Literature summary for 3.1.2.B5 extracted from

Song, F.; Zhuang, Z.; Finci, L.; Dunaway-Mariano, D.; Kniewel, R.; Buglino, J.A.; Solorzano, V.; Wu, J.; Lima, C.D.
Structure, function, and mechanism of the phenylacetate pathway hot dog-fold thioesterase PaaI (2006), J. Biol. Chem., 281, 11028-11038.

Crystallization (Commentary)

Crystallization (Comment)	Organism
C-terminal His-tagged enzyme is prepared for crystallization. The N-terminal Met is replaced by Ser-Leu. Crystals of the selenomethionine enzyme are grown in 13% polyethylene glycol, 0.1 M [bis(2-hydroxyethyl)imino]-tris(hydroxymethyl)methane (pH 6.0) and 0.2 M lithium sulfate at 18°C using the hanging drop vapor diffusion method. They are incubated in the presence of 30% glycerol before being flash-frozen in liquid nitrogen. The crystals belong to trigonal space group P3(1)21 with a = b = 69.9 A and c = 117.2 A	Escherichia coli

Crystallization (Comment)

Organism

C-terminal His-tagged enzyme is prepared for crystallization. The N-terminal Met is replaced by Ser-Leu. Crystals of the selenomethionine enzyme are grown in 13% polyethylene glycol, 0.1 M [bis(2-hydroxyethyl)imino]-tris(hydroxymethyl)methane (pH 6.0) and 0.2 M lithium sulfate at 18°C using the hanging drop vapor diffusion method. They are incubated in the presence of 30% glycerol before being flash-frozen in liquid nitrogen. The crystals belong to trigonal space group P3(1)21 with a = b = 69.9 A and c = 117.2 A

Escherichia coli

Protein Variants

Protein Variants	Comment	Organism
D16A	significantly reduced activity	Escherichia coli
D61A	small but detectable level of catalytic activity	Escherichia coli
D75A	no activity	Aromatoleum evansii
E14A	reduced activity	Escherichia coli
H52A	1000fold decrease in the kcat/Km is observed with the 4-hydroxyphenylacetyl-CoA, and a 100fold decrease in the kcat/Km value is observed with the 3-hydroxyphenylacetyl-CoA or 3,4-dihydroxyphenylacetyl-CoA	Escherichia coli
N15A	reduced activity	Escherichia coli
N46A	significantly reduced activity	Escherichia coli
N46Q/D61E	no activity	Escherichia coli
N60A	significantly reduced activity	Aromatoleum evansii
N60D	significantly reduced activity	Aromatoleum evansii

Inhibitors

Inhibitors	Comment	Organism	Structure
3-hydroxyphenylacyl-CoA	-	Aromatoleum evansii
4-hydroxyphenylacyl-CoA	competitive	Aromatoleum evansii

