Literature summary for 3.8.1.8 extracted from

Seffernick, J.L.; Reynolds, E.; Fedorov, A.A.; Fedorov, E.; Almo, S.C.; Sadowsky, M.J.; Wackett, L.P.
X-ray structure and mutational analysis of the atrazine chlorohydrolase TrzN (2010), J. Biol. Chem., 285, 30606-30614.

Cloned(Commentary)

Cloned (Comment)	Organism
expression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)	Paenarthrobacter aurescens

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified recombinant Zn2+-bound wild-type TrzN, and zinc-bound TrzN mutant E241Q complexed with ametryn or atratone, hanging drop method, for the wild-typeenzyme: 21 mg/ml TrzN in 25 mM MOPS, pH 5.5, and 1mM ZnCl2 with precipitant solution containing 25% PEG 3350, 0.1 M Bis-Tris, pH 5.5, 0.2 M ammonium sulfate, and 1 mM ZnCl2, 7-8 days at 25°C. For the mutant enzyme: 6.6 mg/ml TrzN-E241Q in 25 mM MOPS, pH 5.5, and 1 mM ZnCl2 with precipitant containing 30% PEG 4000, 0.1 M sodium citrate, pH 5.6, 0.2 M ammonium acetate, and 1 mM ZnCl2, 2 weeks at 25°C, soaking of crystals for 1 h in cryobuffer composed of its mother liquid, 15% glycerol, and excess of the ametryn or atratone powder, X-ray diffraction structure determination and analysis at 1.40 A, 1.93 A, and 1.64 A resolutions, respectively, modeling	Paenarthrobacter aurescens

Crystallization (Comment)

Organism

purified recombinant Zn2+-bound wild-type TrzN, and zinc-bound TrzN mutant E241Q complexed with ametryn or atratone, hanging drop method, for the wild-typeenzyme: 21 mg/ml TrzN in 25 mM MOPS, pH 5.5, and 1mM ZnCl2 with precipitant solution containing 25% PEG 3350, 0.1 M Bis-Tris, pH 5.5, 0.2 M ammonium sulfate, and 1 mM ZnCl2, 7-8 days at 25°C. For the mutant enzyme: 6.6 mg/ml TrzN-E241Q in 25 mM MOPS, pH 5.5, and 1 mM ZnCl2 with precipitant containing 30% PEG 4000, 0.1 M sodium citrate, pH 5.6, 0.2 M ammonium acetate, and 1 mM ZnCl2, 2 weeks at 25°C, soaking of crystals for 1 h in cryobuffer composed of its mother liquid, 15% glycerol, and excess of the ametryn or atratone powder, X-ray diffraction structure determination and analysis at 1.40 A, 1.93 A, and 1.64 A resolutions, respectively, modeling

Paenarthrobacter aurescens

Protein Variants

Protein Variants	Comment	Organism
E241Q	site-directed mutagenesis, isosteric substitution of the active site glutamate, the mutant shows a large diminution in activity with ametryn, no detectable activity with atratone, and a 10fold decrease with atrazine compared to the wild-type TrzN. Activity of the mutant is nearly constant from pH 6.0 to pH 10.0, consistent with the loss of a proton donating group	Paenarthrobacter aurescens
R325S	site-directed mutagenesis, the mutation results in a 12fold decrease in kcat/Km, largely due to kcat	Paenarthrobacter aurescens
T325D	site-directed mutagenesis, inactive active site mutant, altered active site structure compared to the wild-type enzyme	Paenarthrobacter aurescens
T325E	site-directed mutagenesis, inactive active site mutant, altered active site structure compared to the wild-type enzyme	Paenarthrobacter aurescens

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
3.1	-	Atrazine	mutant E241Q, pH and temperature not specified in the publication	Paenarthrobacter aurescens
3.9	-	ametryn	wild-type enzyme, pH 7.0, temperature not specified in the publication	Paenarthrobacter aurescens
7.8	-	ametryn	mutant R325, pH and temperature not specified in the publicationS	Paenarthrobacter aurescens
13.3	-	Atrazine	mutant R325S, pH and temperature not specified in the publication	Paenarthrobacter aurescens
19	-	Atrazine	wild-type enzyme, pH 7.0, temperature not specified in the publication	Paenarthrobacter aurescens

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Zn2+	required	Paenarthrobacter aurescens

