Cloned (Comment) | Organism |
---|---|
gene 3HB6H is encoded in a cluster that potentially encodes for gentisic acid degradation, sequence comparisons and phylogenetic analysis and tree recombinant expression of N-terminally His-tagged enzyme in Escherichia coli strain BL21(DE3) | Martelella sp. AD-3 |
Protein Variants | Comment | Organism |
---|---|---|
A308G | site-directed mutagenesis, the mutant shows reduced activity compared to wild-type enzyme, comparison of FAD contents | Martelella sp. AD-3 |
Q305P | site-directed mutagenesis, the mutant shows reduced activity compared to wild-type enzyme, comparison of FAD contents | Martelella sp. AD-3 |
Y221F | site-directed mutagenesis, the mutant shows reduced activity compared to wild-type enzyme, comparison of FAD contents | Martelella sp. AD-3 |
Y306H | site-directed mutagenesis, the mutant shows reduced activity compared to wild-type enzyme, comparison of FAD contents | Martelella sp. AD-3 |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
Co2+ | inhibits the enzyme activity at 0.5 mM by about 30% | Martelella sp. AD-3 | |
Cu2+ | inhibits the enzyme activity at 0.5 mM by 83.2% | Martelella sp. AD-3 | |
Fe3+ | inhibits the enzyme activity at 0.5 mM by about 50% | Martelella sp. AD-3 | |
Mg2+ | inhibits the enzyme activity at 0.5 mM by about 50% | Martelella sp. AD-3 | |
Zn2+ | inhibits the enzyme activity at 0.5 mM by 77.4% | Martelella sp. AD-3 |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | purified 3HB6H displays typical Michaelis-Menten kinetics, Lineweaver-Burk plots | Martelella sp. AD-3 | |
0.0337 | - |
NADH | pH 8.0, 30°C, mutant A308G | Martelella sp. AD-3 | |
0.038 | - |
3-hydroxybenzoate | pH 8.0, 30°C, mutant A308G | Martelella sp. AD-3 | |
0.0463 | - |
3-hydroxybenzoate | pH 8.0, 30°C, mutant Y306H | Martelella sp. AD-3 | |
0.0726 | - |
3-hydroxybenzoate | pH 8.0, 30°C, recombinant wild-type enzyme | Martelella sp. AD-3 | |
0.0867 | - |
NADH | pH 8.0, 30°C, mutant Y221F | Martelella sp. AD-3 | |
0.0913 | - |
3-hydroxybenzoate | pH 8.0, 30°C, mutant Q305P | Martelella sp. AD-3 | |
0.0959 | - |
NADH | pH 8.0, 30°C, mutant Q305P | Martelella sp. AD-3 | |
0.1037 | - |
NADH | pH 8.0, 30°C, mutant Y306H | Martelella sp. AD-3 | |
0.1041 | - |
NADH | pH 8.0, 30°C, recombinant enzyme | Martelella sp. AD-3 | |
0.1642 | - |
3-hydroxybenzoate | pH 8.0, 30°C, mutant Y221F | Martelella sp. AD-3 |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
additional information | enzyme activity is highest without salinity and metal salts. Ca2+ and Mn2+ have a poor effect on enzyme activity at 0.5 mM. With salinity ranging from 0 to 85.56 mM, the increase in salinity clearly causes decreased enzyme activity, 3% activity remaining at 85.56 mM salinity | Martelella sp. AD-3 |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
3-hydroxybenzoate + NADH + H+ + O2 | Martelella sp. AD-3 | - |
2,5-dihydroxybenzoate + NAD+ + H2O | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Martelella sp. AD-3 | - |
- |
- |
Posttranslational Modification | Comment | Organism |
---|---|---|
lipoprotein | the recombinant 3HB6H expressed in Escherichia coil contains phosphatidylglycerol, while phosphatidylethanolamine is not detected | Martelella sp. AD-3 |
Purification (Comment) | Organism |
---|---|
recombinant N-terminally His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography | Martelella sp. AD-3 |
Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
---|---|---|---|
25.2 | - |
purified recombinant enzyme, pH 8.0, 37°C | Martelella sp. AD-3 |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
3-hydroxybenzoate + NADH + H+ + O2 | - |
Martelella sp. AD-3 | 2,5-dihydroxybenzoate + NAD+ + H2O | - |
? |
Subunits | Comment | Organism |
---|---|---|
