Literature summary for 1.7.1.B1 extracted from

Van Dillewijn, P.; Wittich, R.M.; Caballero, A.; Ramos, J.L.
Subfunctionality of hydride transferases of the old yellow enzyme family of flavoproteins of Pseudomonas putida (2008), Appl. Environ. Microbiol., 74, 6703-6708.

Cloned(Commentary)

Cloned (Comment)	Organism
xenB gene is amplified using appropriate primers with BamHI and HindIII sites and Pseudomonas putida KT2440 chromosomal DNA as a template. After digestion with restriction enzymes, the PCR product is ligated into the pET28b(+) vector. Resulting plasmid contains the coding sequence in frame with a DNA sequence encoding a His-tag, which resulted in a hexahistidine tail. For protein-His6 expression, plasmid is transformed into Escherichia coli BL21.	Pseudomonas putida

Cloned (Comment)

Organism

xenB gene is amplified using appropriate primers with BamHI and HindIII sites and Pseudomonas putida KT2440 chromosomal DNA as a template. After digestion with restriction enzymes, the PCR product is ligated into the pET28b(+) vector. Resulting plasmid contains the coding sequence in frame with a DNA sequence encoding a His-tag, which resulted in a hexahistidine tail. For protein-His6 expression, plasmid is transformed into Escherichia coli BL21.

Pseudomonas putida

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
0.027	-	glycerol trinitrate	commentary	Pseudomonas putida
0.037	-	2,4,6-trinitrotoluene	-	Pseudomonas putida
0.044	-	N-ethylmaleimide	commentary	Pseudomonas putida
0.077	-	NADPH	-	Pseudomonas putida
0.158	-	cyclohexenone	commentary	Pseudomonas putida

Molecular Weight [Da]

Molecular Weight [Da]	Molecular Weight Maximum [Da]	Comment	Organism
additional information	-	determined by gel filtration, molecular masses of the His6-tagged proteins XenA to XenF are in the range of 40900 to 42600 Da	Pseudomonas putida

Organism

Organism	UniProt	Comment	Textmining
Pseudomonas putida	Q88PD0	-	-
Pseudomonas putida KT 2240	Q88PD0	-	-

Purification (Commentary)

Purification (Comment)	Organism
His6-tagged protein is purified by nickel affinity chromatography and eluted with a continuous imidazole gradient.	Pseudomonas putida

Reaction

Reaction	Comment	Organism	Reaction ID
2,4,6-trinitrotoluene + 2 NADPH + 2 H+ = N-hydroxy-2-methyl-3,5-dinitroaniline + 2 NADP+ + H2O	The amount of N-hydroxy-2-methyl-3,5-dinitroaniline is larger than the amount of N-hydroxy-4-methyl-3,5-dinitroaniline formed. In a further nonenzymatic reaction nitrite is released and N-(2-methyl-3,5-dinitrophenyl)-N-4-methyl-3,5-dinitrophenyl-hydroxylamine and eventually N-(2-methyl-3,5-dinitrophenyl)-N-4-methyl-3,5-dinitroaniline are produced. In adidtion the aromatic ring of 2,4,6-trinitrotoluene is susceptible to nucleophilic attack by hydride ions to form Meisenheimer complex intermediates (monohydride and dihydride complexes) which also form the secondary diaryl hydroxylamines and the secondary diarylamine with release of nitrite	Pseudomonas putida	a
2,4,6-trinitrotoluene + 2 NADPH + 2 H+ = N-hydroxy-4-methyl-3,5-dinitroaniline + 2 NADP+ + H2O	The amount of N-hydroxy-4-methyl-3,5-dinitroaniline is larger than the amount of N-hydroxy-2-methyl-3,5-dinitroaniline formed. In a further nonenzymatic reaction nitrite is released and N,N-bis-(3,5-dinitrotolyl)-hydroxylamine and eventually N,N-bis-(3,5-dinitrotolyl)-amine are produced. In addition the aromatic ring of 2,4,6-trinitrotoluene is susceptible to nucleophilic attack by hydride ions to form Meisenheimer complex intermediates (monohydride and dihydride complexes) which also form the secondary diaryl hydroxylamines and the secondary diarylamine with release of nitrite	Pseudomonas putida	a

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg]	Specific Activity Maximum [µmol/min/mg]	Comment	Organism
additional information	-	Vmax is denoted with 112 micromol/min/mg protein, XenB exhibits the highest Vmax values and the most favorable Vmax/Km relationship for 2,4,6-trinitrotoluene compared to those of the other active xenobiotic reductases of Pseudomonas putida KT2440	Pseudomonas putida
1.88	-	highest specific activity within xenobiotic reductases A to F	Pseudomonas putida

