4-methyl-2-oxoglutarate	mutant enzyme H75A, in the absence of phosphate, at pH 8.0 and 25°C	Pseudomonas putida
0.741	-	oxaloacetate	wild type enzyme, in the presence of 2 mM phosphate, at pH 8.0 and 25°C	Pseudomonas putida
0.796	-	4-hydroxy-4-methyl-2-oxoglutarate	mutant enzyme N71A, in the absence of phosphate, at pH 8.0 and 25°C	Pseudomonas putida
1.08	-	4-hydroxy-4-methyl-2-oxoglutarate	mutant enzyme T145A, in the absence of phosphate, at pH 8.0 and 25°C	Pseudomonas putida
1.11	-	4-hydroxy-4-methyl-2-oxoglutarate	mutant enzyme N71A, in the presence of 2 mM phosphate, at pH 8.0 and 25°C	Pseudomonas putida
1.21	-	4-hydroxy-4-methyl-2-oxoglutarate	mutant enzyme H75A, in the presence of 2 mM phosphate, at pH 8.0 and 25°C	Pseudomonas putida
1.36	-	4-hydroxy-4-methyl-2-oxoglutarate	wild type enzyme, in the presence of 2 mM phosphate, at pH 8.0 and 25°C	Pseudomonas putida
1.37	-	4-hydroxy-4-methyl-2-oxoglutarate	mutant enzyme R195A, in the absence of phosphate, at pH 8.0 and 25°C	Pseudomonas putida
1.39	-	4-hydroxy-4-methyl-2-oxoglutarate	mutant enzyme G144V, in the absence of phosphate, at pH 8.0 and 25°C	Pseudomonas putida
1.78	-	4-hydroxy-4-methyl-2-oxoglutarate	mutant enzyme T145A, in the presence of 2 mM phosphate, at pH 8.0 and 25°C	Pseudomonas putida
2.01	-	4-hydroxy-4-methyl-2-oxoglutarate	mutant enzyme K147A, in the absence of phosphate, at pH 8.0 and 25°C	Pseudomonas putida
2.89	-	4-hydroxy-4-methyl-2-oxoglutarate	mutant enzyme R195A, in the presence of 2 mM phosphate, at pH 8.0 and 25°C	Pseudomonas putida
3.21	-	4-hydroxy-4-methyl-2-oxoglutarate	mutant enzyme R123K, in the absence of phosphate, at pH 8.0 and 25°C	Pseudomonas putida
3.26	-	4-hydroxy-4-methyl-2-oxoglutarate	mutant enzyme R40A, in the presence of 2 mM phosphate, at pH 8.0 and 25°C	Pseudomonas putida
3.72	-	4-hydroxy-4-methyl-2-oxoglutarate	mutant enzyme K147A, in the presence of 2 mM phosphate, at pH 8.0 and 25°C	Pseudomonas putida
3.97	-	4-hydroxy-4-methyl-2-oxoglutarate	mutant enzyme R123K, in the presence of 2 mM phosphate, at pH 8.0 and 25°C	Pseudomonas putida
6.83	-	4-hydroxy-4-methyl-2-oxoglutarate	mutant enzyme G144V, in the presence of 2 mM phosphate, at pH 8.0 and 25°C	Pseudomonas putida

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
4-carboxy-4-hydroxy-2-oxoadipate	Pseudomonas putida	-	oxaloacetate + pyruvate	-	r
4-hydroxy-4-methyl-2-oxoglutarate	Pseudomonas putida	-	2 pyruvate	-	r

Organism

Organism	UniProt	Comment	Textmining
Pseudomonas putida	A5W059	-	-

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
4-carboxy-4-hydroxy-2-oxoadipate	-	Pseudomonas putida	oxaloacetate + pyruvate	-	r
4-hydroxy-4-methyl-2-oxoglutarate	-	Pseudomonas putida	2 pyruvate	-	r
oxaloacetate + pyruvate	-	Pseudomonas putida	4-carboxy-4-hydroxy-2-oxoadipate	-	r

Synonyms

Synonyms	Comment	Organism
4-hydroxy-4-methyl-2-oxoglutarate/4-carboxy-4-hydroxy-2-oxoadipate aldolase	-	Pseudomonas putida
HMG/CHA aldolase	-	Pseudomonas putida