homodimer | 2 * 46000, recombinant His-tagged enzyme SDS-PAGE | Martelella sp. AD-3 |
Synonyms | Comment | Organism |
---|---|---|
3-hydroxybenzoate 6-hydroxylase | - |
Martelella sp. AD-3 |
3HB6H | - |
Martelella sp. AD-3 |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
37 | - |
recombinant enzyme | Martelella sp. AD-3 |
Temperature Stability Minimum [°C] | Temperature Stability Maximum [°C] | Comment | Organism |
---|---|---|---|
16 | 50 | enzyme activity is maintained at 16-37°C, and quickly lost at higher temperatures. The enzyme precipitates initially at 42°C and precipitates completely at 50°C | Martelella sp. AD-3 |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
1 | - |
NADH | pH 8.0, 30°C, mutant Y221F | Martelella sp. AD-3 | |
1.5 | - |
3-hydroxybenzoate | pH 8.0, 30°C, mutant Y306H | Martelella sp. AD-3 | |
1.6 | - |
NADH | pH 8.0, 30°C, mutant Q305P | Martelella sp. AD-3 | |
1.6 | - |
3-hydroxybenzoate | pH 8.0, 30°C, mutant Q305P | Martelella sp. AD-3 | |
1.6 | - |
NADH | pH 8.0, 30°C, mutant Y306H | Martelella sp. AD-3 | |
1.7 | - |
3-hydroxybenzoate | pH 8.0, 30°C, mutant Y221F | Martelella sp. AD-3 | |
2 | - |
NADH | pH 8.0, 30°C, mutant A308G | Martelella sp. AD-3 | |
2.3 | - |
3-hydroxybenzoate | pH 8.0, 30°C, mutant A308G | Martelella sp. AD-3 | |
5.7 | - |
3-hydroxybenzoate | pH 8.0, 30°C, recombinant wild-type enzyme | Martelella sp. AD-3 | |
7.8 | - |
NADH | pH 8.0, 30°C, recombinant enzyme | Martelella sp. AD-3 |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
8 | - |
recombinant enzyme, phosphate buffer | Martelella sp. AD-3 |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
FAD | residues 305, 306 and 308 are important for FAD binding. FAD concentration in the purified 3HB6H (0.29 mM) is 0.30 mM, and FAD/enzyme ratio is 1.02/0.05 determined by HPLC analysis | Martelella sp. AD-3 | |
NADH | - |
Martelella sp. AD-3 |
General Information | Comment | Organism |
---|---|---|
evolution | sequence comparisons and phylogenetic analysis, phylogenetic tree is constructed with 3-hydroxybenzoate 6-hydroxylases, salicylate 1-hydroxylases and 3-hydroxybenzoate 4-hydroxylases from other 17 strains and confirmed that it is most closely related to 3HB6H from Pseudomonas alcaligenes strain NCIMB 9867 | Martelella sp. AD-3 |
metabolism | 3-hydroxybenzoate 6-hydroxylase can convert 3-hydroxybenzoate which is an important intermediate in the biodegradation of many aromatic hydrocarbons. 3-Hydroxybenzoate is metabolized by entering the TCA cycle through the gentisate pathway | Martelella sp. AD-3 |
additional information | residues Tyr221 and Gln305 of 3HB6H from Martelella sp. strain AD-3 are involved in substrate binding | Martelella sp. AD-3 |
physiological function | 3-hydroxybenzoate 6-hydroxylase, an NADH-dependent flavoprotein, can convert 3-hydroxybenzoate which is an important intermediate in the biodegradation of many aromatic hydrocarbons | Martelella sp. AD-3 |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
10.35 | - |
3-hydroxybenzoate | pH 8.0, 30°C, mutant Y221F | Martelella sp. AD-3 | |
11.53 | - |
NADH | pH 8.0, 30°C, mutant Y221F | Martelella sp. AD-3 | |
15.43 | - |
NADH | pH 8.0, 30°C, mutant Y306H | Martelella sp. AD-3 | |
16.68 | - |
NADH | pH 8.0, 30°C, mutant Q305P | Martelella sp. AD-3 | |
32.4 | - |
3-hydroxybenzoate | pH 8.0, 30°C, mutant Y306H | Martelella sp. AD-3 | |
59.35 | - |
NADH | pH 8.0, 30°C, mutant A308G | Martelella sp. AD-3 | |
60.53 | - |
3-hydroxybenzoate | pH 8.0, 30°C, mutant A308G | Martelella sp. AD-3 | |
62.61 | - |
3-hydroxybenzoate | pH 8.0, 30°C, mutant Q305P | Martelella sp. AD-3 | |
74.93 | - |
NADH | pH 8.0, 30°C, recombinant enzyme | Martelella sp. AD-3 | |
78.51 | - |
3-hydroxybenzoate | pH 8.0, 30°C, recombinant wild-type enzyme | Martelella sp. AD-3 |