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
2,4,6-trinitrotoluene + 2 NADPH + 2 H+	-	Pseudomonas putida	N-hydroxy-2-methyl-3,5-dinitroaniline + 2 NADP+ + H2O	i.e. 2-hydroxylamino-2,6-dinitrotoluene. The amount of N-hydroxy-2-methyl-3,5-dinitroaniline is larger than the amount of N-hydroxy-4-methyl-3,5-dinitroaniline formed. In a further nonenzymatic reaction nitrite is released and N-(2-methyl-3,5-dinitrophenyl)-N-4-methyl-3,5-dinitrophenyl-hydroxylamine and eventually N-(2-methyl-3,5-dinitrophenyl)-N-4-methyl-3,5-dinitroaniline are produced. In addition the aromatic ring of 2,4,6-trinitrotoluene is susceptibe to nucleophilic attack by hydride ions to form Meisenheimer complex intermediates (monohydride and dihydride complexes) which also form secondary diaryl hydroxylamines and secondary diarylamine with release of nitrite	?
2,4,6-trinitrotoluene + 2 NADPH + 2 H+	-	Pseudomonas putida KT 2240	N-hydroxy-2-methyl-3,5-dinitroaniline + 2 NADP+ + H2O	i.e. 2-hydroxylamino-2,6-dinitrotoluene. The amount of N-hydroxy-2-methyl-3,5-dinitroaniline is larger than the amount of N-hydroxy-4-methyl-3,5-dinitroaniline formed. In a further nonenzymatic reaction nitrite is released and N-(2-methyl-3,5-dinitrophenyl)-N-4-methyl-3,5-dinitrophenyl-hydroxylamine and eventually N-(2-methyl-3,5-dinitrophenyl)-N-4-methyl-3,5-dinitroaniline are produced. In addition the aromatic ring of 2,4,6-trinitrotoluene is susceptibe to nucleophilic attack by hydride ions to form Meisenheimer complex intermediates (monohydride and dihydride complexes) which also form secondary diaryl hydroxylamines and secondary diarylamine with release of nitrite	?
2,4,6-trinitrotoluene + 2 NADPH + 2 H+	-	Pseudomonas putida	N-hydroxy-4-methyl-3,5-dinitroaniline + 2 NADP+ + H2O	i.e. 4-hydroxylamino-2,6-dinitrotoluene. The amount of N-hydroxy-4-methyl-3,5-dinitroaniline is larger than the amount of N-hydroxy-2-methyl-3,5-dinitroaniline formed. In a further nonenzymatic reaction nitrite is released and N,N-bis-(3,5-dinitrotolyl)-hydroxylamine and eventually N,N-bis-(3,5-dinitrotolyl)-amine are produced. In addition the aromatic ring of 2,4,6-trinitrotoluene is susceptible to nucleophilic attack by hydride ions to form Meisenheimer complex intermediates (monohydride and dihydride complexes) which also form the secondary diaryl hydroxylamines and the secondary diarylamine with release of nitrite	?
2,4,6-trinitrotoluene + 2 NADPH + 2 H+	-	Pseudomonas putida KT 2240	N-hydroxy-4-methyl-3,5-dinitroaniline + 2 NADP+ + H2O	i.e. 4-hydroxylamino-2,6-dinitrotoluene. The amount of N-hydroxy-4-methyl-3,5-dinitroaniline is larger than the amount of N-hydroxy-2-methyl-3,5-dinitroaniline formed. In a further nonenzymatic reaction nitrite is released and N,N-bis-(3,5-dinitrotolyl)-hydroxylamine and eventually N,N-bis-(3,5-dinitrotolyl)-amine are produced. In addition the aromatic ring of 2,4,6-trinitrotoluene is susceptible to nucleophilic attack by hydride ions to form Meisenheimer complex intermediates (monohydride and dihydride complexes) which also form the secondary diaryl hydroxylamines and the secondary diarylamine with release of nitrite	?
cyclohexenone + NADPH + H+	-	Pseudomonas putida	?	-	?
cyclohexenone + NADPH + H+	-	Pseudomonas putida KT 2240	?	-	?
glycerol trinitrate + NADPH + H+	-	Pseudomonas putida	?	-	?
glycerol trinitrate + NADPH + H+	-	Pseudomonas putida KT 2240	?	-	?
additional information	ability to reduce nitroaromatic compounds, enzyme demonstrates type I and type II hydride transferase activity and reduced the nitro groups of 2,4,6-trinitrotoluene to hydroxylaminodinitrotoluene derivatives. The condensations of the primary products of type I and type II hydride transferases react with each other to yield diarylamines and nitrite, the latter can be further reduced to ammonium and serves as a nitrogen source for microorganisms in vivo	Pseudomonas putida	?	-	?
additional information	ability to reduce nitroaromatic compounds, enzyme demonstrates type I and type II hydride transferase activity and reduced the nitro groups of 2,4,6-trinitrotoluene to hydroxylaminodinitrotoluene derivatives. The condensations of the primary products of type I and type II hydride transferases react with each other to yield diarylamines and nitrite, the latter can be further reduced to ammonium and serves as a nitrogen source for microorganisms in vivo	Pseudomonas putida KT 2240	?	-	?
N-ethylmaleimide + NADPH + H+	-	Pseudomonas putida	?	-	?

Subunits

Subunits	Comment	Organism
monomer	in solution	Pseudomonas putida

Synonyms

Synonyms	Comment	Organism
old yellow enzyme	-	Pseudomonas putida
OYE	-	Pseudomonas putida
XenB	-	Pseudomonas putida
xenobiotic reductase B	family of flavoproteins	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	-	assay at	Pseudomonas putida

Cofactor

Cofactor	Comment	Organism	Structure
FMN	in the primary sequence of these proteins, a number of residues involved in interactions with the FMN cofactor is found	Pseudomonas putida
NAD(P)H	enzyme preferentially uses NADPH as a cofactor	Pseudomonas putida