Cloned (Comment) | Organism |
---|---|
gene cat-2, recombinant expression of N-terminally His6-tagged enzyme in Escherichia coli strain M15/pREP4, recombinant expression of C-terminally intein-tagged enzyme in Escherichia coli | Neurospora crassa |
Crystallization (Comment) | Organism |
---|---|
purified recombinant His6-tagged enzyme CAT-2, (1) microbatch method, mixing 800 nl of 20 mg/ml protein in 50 mM phosphate Na/K, pH 7.0, 150 mM NaCl, and 0.5% noctyl-beta-D-glycoside with 800 nl of crystallization solution containing 0.2 M calcium acetate and 20% PEG 3350, the drops are covered with 0.01 ml of paraffin oil for 7 days at 18°C, (2) sitting drop vapor diffusion technique, mixing of 200 nl of 42 mg/ml protein in 50 mM arginine/glutamate, pH 7.5, with 200 nl of crystallization solution containing 9% PEG 8000, 225 mM magnesium chloride, and 100 mM Tris/HCl, pH 7.0, at 18°C, 3-4 weeks. X-ray diffraction structure determination and analysis at 2.9 A and 2.6 A resolution, respectively, structure modeling | Neurospora crassa |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | Michaelis-Menten kinetics | Neurospora crassa | |
53 | - |
H2O2 | pH 5.5, 25°C, recombinant intein-tagged enzyme | Neurospora crassa |
Localization | Comment | Organism | GeneOntology No. | Textmining |
---|---|---|---|---|
cytosol | CAT-2 is mainly if not entirely cytoplasmic | Neurospora crassa | 5829 | - |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Fe2+ | in the heme group | Neurospora crassa | |
K+ | the CAT-2 homodimer crystallographic structure contains two K+ ions bound by Glu107 residues, binding structure, modeling, overview | Neurospora crassa |
Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
---|---|---|---|
177000 | - |
recombinant intein-tagged enzyme, gel filtration | Neurospora crassa |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
2 H2O2 | Neurospora crassa | - |
O2 + 2 H2O | - |
? | |
2 H2O2 | Neurospora crassa CBS 708.71 | - |
O2 + 2 H2O | - |
? | |
2 H2O2 | Neurospora crassa 74-OR23-1A | - |
O2 + 2 H2O | - |
? | |
2 H2O2 | Neurospora crassa DSM 1257 | - |
O2 + 2 H2O | - |
? | |
2 H2O2 | Neurospora crassa ATCC 24698 | - |
O2 + 2 H2O | - |
? | |
2 H2O2 | Neurospora crassa FGSC 987 | - |
O2 + 2 H2O | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Neurospora crassa | Q8X182 | - |
- |
Neurospora crassa 74-OR23-1A | Q8X182 | - |
- |
Neurospora crassa ATCC 24698 | Q8X182 | - |
- |
Neurospora crassa CBS 708.71 | Q8X182 | - |
- |
Neurospora crassa DSM 1257 | Q8X182 | - |
- |
Neurospora crassa FGSC 987 | Q8X182 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant N-terminally His6-tagged enzyme 5.1fold (peroxidase activity) from Escherichia coli strain M15/pREP4 by nickel affinity chromatography, ultrafiltration, and gel filtration. Recombinant C-terminally intein-tagged enzyme 22.5fold from Escherichia coli by chitin affinity chromatography and treatment thereafter with a high concentration of DTT to excise the intein | Neurospora crassa |
Source Tissue | Comment | Organism | Textmining |
---|---|---|---|
hypha | - |
Neurospora crassa | - |
additional information | the catalase-peroxidase in Neurospora crassa, CAT-2, is a developmentally regulated enzyme: both gene expression and CAT-2 activity are induced at the stationary growth phase and during adhesion of hyphae, aerial hyphae growth and conidia formation. gene expression and CAT-2 activity are induced when the fungus is deprived of a carbon source, or when glucose is replaced by a poor carbon sources | Neurospora crassa | - |
mycelium | - |
Neurospora crassa | - |
Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
---|---|---|---|
14.9 | - |
purified recombinant intein-tagged enzyme, pH 5.5, 25°C, peroxidase activity | Neurospora crassa |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
2 H2O2 | - |
Neurospora crassa | O2 + 2 H2O | - |
? | |
2 H2O2 | - |
