Any feedback?
Please rate this page
(literature.php)
(0/150)

BRENDA support

Literature summary for 1.14.13.20 extracted from

  • Wang, Y.; Zhang, C.; An, S.; Fang, X.; Yu, D.
    Engineering substrate promiscuity in 2,4-dichlorophenol hydroxylase by in silico design (2018), RSC Adv., 8, 21184-21190 .
    View publication on PubMedView publication on EuropePMC
Search for more literature for 1.14.13.20

Cloned(Commentary)

Cloned (Comment) Organism
recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain DH5alpha uncultured bacterium

Protein Variants

Protein Variants Comment Organism
F424A site-directed mutagenesis, analysis of substrate binding by the mutant and docking study in comparison to the wild-type enzyme uncultured bacterium
H47A site-directed mutagenesis, analysis of substrate binding by the mutant and docking study in comparison to the wild-type enzyme uncultured bacterium
I48A site-directed mutagenesis, analysis of substrate binding by the mutant and docking study in comparison to the wild-type enzyme uncultured bacterium
additional information enzyme TfdB-JLU is engineered by rational design to further broaden its substrate scope towards chlorophenols (CPs). Dissection of the architectures of enzymes from oxidoreductase families to discover their underlying structural sources of substrate promiscuity, and homology modeling of TfdB-JLU. Docking experiments of this homology model with its natural substrate 2,4-dichlorophenol reveals that the phenyl rings of 2,4-DCP form strong interactions with residues His47, Ile48, Trp222, Pro316, and Phe424. These residues are found to be important for substrate binding in the active site. Site-directed mutagenesis strategy is applied for redesigning substrate promiscuity in enzyme TfdB-JLU. P316 is the key residue for enzyme functional engineering uncultured bacterium
P316A site-directed mutagenesis, analysis of substrate binding by the mutant and docking study in comparison to the wild-type enzyme uncultured bacterium
P316Q site-directed mutagenesis, the TfdB-JLU variant shows a significant enhancement of activity (up to 3.4fold) toward 10 CP congeners compared to wild-type TfdB-JLU. The active improvements of TfdB-JLU-P316Q toward CP congeners show significant difference, especially for active improvements of positional congeners such as 3-CP (1.1fold) compared to 4-CP (3.0fold), as well as 2,3-DCP (1.2fold) compared to 2,5-DCP (3.4fold). Structural analysis results indicate that the improvement in substrate promiscuity of the variant enzyme compared to the wild-type enzyme is possibly due to the increase of non-bonding interaction uncultured bacterium
W222A site-directed mutagenesis, analysis of substrate binding by the mutant and docking study in comparison to the wild-type enzyme uncultured bacterium

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
2,4-dichlorophenol + NADPH + H+ + O2 uncultured bacterium
-
 3,5-dichlorocatechol + NADP+ + H2O
-
 ?

Organism

Organism UniProt Comment Textmining
uncultured bacterium
-
 from soil
-

Purification (Commentary)

Purification (Comment) Organism
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain DH5alpha by nickel affinity chromatography and ultrafiltration uncultured bacterium

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2,3-dichlorophenol + NADPH + H+ + O2
-
 uncultured bacterium ? + NADP+ + H2O
-
 ?
2,4,5-trichlorophenol + NADPH + H+ + O2 low activity uncultured bacterium ? + NADP+ + H2O
-
 ?
2,4-dichlorophenol + NADPH + H+ + O2
-
 uncultured bacterium 3,5-dichlorocatechol + NADP+ + H2O
-
 ?
2,5-dichlorophenol + NADPH + H+ + O2
-
 uncultured bacterium ? + NADP+ + H2O
-
 ?
2,6-dichlorophenol + NADPH + H+ + O2
-
 uncultured bacterium ? + NADP+ + H2O
-
 ?
3,4-dichlorophenol + NADPH + H+ + O2 low activity uncultured bacterium ? + NADP+ + H2O
-
 ?
3,5-dichlorophenol + NADPH + H+ + O2 low activity uncultured bacterium ? + NADP+ + H2O
-
 ?
3-chlorophenol + NADPH + H+ + O2 best substrate uncultured bacterium ? + NADP+ + H2O
-
 ?
4-chlorophenol + NADPH + H+ + O2
-
 uncultured bacterium ? + NADP+ + H2O
-
 ?
additional information the 2,4-DCP hydroxylase (TfdB-JLU) exhibits broad substrate specificity for chlorophenols (CPs) and their homologues. Substrate specificity of wild-type and mutant P316Q enzymes, overview uncultured bacterium ?
-
-

Synonyms

Synonyms Comment Organism
2,4-DCP hydroxylase
-
 uncultured bacterium
2,4-dichlorophenol hydroxylase
-
 uncultured bacterium
TfdB-JLU
-
 uncultured bacterium

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
25
-
 assay at uncultured bacterium

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.5
-
 assay at uncultured bacterium

Cofactor

Cofactor Comment Organism Structure
FAD
-
 uncultured bacterium
NADPH
-
 uncultured bacterium

General Information

General Information Comment Organism
malfunction analysis of substrate binding by the mutant P316Q and the wild-type enzyme, docking study, overview uncultured bacterium
additional information molecular docking experiments of the enzyme TfdB-JLU's homology model with its natural substrate 2,4-dichlorophenol (with template PDB ID 5brt) reveals that the phenyl rings of 2,4-DCP form strong interactions with residues His47, Ile48, Trp222, Pro316, and Phe424. These residues are found to be important for substrate binding in the active site uncultured bacterium
physiological function 2,4-dichlorophenol hydroxylase (2,4-DCP hydroxylase) is a key enzyme in the degradation of 2,4-dichlorophenoxyacetic acid through the hydroxylation step in many bacteria uncultured bacterium