Cloned (Comment) | Organism |
---|---|
recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain DH5alpha | uncultured bacterium |
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 | 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 | UniProt | Comment | Textmining |
---|---|---|---|
uncultured bacterium | - |
from soil | - |
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 | 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 | Comment | Organism |
---|---|---|
2,4-DCP hydroxylase | - |
uncultured bacterium |
2,4-dichlorophenol hydroxylase | - |
uncultured bacterium |
TfdB-JLU | - |
uncultured bacterium |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
25 | - |
assay at | uncultured bacterium |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.5 | - |
assay at | uncultured bacterium |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
FAD | - |
uncultured bacterium | |
NADPH | - |
uncultured bacterium |
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 |