Neurospora crassa CBS 708.71 | O2 + 2 H2O | - |
? | |
2 H2O2 | - |
Neurospora crassa 74-OR23-1A | O2 + 2 H2O | - |
? | |
2 H2O2 | - |
Neurospora crassa DSM 1257 | O2 + 2 H2O | - |
? | |
2 H2O2 | - |
Neurospora crassa ATCC 24698 | O2 + 2 H2O | - |
? | |
2 H2O2 | - |
Neurospora crassa FGSC 987 | O2 + 2 H2O | - |
? | |
additional information | CAT-2 requires the Met-Tyr-Trp adduct for catalase activity. CAT-2 Arg426 is oriented towards the M-Y-W adduct, interacting with the deprotonated Tyr238 hydroxyl group. The second-order rate constant is one order of magnitude higher for CAT-2_intein compared to CAT-2_His6 besides the CAT-2_His6 enzyme does not saturate with o-dianisidine. Because the N-terminal end is close to the entrance channel, its increased size due to the histidine tag can possibly interfere with the channel | Neurospora crassa | ? | - |
- |
|
additional information | CAT-2 requires the Met-Tyr-Trp adduct for catalase activity. CAT-2 Arg426 is oriented towards the M-Y-W adduct, interacting with the deprotonated Tyr238 hydroxyl group. The second-order rate constant is one order of magnitude higher for CAT-2_intein compared to CAT-2_His6 besides the CAT-2_His6 enzyme does not saturate with o-dianisidine. Because the N-terminal end is close to the entrance channel, its increased size due to the histidine tag can possibly interfere with the channel | Neurospora crassa CBS 708.71 | ? | - |
- |
|
additional information | CAT-2 requires the Met-Tyr-Trp adduct for catalase activity. CAT-2 Arg426 is oriented towards the M-Y-W adduct, interacting with the deprotonated Tyr238 hydroxyl group. The second-order rate constant is one order of magnitude higher for CAT-2_intein compared to CAT-2_His6 besides the CAT-2_His6 enzyme does not saturate with o-dianisidine. Because the N-terminal end is close to the entrance channel, its increased size due to the histidine tag can possibly interfere with the channel | Neurospora crassa 74-OR23-1A | ? | - |
- |
|
additional information | CAT-2 requires the Met-Tyr-Trp adduct for catalase activity. CAT-2 Arg426 is oriented towards the M-Y-W adduct, interacting with the deprotonated Tyr238 hydroxyl group. The second-order rate constant is one order of magnitude higher for CAT-2_intein compared to CAT-2_His6 besides the CAT-2_His6 enzyme does not saturate with o-dianisidine. Because the N-terminal end is close to the entrance channel, its increased size due to the histidine tag can possibly interfere with the channel | Neurospora crassa DSM 1257 | ? | - |
- |
|
additional information | CAT-2 requires the Met-Tyr-Trp adduct for catalase activity. CAT-2 Arg426 is oriented towards the M-Y-W adduct, interacting with the deprotonated Tyr238 hydroxyl group. The second-order rate constant is one order of magnitude higher for CAT-2_intein compared to CAT-2_His6 besides the CAT-2_His6 enzyme does not saturate with o-dianisidine. Because the N-terminal end is close to the entrance channel, its increased size due to the histidine tag can possibly interfere with the channel | Neurospora crassa ATCC 24698 | ? | - |
- |
|
additional information | CAT-2 requires the Met-Tyr-Trp adduct for catalase activity. CAT-2 Arg426 is oriented towards the M-Y-W adduct, interacting with the deprotonated Tyr238 hydroxyl group. The second-order rate constant is one order of magnitude higher for CAT-2_intein compared to CAT-2_His6 besides the CAT-2_His6 enzyme does not saturate with o-dianisidine. Because the N-terminal end is close to the entrance channel, its increased size due to the histidine tag can possibly interfere with the channel | Neurospora crassa FGSC 987 | ? | - |
- |
|
o-dianisidine + H2O2 | - |
Neurospora crassa | oxidized o-dianisidine + 2 H2O | - |
? | |
o-dianisidine + H2O2 | - |
Neurospora crassa CBS 708.71 | oxidized o-dianisidine + 2 H2O | - |
? | |
o-dianisidine + H2O2 | - |
Neurospora crassa 74-OR23-1A | oxidized o-dianisidine + 2 H2O | - |