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
0.0086	-	3,4-dihydroxyphenylacetyl-CoA	pH 8.1, 25°C, mutant enzyme N60A	Aromatoleum evansii
0.0096	-	phenylacetyl-CoA	pH 8.1, 25°C, wild-type enzyme	Escherichia coli
0.016	-	3,4-dihydroxyphenylacetyl-CoA	pH 8.1, 25°C, wild-type enzyme	Escherichia coli
0.018	-	4-hydroxyphenylacetyl-CoA	pH 8.1, 25°C, mutant enzyme N15A	Escherichia coli
0.021	-	3-hydroxyphenylacetyl-CoA	pH 8.1, 25°C, wild-type enzyme	Escherichia coli
0.024	-	3,4-dihydroxyphenylacetyl-CoA	pH 8.1, 25°C, mutant enzyme N46A	Escherichia coli
0.028	-	4-hydroxyphenylacetyl-CoA	pH 8.1, 25°C, mutant enzyme H52A	Escherichia coli
0.028	-	3,5-dihydroxyphenylacetyl-CoA	pH 8.1, 25°C, wild-type enzyme	Aromatoleum evansii
0.032	-	3-hydroxyphenylacetyl-CoA	pH 8.1, 25°C, mutant enzyme H52A	Escherichia coli
0.033	-	3,4-dihydroxyphenylacetyl-CoA	pH 8.1, 25°C, mutant enzyme D16A	Escherichia coli
0.034	-	3-hydroxyphenylacetyl-CoA	pH 8.1, 25°C, mutant enzyme N15A	Escherichia coli
0.035	-	4-hydroxyphenylacetyl-CoA	pH 8.1, 25°C, wild-type enzyme	Escherichia coli
0.045	-	3,4-dihydroxyphenylacetyl-CoA	pH 8.1, 25°C, mutant enzyme H52A	Escherichia coli
0.047	-	3,4-dihydroxyphenylacetyl-CoA	pH 8.1, 25°C, mutant enzyme N15A	Escherichia coli
0.048	-	3,4-dihydroxyphenylacetyl-CoA	pH 8.1, 25°C, mutant enzyme D75A	Aromatoleum evansii
0.051	-	3,4-dihydroxyphenylacetyl-CoA	pH 8.1, 25°C, wild-type enzyme	Aromatoleum evansii
0.055	-	4-hydroxyphenylacetyl-CoA	pH 8.1, 25°C, wild-type enzyme	Aromatoleum evansii
0.055	-	3,4-dihydroxyphenylacetyl-CoA	pH 8.1, 25°C, mutant enzyme N60D	Aromatoleum evansii
0.08	-	3-hydroxyphenylacetyl-CoA	pH 8.1, 25°C, mutant enzyme E14A	Escherichia coli
0.08	-	3-hydroxyphenylacetyl-CoA	pH 8.1, 25°C, wild-type enzyme	Aromatoleum evansii
0.09	-	4-hydroxyphenylacetyl-CoA	pH 8.1, 25°C, mutant enzyme E14A	Escherichia coli
0.09	-	3-hydroxyphenylacetyl-CoA	pH 8.1, 25°C, mutant enzyme D16A	Escherichia coli
0.13	-	3,4-dihydroxyphenylacetyl-CoA	pH 8.1, 25°C, mutant enzyme E14A	Escherichia coli
0.2	-	3,5-dihydroxyphenylacetyl-CoA	pH 8.1, 25°C, wild-type enzyme	Escherichia coli
0.23	-	3-hydroxyphenylacetyl-CoA	pH 8.1, 25°C, mutant enzyme N46A	Escherichia coli
0.25	-	4-hydroxyphenylacetyl-CoA	pH 8.1, 25°C, mutant enzyme D16A	Escherichia coli
0.28	-	4-hydroxyphenylacetyl-CoA	pH 8.1, 25°C, mutant enzyme N46A	Escherichia coli
0.39	-	phenylacetyl-CoA	pH 8.1, 25°C, wild-type enzyme	Aromatoleum evansii

Molecular Weight [Da]

Molecular Weight [Da]	Molecular Weight Maximum [Da]	Comment	Organism
16419	-	4 * 16419, ESI mass spectrometry	Aromatoleum evansii
58000	-	gel filtration	Escherichia coli
67000	-	gel filtration	Aromatoleum evansii

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.
3,4-dihydroxyphenylacetyl-CoA + H2O	Escherichia coli	the enzyme can liberate CoA from phenylacetyl-CoA and from hydroxyphenylacetyl-CoA substrates that are formed in the phenylacetate pathway	3,4-dihydroxyphenylacetate + CoA	-	?
3,4-dihydroxyphenylacetyl-CoA + H2O	Aromatoleum evansii	the enzyme can liberate CoA from phenylacetyl-CoA and from hydroxyphenylacetyl-CoA substrates that form from the phenylacetate pathway	3,4-dihydroxyphenylacetate + CoA	-	?
3,4-dihydroxyphenylacetyl-CoA + H2O	Aromatoleum evansii DSM 6898	the enzyme can liberate CoA from phenylacetyl-CoA and from hydroxyphenylacetyl-CoA substrates that form from the phenylacetate pathway	3,4-dihydroxyphenylacetate + CoA	-	?

