Literature summary for 1.1.3.9 extracted from

Paukner, R.; Staudigl, P.; Choosri, W.; Sygmund, C.; Halada, P.; Haltrich, D.; Leitner, C.
Galactose oxidase from Fusarium oxysporum - expression in E. coli and P. pastoris and biochemical characterization (2014), PLoS ONE, 9, e100116 .

Cloned(Commentary)

Cloned (Comment)	Organism
gene GAOA, sequence comparison to the Fusarium graminearum enzyne, sucessful recombinant expression of C-terminally His-tagged enzyme in Escherichia coli even without codon optimization or further amino acid substitutions. The C-terminal His-tag added to recombinant GalOx from Fusarium oxysporum does not interfere with the catalytic activity. Sucessful recombinant expression of C-terminally His-tagged full-length enzyme and enzyme with the alpha-factor signal sequence of Saccharomyces cerevisiae in Pichia pastoris	Fusarium oxysporum

Cloned (Comment)

Organism

gene GAOA, sequence comparison to the Fusarium graminearum enzyne, sucessful recombinant expression of C-terminally His-tagged enzyme in Escherichia coli even without codon optimization or further amino acid substitutions. The C-terminal His-tag added to recombinant GalOx from Fusarium oxysporum does not interfere with the catalytic activity. Sucessful recombinant expression of C-terminally His-tagged full-length enzyme and enzyme with the alpha-factor signal sequence of Saccharomyces cerevisiae in Pichia pastoris

Fusarium oxysporum

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism	Structure
47	-	D-galactose	pH 7.0, 40°C	Fusarium oxysporum

Localization

Localization	Comment	Organism	GeneOntology No.	Textmining
extracellular	-	Fusarium oxysporum	-	-

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Cu2+	the enzyme contains a unique metalloradical complex consisting of a copper atom and a tyrosine residue covalently attached to the sulfur of a cysteine	Fusarium oxysporum
additional information	GalOx is unusual among metalloenzymes in catalyzing a two-electron redox chemistry at a mononuclear metal ion active site	Fusarium oxysporum

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
D-galactose + O2	Fusarium oxysporum	-	D-galacto-hexodialdose + H2O2	-	?
D-galactose + O2	Fusarium oxysporum G12	-	D-galacto-hexodialdose + H2O2	-	?

Organism

Organism	UniProt	Comment	Textmining
Fusarium oxysporum	V5NQ89	-	-
Fusarium oxysporum G12	V5NQ89	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant C-terminally His-tagged enzyme 28fold from Escherichia coli, recombinant C-terminally His-tagged full-length enzyme (including the native prepro signal sequence) 6fold from Pichia, and recombinant C-terminally His-tagged enzyme with alpha-factor signal sequence 22fold from Pichia pastoris	Fusarium oxysporum

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg]	Specific Activity Maximum [µmol/min/mg]	Comment	Organism
61	-	purified recombinant His-tagged full-length enzyme, expressed from Pichia pastoris using the alpha-factor signal of Saccharomyces cerevisiae, pH 7.0, 40°C	Fusarium oxysporum
63.2	-	purified recombinant His-tagged full-length enzyme, expressed from Pichia pastoris, pH 7.0, 40°C	Fusarium oxysporum
65.4	-	purified recombinant His-tagged enzyme, expressed from Escherichia coli, pH 7.0, 40°C	Fusarium oxysporum

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
1-methyl-beta-D-galactopyranoside + O2	best substrate	Fusarium oxysporum	? + H2O2	-	?
1-methyl-beta-D-galactopyranoside + O2	best substrate	Fusarium oxysporum G12	? + H2O2	-	?
D-galactose + O2	-	Fusarium oxysporum	D-galacto-hexodialdose + H2O2	-	?
D-galactose + O2	-	Fusarium oxysporum G12	D-galacto-hexodialdose + H2O2	-	?
additional information	GalOx catalyzes the oxidation of primary alcohols (e.g. the hydroxyl group at the C6 position in D-galactose) to aldehydes, accompanied by the reduction of molecular oxygen to hydrogen peroxide, and shows a broad substrate tolerance, yet strict stereospecificity, for various alcohol substrates. Because of the high sensitivity of GalOx to the stereo configuration of the C4 hydroxyl group D-glucose is not a substrate for the enzyme	Fusarium oxysporum	?	-	?
additional information	GalOx catalyzes the oxidation of primary alcohols (e.g. the hydroxyl group at the C6 position in D-galactose) to aldehydes, accompanied by the reduction of molecular oxygen to hydrogen peroxide, and shows a broad substrate tolerance, yet strict stereospecificity, for various alcohol substrates. Because of the high sensitivity of GalOx to the stereo configuration of the C4 hydroxyl group D-glucose is not a substrate for the enzyme	Fusarium oxysporum G12	?	-	?

Subunits

Subunits	Comment	Organism
monomer	1 * 72000, recombinant His-tagged enzyme, SDS-PAGE, structure analysis, overview	Fusarium oxysporum
More	trypsin digestion and peptide mapping, mass spectrometric analysis	Fusarium oxysporum

Synonyms

Synonyms	Comment	Organism
galactose 6-oxidase	-	Fusarium oxysporum
GalOx	-	Fusarium oxysporum

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
40	-	-	Fusarium oxysporum

Temperature Stability [°C]

Temperature Stability Minimum [°C]	Temperature Stability Maximum [°C]	Comment	Organism
additional information	-	the thermostability of GalOx from Fusarium oxysporum expressed in Escherichia coli is significantly lower than the thermostability of enzymes from other Fusarium strains	Fusarium oxysporum
30	-	purified recombinant enzyme, pH 7.0, completely stable for at least 24 h	Fusarium oxysporum
40	-	purified recombinant enzyme, pH 7.0, loss of 80% activity after 24 h	Fusarium oxysporum
50	-	purified recombinant enzyme, pH 7.0, inactivation within 2 h	Fusarium oxysporum

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7	-	-	Fusarium oxysporum

pH Range

pH Minimum	pH Maximum	Comment	Organism
6	9	more than 80% of the maximum activity between pH 6.0 and 9.0, maximum activity occurs at pH 7.0 (phosphate buffer) and 8.0 (Tris buffer), respectively. At pH values below pH 5.0 the enzyme loses its activity	Fusarium oxysporum

Cofactor

Cofactor	Comment	Organism	Structure
Cys-Tyr cofactor	the enzyme contains a unique metalloradical complex consisting of a copper atom and a tyrosine residue covalently attached to the sulfur of a cysteine. The Tyr-Cys crosslink is essential for the catalytic activity of GalOx. The formation of the TyrN-Cys redox cofactor in GalOx is a self-processing reaction requiring only the apoprotein, copper, and dioxygen; no other proteins or enzymes are required for the processing and assembly of the catalytically active enzyme	Fusarium oxysporum

General Information

General Information	Comment	Organism
evolution	galactose oxidase is a member of the radical copper oxidase family and is classified as a member of the carbohydrate active-enzyme family AA5, subfamiliy 2	Fusarium oxysporum
additional information	GalOx is unusual among metalloenzymes in catalyzing a two-electron redox chemistry at a mononuclear metal ion active site	Fusarium oxysporum

kcat/KM [mM/s]

kcat/KM Value [1/mMs^-1]	kcat/KM Value Maximum [1/mMs^-1]	Substrate	Comment	Organism	Structure
2.2	-	1-methyl-beta-D-galactopyranoside	pH 7.0, 40°C	Fusarium oxysporum