Literature summary for 1.11.1.21 extracted from

Vega-Garcia, V.; Diaz-Vilchis, A.; Saucedo-Vazquez, J.P.; Solano-Peralta, A.; Rudino-Pinera, E.; Hansberg, W.
Structure, kinetics, molecular and redox properties of a cytosolic and developmentally regulated fungal catalase-peroxidase (2018), Arch. Biochem. Biophys., 640, 17-26 .

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Cloned(Commentary)

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 (Commentary)

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

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 [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

Localization	Comment	Organism	GeneOntology No.	Textmining
cytosol	CAT-2 is mainly if not entirely cytoplasmic	Neurospora crassa	5829	-

Metals/Ions

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 [Da]	Molecular Weight Maximum [Da]	Comment	Organism
177000	-	recombinant intein-tagged enzyme, gel filtration	Neurospora crassa

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.
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

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 (Commentary)

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

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 [micromol/min/mg]

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 and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
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

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

Synonyms	Comment	Organism
CAT-2	-	Neurospora crassa
KatG	-	Neurospora crassa

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
25	-	assay at	Neurospora crassa

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
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

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
5	-	peroxidase activity	Neurospora crassa
5.5	-	catalyase activity	Neurospora crassa

Cofactor

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

pI Value

Organism	Comment	pI Value Maximum	pI Value
Neurospora crassa	isoelectric focusing, recombinant intein-tagged enzyme	-	6.57

Expression

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

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