Organism

Organism	UniProt	Comment	Textmining
Aromatoleum evansii	Q9F9V0	-	-
Aromatoleum evansii DSM 6898	Q9F9V0	-	-
Escherichia coli	P76084	-	-

Storage Stability

Storage Stability	Organism
-80°C, stable for storage in 10 mM K+/Hepes buffer (pH 7.5) or in 10 mM Tris buffer (pH 8.1) containing 10 mM KCl for several months	Aromatoleum evansii
0°C, pH 8.1, stored in 10 mM Tris buffer for several weeks without significant loss of activity	Escherichia coli

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
3,4-dihydroxyphenylacetyl-CoA + H2O	the enzyme can liberate CoA from phenylacetyl-CoA and from hydroxyphenylacetyl-CoA substrates that are formed in the phenylacetate pathway	Escherichia coli	3,4-dihydroxyphenylacetate + CoA	-	?
3,4-dihydroxyphenylacetyl-CoA + H2O	the enzyme can liberate CoA from phenylacetyl-CoA and from hydroxyphenylacetyl-CoA substrates that form from the phenylacetate pathway	Aromatoleum evansii	3,4-dihydroxyphenylacetate + CoA	-	?
3,4-dihydroxyphenylacetyl-CoA + H2O	-	Escherichia coli	3,4-dihydroxyphenylacetate + CoA	-	?
3,4-dihydroxyphenylacetyl-CoA + H2O	-	Aromatoleum evansii	3,4-dihydroxyphenylacetate + CoA	-	?
3,4-dihydroxyphenylacetyl-CoA + H2O	the enzyme can liberate CoA from phenylacetyl-CoA and from hydroxyphenylacetyl-CoA substrates that form from the phenylacetate pathway	Aromatoleum evansii DSM 6898	3,4-dihydroxyphenylacetate + CoA	-	?
3,4-dihydroxyphenylacetyl-CoA + H2O	-	Aromatoleum evansii DSM 6898	3,4-dihydroxyphenylacetate + CoA	-	?
3,5-dihydroxyphenylacetyl-CoA + H2O	-	Escherichia coli	3,5-dihydroxyphenylacetate + CoA	-	?
3,5-dihydroxyphenylacetyl-CoA + H2O	-	Aromatoleum evansii	3,5-dihydroxyphenylacetate + CoA	-	?
3,5-dihydroxyphenylacetyl-CoA + H2O	-	Aromatoleum evansii DSM 6898	3,5-dihydroxyphenylacetate + CoA	-	?
3-hydroxyphenylacetyl-CoA + H2O	-	Escherichia coli	3-hydroxyphenylacetate + CoA	-	?
3-hydroxyphenylacetyl-CoA + H2O	-	Aromatoleum evansii	3-hydroxyphenylacetate + CoA	-	?
3-hydroxyphenylacetyl-CoA + H2O	-	Aromatoleum evansii DSM 6898	3-hydroxyphenylacetate + CoA	-	?
4-hydroxyphenylacetyl-CoA + H2O	-	Escherichia coli	4-hydroxyphenylacetate + CoA	-	?
4-hydroxyphenylacetyl-CoA + H2O	-	Aromatoleum evansii	4-hydroxyphenylacetate + CoA	-	?
4-hydroxyphenylacetyl-CoA + H2O	-	Aromatoleum evansii DSM 6898	4-hydroxyphenylacetate + CoA	-	?
additional information	no activity with 4-hydroxybenzoyl-CoA, 4-hydroxybenzyl-CoA, 4-hydroxyphenacyl-CoA	Escherichia coli	?	-	?
additional information	no activity with 4-hydroxybenzoyl-CoA, 3-hydroxybenzoyl-CoA, 4-hydroxybenzyl-CoA, 4-hydroxyphenacyl-CoA, 3-hydroxyphenacyl-CoA, 4-chlorobenzoyl-CoA, 4-methoxybenzoyl-CoA. Low activity with acetyl-CoA, n-hexanoyl-CoA, n-butyryl-CoA, isobutyryl-CoA, beta-hydroxybutyryl-CoA, methylmalonyl-CoA, crotonyl-CoA	Aromatoleum evansii	?	-	?
phenylacetyl-CoA + H2O	-	Escherichia coli	phenylacetate + CoA	-	?
phenylacetyl-CoA + H2O	-	Aromatoleum evansii	phenylacetate + CoA	-	?