Organism

Organism	UniProt	Comment	Textmining
Paenarthrobacter aurescens	-	gene trzN	-
Paenarthrobacter aurescens TC1	-	gene trzN	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant wild-type and mutant enzymes from Escherichia coli strain Bl21(DE3)	Paenarthrobacter aurescens

Reaction

Reaction	Comment	Organism	Reaction ID
atrazine + H2O = hydroxyatrazine + chloride	catalytic mechanism, the Glu241 side chain provides a proton to N-1 of the s-triazine substrate to facilitate nucleophilic displacement at the adjacent C-2, overview	Paenarthrobacter aurescens	a

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
ametryn + H2O	i.e. 2-thiomethyl-4-isopropylamino-6-ethylamino-1,3,5-triazine, a s-triazine herbicide	Paenarthrobacter aurescens	4-(ethylamino)-2-hydroxy-6-(isopropylamino)-1,3,5-triazine + H-S-CH3	-	?
ametryn + H2O	i.e. 2-thiomethyl-4-isopropylamino-6-ethylamino-1,3,5-triazine, a s-triazine herbicide	Paenarthrobacter aurescens TC1	4-(ethylamino)-2-hydroxy-6-(isopropylamino)-1,3,5-triazine + H-S-CH3	-	?
atratone + H2O	-	Paenarthrobacter aurescens	4-(ethylamino)-2-hydroxy-6-(isopropylamino)-1,3,5-triazine + methanol	-	?
atratone + H2O	-	Paenarthrobacter aurescens TC1	4-(ethylamino)-2-hydroxy-6-(isopropylamino)-1,3,5-triazine + methanol	-	?
atrazine + H2O	i.e. 2-chloro-4-isopropylamino-6-ethylamino-1,3,5-triazine, a s-triazine herbicide	Paenarthrobacter aurescens	4-(ethylamino)-2-hydroxy-6-(isopropylamino)-1,3,5-triazine + chloride	-	?
atrazine + H2O	i.e. 2-chloro-4-isopropylamino-6-ethylamino-1,3,5-triazine, a s-triazine herbicide	Paenarthrobacter aurescens TC1	4-(ethylamino)-2-hydroxy-6-(isopropylamino)-1,3,5-triazine + chloride	-	?

Subunits

Subunits	Comment	Organism
homodimer	(betaalpha)8 barrel enzyme structure	Paenarthrobacter aurescens

Synonyms

Synonyms	Comment	Organism
atrazine chlorohydrolase 2	-	Paenarthrobacter aurescens
triazine hydrolase	-	Paenarthrobacter aurescens
TrZN	-	Paenarthrobacter aurescens

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
0.0869	-	Atrazine	mutant R325S, pH and temperature not specified in the publication	Paenarthrobacter aurescens
0.137	-	Atrazine	mutant E241Q, pH and temperature not specified in the publication	Paenarthrobacter aurescens
0.467	-	ametryn	mutant R325S, pH and temperature not specified in the publication	Paenarthrobacter aurescens
1.49	-	Atrazine	wild-type enzyme, pH 7.0, temperature not specified in the publication	Paenarthrobacter aurescens
6.04	-	ametryn	wild-type enzyme, pH 7.0, temperature not specified in the publication	Paenarthrobacter aurescens

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
6	10	mutant E241Q	Paenarthrobacter aurescens
7	-	wild-type enzyme	Paenarthrobacter aurescens

General Information

General Information	Comment	Organism
evolution	TrzN uniquely positions threonine 325 in place of a conserved aspartate that ligates the metal in most mononuclear amidohydrolases superfamily members	Paenarthrobacter aurescens
physiological function	atrazine chlorohydrolase 2 initiates bacterial metabolism of the herbicide atrazine by hydrolytic displacement of a chlorine substituent from the s-triazine ring	Paenarthrobacter aurescens

kcat/KM [mM/s]

kcat/KM Value [1/mMs^-1]	kcat/KM Value Maximum [1/mMs^-1]	Substrate	Comment	Organism
6.5	-	Atrazine	mutant R325S, pH and temperature not specified in the publication	Paenarthrobacter aurescens
44	-	Atrazine	mutant E241Q, pH and temperature not specified in the publication	Paenarthrobacter aurescens
60	-	ametryn	mutant R325S, pH and temperature not specified in the publication	Paenarthrobacter aurescens
78	-	Atrazine	wild-type enzyme, pH 7.0, temperature not specified in the publication	Paenarthrobacter aurescens
1530	-	ametryn	wild-type enzyme, pH 7.0, temperature not specified in the publication	Paenarthrobacter aurescens