? | |
o-dianisidine + H2O2 | - |
Neurospora crassa DSM 1257 | oxidized o-dianisidine + 2 H2O | - |
? | |
o-dianisidine + H2O2 | - |
Neurospora crassa ATCC 24698 | oxidized o-dianisidine + 2 H2O | - |
? | |
o-dianisidine + H2O2 | - |
Neurospora crassa FGSC 987 | oxidized o-dianisidine + 2 H2O | - |
? |
Subunits | Comment | Organism |
---|---|---|
homodimer | 2 * 88500, SDS-PAGE | Neurospora crassa |
More | the N-terminal domain sequence is well conserved, it contains heme b and is enzymatically active. The heme-less inactive C-terminal domain sequence is less well conserved, but is required for dimerization of the enzyme and for the conformation of the active heme cavity of the N-terminal half | Neurospora crassa |
Synonyms | Comment | Organism |
---|---|---|
CAT-2 | - |
Neurospora crassa |
KatG | - |
Neurospora crassa |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
25 | - |
assay at | Neurospora crassa |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
44 | - |
o-Dianisidine | pH 5.0, 25°C, recombinant intein-tagged enzyme | Neurospora crassa | |
98 | - |
H2O2 | pH 5.0, 25°C, with o-dianisidine, recombinant intein-tagged enzyme | Neurospora crassa | |
551 | - |
H2O2 | pH 5.5, 25°C, recombinant intein-tagged enzyme | Neurospora crassa |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
5 | - |
peroxidase activity | Neurospora crassa |
5.5 | - |
catalyase activity | Neurospora crassa |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
heme | The amino acids residues that are essential for both activities are His91, Asn121 and Arg87 of the distal side of the heme cavity and His279, Asp389 and Trp330 of the proximal side, together with the M-Y-W adduct, Arg426 and Asp120, which are only required for the catalase reaction | Neurospora crassa |
Organism | Comment | pI Value Maximum | pI Value |
---|---|---|---|
Neurospora crassa | isoelectric focusing, recombinant intein-tagged enzyme | - |
6.57 |
Organism | Comment | Expression |
---|---|---|
Neurospora crassa | the catalase-peroxidase in Neurospora crassa, CAT-2, is a developmentally regulated enzyme: both gene expression and CAT-2 activity are induced at the stationary growth phase and during adhesion of hyphae, aerial hyphae growth and conidia formation. Gene expression and CAT-2 activity are induced when the fungus is deprived of a carbon source, or when glucose is replaced by a poor carbon sources | up |
General Information | Comment | Organism |
---|---|---|
additional information | CAT-2 Arg426 is oriented towards the M-Y-W adduct, interacting with the deprotonated Tyr238 hydroxyl group. A perhydroxy modification of the indole nitrogen of Trp90 is oriented toward the catalytic His91. In contrast to cytochrome c peroxidase and ascorbate peroxidase, the catalase-peroxidase heme propionates are not exposed to the solvent. Together with other Nueorspora crassa enzymes that utilize H2O2 as a substrate, CAT-2 has many tryptophan and proline residues at its surface, probably related to H2O2 selection in water. Potentiometric titration of CAT-2 metalloenzyme sample in phosphate buffer, pH 7.0, at 25°C, CAT-2 is reduced with sodium dithionite and reoxidized with potassium ferricyanide following the changes with a spectrophotometer. The amino acids residues that are essential for both activities are His91, Asn121 and Arg87 of the distal side of the heme cavity and His279, Asp389 and Trp330 of the proximal side, together with the M-Y-W adduct, Arg426 and Asp120, which are only required for the catalase reaction. Structure comparisons, overview | Neurospora crassa |
physiological function | CAT-2 is a fungal catalase-peroxidase (CP). In contrast to catalases, CPs are bi-functional enzymes having both catalase activity and peroxidase activity. CPs are homodimeric heme-oxidoreductases. CAT-2 is induced during asexual spore formation in Neurospora crassa | Neurospora crassa |