Subunits

Subunits	Comment	Organism
homotetramer	4 *14720, ESI mass spectrometry	Escherichia coli
homotetramer	4 * 16419, ESI mass spectrometry	Aromatoleum evansii

Synonyms

Synonyms	Comment	Organism
PaaI	-	Escherichia coli
PaaI	-	Aromatoleum evansii

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
0.0065	-	3,4-dihydroxyphenylacetyl-CoA	pH 8.1, 25°C, mutant enzyme D75A	Aromatoleum evansii
0.012	-	3,4-dihydroxyphenylacetyl-CoA	pH 8.1, 25°C, mutant enzyme N60A	Aromatoleum evansii
0.065	-	3-hydroxyphenylacetyl-CoA	pH 8.1, 25°C, mutant enzyme D16A	Escherichia coli
0.076	-	3,4-dihydroxyphenylacetyl-CoA	pH 8.1, 25°C, mutant enzyme N46A	Escherichia coli
0.11	-	3,4-dihydroxyphenylacetyl-CoA	pH 8.1, 25°C, mutant enzyme D16A	Escherichia coli
0.17	-	4-hydroxyphenylacetyl-CoA	pH 8.1, 25°C, mutant enzyme D16A	Escherichia coli
0.18	-	3,4-dihydroxyphenylacetyl-CoA	pH 8.1, 25°C, mutant enzyme N60D	Aromatoleum evansii
0.26	-	4-hydroxyphenylacetyl-CoA	pH 8.1, 25°C, mutant enzyme H52A	Escherichia coli
0.31	-	4-hydroxyphenylacetyl-CoA	pH 8.1, 25°C, mutant enzyme N46A	Escherichia coli
0.41	-	phenylacetyl-CoA	pH 8.1, 25°C, wild-type enzyme	Escherichia coli
0.81	-	3-hydroxyphenylacetyl-CoA	pH 8.1, 25°C, mutant enzyme N46A	Escherichia coli
0.89	-	3-hydroxyphenylacetyl-CoA	pH 8.1, 25°C, mutant enzyme H52A	Escherichia coli
2.3	-	3,5-dihydroxyphenylacetyl-CoA	pH 8.1, 25°C, wild-type enzyme	Aromatoleum evansii
2.7	-	3,4-dihydroxyphenylacetyl-CoA	pH 8.1, 25°C, mutant enzyme H52A	Escherichia coli
3.2	-	4-hydroxyphenylacetyl-CoA	pH 8.1, 25°C, mutant enzyme N15A	Escherichia coli
3.7	-	phenylacetyl-CoA	pH 8.1, 25°C, wild-type enzyme	Aromatoleum evansii
8.2	-	3-hydroxyphenylacetyl-CoA	pH 8.1, 25°C, wild-type enzyme	Aromatoleum evansii
9	-	3,5-dihydroxyphenylacetyl-CoA	pH 8.1, 25°C, wild-type enzyme	Escherichia coli
11	-	4-hydroxyphenylacetyl-CoA	pH 8.1, 25°C, wild-type enzyme	Aromatoleum evansii
15	-	3-hydroxyphenylacetyl-CoA	pH 8.1, 25°C, mutant enzyme E14A	Escherichia coli
18	-	3,4-dihydroxyphenylacetyl-CoA	pH 8.1, 25°C, wild-type enzyme	Aromatoleum evansii
19	-	4-hydroxyphenylacetyl-CoA	pH 8.1, 25°C, mutant enzyme E14A	Escherichia coli
20	-	3,4-dihydroxyphenylacetyl-CoA	pH 8.1, 25°C, mutant enzyme E14A	Escherichia coli
26	-	3,4-dihydroxyphenylacetyl-CoA	pH 8.1, 25°C, mutant enzyme N15A	Escherichia coli
30	-	3-hydroxyphenylacetyl-CoA	pH 8.1, 25°C, mutant enzyme N15A	Escherichia coli
79	-	4-hydroxyphenylacetyl-CoA	pH 8.1, 25°C, wild-type enzyme	Escherichia coli
86	-	3-hydroxyphenylacetyl-CoA	pH 8.1, 25°C, wild-type enzyme	Escherichia coli
100	-	3,4-dihydroxyphenylacetyl-CoA	pH 8.1, 25°C, wild-type enzyme	Escherichia coli

Ki Value [mM]