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
0.014	-	4-hydroxy-4-methyl-2-oxoglutarate	mutant enzyme K147A, in the absence of phosphate, at pH 8.0 and 25°C	Pseudomonas putida
0.014	-	4-hydroxy-4-methyl-2-oxoglutarate	mutant enzyme N71A, in the absence of phosphate, at pH 8.0 and 25°C	Pseudomonas putida
0.0184	-	4-hydroxy-4-methyl-2-oxoglutarate	mutant enzyme N71A, in the presence of 2 mM phosphate, at pH 8.0 and 25°C	Pseudomonas putida
0.0224	-	4-hydroxy-4-methyl-2-oxoglutarate	mutant enzyme K147A, in the presence of 2 mM phosphate, at pH 8.0 and 25°C	Pseudomonas putida
0.07	-	4-hydroxy-4-methyl-2-oxoglutarate	mutant enzyme R123K, in the presence of 2 mM phosphate, at pH 8.0 and 25°C	Pseudomonas putida
0.086	-	4-hydroxy-4-methyl-2-oxoglutarate	mutant enzyme R123K, in the absence of phosphate, at pH 8.0 and 25°C	Pseudomonas putida
0.215	-	4-hydroxy-4-methyl-2-oxoglutarate	mutant enzyme G144V, in the absence of phosphate, at pH 8.0 and 25°C	Pseudomonas putida
0.394	-	4-hydroxy-4-methyl-2-oxoglutarate	mutant enzyme G144V, in the presence of 2 mM phosphate, at pH 8.0 and 25°C	Pseudomonas putida
0.889	-	4-hydroxy-4-methyl-2-oxoglutarate	mutant enzyme T145A, in the absence of phosphate, at pH 8.0 and 25°C	Pseudomonas putida
1.86	-	oxaloacetate	wild type enzyme, in the absence of phosphate, at pH 8.0 and 25°C	Pseudomonas putida
3.16	-	oxaloacetate	wild type enzyme, in the presence of 2 mM phosphate, at pH 8.0 and 25°C	Pseudomonas putida
6.7	-	4-hydroxy-4-methyl-2-oxoglutarate	mutant enzyme R40A, in the absence of phosphate, at pH 8.0 and 25°C	Pseudomonas putida
6.72	-	4-hydroxy-4-methyl-2-oxoglutarate	mutant enzyme R195A, in the absence of phosphate, at pH 8.0 and 25°C	Pseudomonas putida
10.7	-	4-hydroxy-4-methyl-2-oxoglutarate	mutant enzyme T145A, in the presence of 2 mM phosphate, at pH 8.0 and 25°C	Pseudomonas putida
11.5	-	4-hydroxy-4-methyl-2-oxoglutarate	mutant enzyme R195A, in the presence of 2 mM phosphate, at pH 8.0 and 25°C	Pseudomonas putida
13.4	-	4-carboxy-4-hydroxy-2-oxoadipate	wild type enzyme, in the absence of phosphate, at pH 8.0 and 25°C	Pseudomonas putida
15.6	-	4-hydroxy-4-methyl-2-oxoglutarate	wild type enzyme, in the absence of phosphate, at pH 8.0 and 25°C	Pseudomonas putida
22.3	-	4-hydroxy-4-methyl-2-oxoglutarate	mutant enzyme H75A, in the absence of phosphate, at pH 8.0 and 25°C	Pseudomonas putida
36.6	-	4-hydroxy-4-methyl-2-oxoglutarate	mutant enzyme R40A, in the presence of 2 mM phosphate, at pH 8.0 and 25°C	Pseudomonas putida
53.5	-	4-hydroxy-4-methyl-2-oxoglutarate	mutant enzyme H75A, in the presence of 2 mM phosphate, at pH 8.0 and 25°C	Pseudomonas putida
134	-	4-hydroxy-4-methyl-2-oxoglutarate	wild type enzyme, in the presence of 2 mM phosphate, at pH 8.0 and 25°C	Pseudomonas putida
145	-	4-carboxy-4-hydroxy-2-oxoadipate	wild type enzyme, in the presence of 2 mM phosphate, at pH 8.0 and 25°C	Pseudomonas putida

kcat/KM [mM/s]

kcat/KM Value [1/mMs^-1]	kcat/KM Value Maximum [1/mMs^-1]	Substrate	Comment	Organism
0.00645	-	4-hydroxy-4-methyl-2-oxoglutarate	mutant enzyme K147A, in the presence of 2 mM phosphate, at pH 8.0 and 25°C	Pseudomonas putida
0.00697	-	4-hydroxy-4-methyl-2-oxoglutarate	mutant enzyme K147A, in the absence of phosphate, at pH 8.0 and 25°C	Pseudomonas putida
0.0165	-	4-hydroxy-4-methyl-2-oxoglutarate	mutant enzyme N71A, in the presence of 2 mM phosphate, at pH 8.0 and 25°C	Pseudomonas putida
0.0176	-	4-hydroxy-4-methyl-2-oxoglutarate	mutant enzyme N71A, in the absence of phosphate, at pH 8.0 and 25°C	Pseudomonas putida
0.0176	-	4-hydroxy-4-methyl-2-oxoglutarate	mutant enzyme R123K, in the presence of 2 mM phosphate, at pH 8.0 and 25°C	Pseudomonas putida
0.0267	-	4-hydroxy-4-methyl-2-oxoglutarate	mutant enzyme R123K, in the absence of phosphate, at pH 8.0 and 25°C	Pseudomonas putida
0.0577	-	4-hydroxy-4-methyl-2-oxoglutarate	mutant enzyme G144V, in the presence of 2 mM phosphate, at pH 8.0 and 25°C	Pseudomonas putida
0.155	-	4-hydroxy-4-methyl-2-oxoglutarate	mutant enzyme G144V, in the absence of phosphate, at pH 8.0 and 25°C	Pseudomonas putida
0.823	-	4-hydroxy-4-methyl-2-oxoglutarate	mutant enzyme T145A, in the absence of phosphate, at pH 8.0 and 25°C	Pseudomonas putida
3.98	-	4-hydroxy-4-methyl-2-oxoglutarate	mutant enzyme R195A, in the presence of 2 mM phosphate, at pH 8.0 and 25°C	Pseudomonas putida
4.26	-	oxaloacetate	wild type enzyme, in the presence of 2 mM phosphate, at pH 8.0 and 25°C	Pseudomonas putida
4.91	-	4-hydroxy-4-methyl-2-oxoglutarate	mutant enzyme R195A, in the absence of phosphate, at pH 8.0 and 25°C	Pseudomonas putida
6.01	-	4-hydroxy-4-methyl-2-oxoglutarate	mutant enzyme T145A, in the presence of 2 mM phosphate, at pH 8.0 and 25°C	Pseudomonas putida
6.24	-	oxaloacetate	wild type enzyme, in the absence of phosphate, at pH 8.0 and 25°C	Pseudomonas putida
11.2	-	4-hydroxy-4-methyl-2-oxoglutarate	mutant enzyme R40A, in the presence of 2 mM phosphate, at pH 8.0 and 25°C	Pseudomonas putida
22	-	4-hydroxy-4-methyl-2-oxoglutarate	mutant enzyme R40A, in the absence of phosphate, at pH 8.0 and 25°C	Pseudomonas putida
44.5	-	4-hydroxy-4-methyl-2-oxoglutarate	mutant enzyme H75A, in the presence of 2 mM phosphate, at pH 8.0 and 25°C	Pseudomonas putida
57.3	-	4-hydroxy-4-methyl-2-oxoglutarate	mutant enzyme H75A, in the absence of phosphate, at pH 8.0 and 25°C	Pseudomonas putida
83	-	4-hydroxy-4-methyl-2-oxoglutarate	wild type enzyme, in the absence of phosphate, at pH 8.0 and 25°C	Pseudomonas putida
98.5	-	4-hydroxy-4-methyl-2-oxoglutarate	wild type enzyme, in the presence of 2 mM phosphate, at pH 8.0 and 25°C	Pseudomonas putida
893	-	4-carboxy-4-hydroxy-2-oxoadipate	wild type enzyme, in the absence of phosphate, at pH 8.0 and 25°C	Pseudomonas putida
1400	-	4-carboxy-4-hydroxy-2-oxoadipate	wild type enzyme, in the presence of 2 mM phosphate, at pH 8.0 and 25°C	Pseudomonas putida