Ki Value [mM]	Ki Value maximum [mM]	Inhibitor	Comment	Organism	Structure
0.029	-	4-hydroxyphenylacyl-CoA	pH 8.1, 25°C, wild-type enzyme	Aromatoleum evansii
0.039	-	4-hydroxyphenylacyl-CoA	pH 8.1, 25°C, wild-type enzyme	Aromatoleum evansii

kcat/KM [mM/s]

kcat/KM Value [1/mMs^-1]	kcat/KM Value Maximum [1/mMs^-1]	Substrate	Comment	Organism
0.1	-	3,4-dihydroxyphenylacetyl-CoA	pH 8.1, 25°C, mutant enzyme D75A	Aromatoleum evansii
0.7	-	4-hydroxyphenylacetyl-CoA	pH 8.1, 25°C, mutant enzyme D16A	Escherichia coli
0.7	-	3-hydroxyphenylacetyl-CoA	pH 8.1, 25°C, mutant enzyme D16A	Escherichia coli
1	-	4-hydroxyphenylacetyl-CoA	pH 8.1, 25°C, mutant enzyme N46A	Escherichia coli
1	-	3,4-dihydroxyphenylacetyl-CoA	pH 8.1, 25°C, mutant enzyme N60A	Aromatoleum evansii
3	-	3,4-dihydroxyphenylacetyl-CoA	pH 8.1, 25°C, mutant enzyme N46A	Escherichia coli
3	-	3,4-dihydroxyphenylacetyl-CoA	pH 8.1, 25°C, mutant enzyme D16A	Escherichia coli
3	-	3,4-dihydroxyphenylacetyl-CoA	pH 8.1, 25°C, mutant enzyme N60D	Aromatoleum evansii
4	-	3-hydroxyphenylacetyl-CoA	pH 8.1, 25°C, mutant enzyme N46A	Escherichia coli
9	-	4-hydroxyphenylacetyl-CoA	pH 8.1, 25°C, mutant enzyme H52A	Escherichia coli
10	-	phenylacetyl-CoA	pH 8.1, 25°C, wild-type enzyme	Aromatoleum evansii
30	-	3-hydroxyphenylacetyl-CoA	pH 8.1, 25°C, mutant enzyme H52A	Escherichia coli
43	-	phenylacetyl-CoA	pH 8.1, 25°C, wild-type enzyme	Escherichia coli
45	-	3,5-dihydroxyphenylacetyl-CoA	pH 8.1, 25°C, wild-type enzyme	Escherichia coli
60	-	3,4-dihydroxyphenylacetyl-CoA	pH 8.1, 25°C, mutant enzyme H52A	Escherichia coli
80	-	3,5-dihydroxyphenylacetyl-CoA	pH 8.1, 25°C, wild-type enzyme	Aromatoleum evansii
100	-	3-hydroxyphenylacetyl-CoA	pH 8.1, 25°C, wild-type enzyme	Aromatoleum evansii
200	-	3,4-dihydroxyphenylacetyl-CoA	pH 8.1, 25°C, mutant enzyme E14A	Escherichia coli
200	-	4-hydroxyphenylacetyl-CoA	pH 8.1, 25°C, mutant enzyme E14A	Escherichia coli
200	-	3-hydroxyphenylacetyl-CoA	pH 8.1, 25°C, mutant enzyme E14A	Escherichia coli
200	-	4-hydroxyphenylacetyl-CoA	pH 8.1, 25°C, mutant enzyme N15A	Escherichia coli
200	-	4-hydroxyphenylacetyl-CoA	pH 8.1, 25°C, wild-type enzyme	Aromatoleum evansii
400	-	3,4-dihydroxyphenylacetyl-CoA	pH 8.1, 25°C, wild-type enzyme	Aromatoleum evansii
600	-	3,4-dihydroxyphenylacetyl-CoA	pH 8.1, 25°C, mutant enzyme N15A	Escherichia coli
900	-	3-hydroxyphenylacetyl-CoA	pH 8.1, 25°C, mutant enzyme N15A	Escherichia coli
2257	-	4-hydroxyphenylacetyl-CoA	pH 8.1, 25°C, wild-type enzyme	Escherichia coli
4095	-	3-hydroxyphenylacetyl-CoA	pH 8.1, 25°C, wild-type enzyme	Escherichia coli
6250	-	3,4-dihydroxyphenylacetyl-CoA	pH 8.1, 25°C, wild-type enzyme	Escherichia coli