Literature summary for 3.1.1.117 extracted from

Huynh, H.; Ishii, N.; Matsuo, I.; Arioka, M.
A novel glucuronoyl esterase from Aspergillus fumigatus - the role of conserved Lys residue in the preference for 4-O-methyl glucuronoyl esters (2018), Appl. Microbiol. Biotechnol., 102, 2191-2201 .

Search for more literature for 3.1.1.117

Activating Compound

Activating Compound	Comment	Organism	Structure
additional information	no significant effect by EDTA, glycerol, acetic anhydride, ascorbic acid, dimethyl sulfoxide, SDS, and urea at 10 mM	Aspergillus fumigatus
Tween 80	activates 2.3fold at 10 mM	Aspergillus fumigatus

Application

Application	Comment	Organism
industry	the enzyme is a potentially industrially applicable enzyme due to its characteristic as a thermophilic enzyme with the favorable temperature of 40-50°C at pH 5	Aspergillus fumigatus

Cloned(Commentary)

Cloned (Comment)	Organism
gene Afu6g14390, recombinant expression of Myc-His-tagged enzyme AfGE in Aspergillus oryzae strain RIB40 from vector pUNA-AfGE-Myc-His under the control of a strong promoter of alpha-amylase (amyB), subcloning of the enzyme in Escherichia coli strain DH5alpha and of the vector in Aspergillus oryzae strain niaD300, the recombinant enzyme is secreted into the medium. Recombinant expression of wild-type and mutant enzymes in Pichia pastoris strain KM71H	Aspergillus fumigatus
heterologously expressed in Aspergillus oryzae RIB40 strain (ATCC42149) under the control of a strong promoter of alpha-amylase (amyB)	Aspergillus fumigatus

Cloned (Comment)

Organism

gene Afu6g14390, recombinant expression of Myc-His-tagged enzyme AfGE in Aspergillus oryzae strain RIB40 from vector pUNA-AfGE-Myc-His under the control of a strong promoter of alpha-amylase (amyB), subcloning of the enzyme in Escherichia coli strain DH5alpha and of the vector in Aspergillus oryzae strain niaD300, the recombinant enzyme is secreted into the medium. Recombinant expression of wild-type and mutant enzymes in Pichia pastoris strain KM71H

Aspergillus fumigatus

heterologously expressed in Aspergillus oryzae RIB40 strain (ATCC42149) under the control of a strong promoter of alpha-amylase (amyB)

Aspergillus fumigatus

Protein Variants

Protein Variants	Comment	Organism
K209A	site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme	Aspergillus fumigatus
K209E	site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme	Aspergillus fumigatus
K209Q	site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzyme	Aspergillus fumigatus
K209R	site-directed mutagenesis, the mutant shows altered substrate specificity compared to the wild-type enzymethe mutant shows altered substrate specificity compared to the wild-type enzyme	Aspergillus fumigatus

Inhibitors

Inhibitors	Comment	Organism
2-mercaptoethanol	10 mM	Aspergillus fumigatus
Al3+	1 mM, slight inhibition; slight inhibition at 1 mM	Aspergillus fumigatus
Ca2+	1 mM, about 40% inhibition; about 40% inhibition at 1 mM	Aspergillus fumigatus
Cu2+	1 mM, slight inhibition; slight inhibition at 1 mM	Aspergillus fumigatus
dithiothreitol	10 mM	Aspergillus fumigatus
Fe2+	1 mM, slight inhibition; slight inhibition at 1 mM	Aspergillus fumigatus
Fe3+	about 40% inhibition at 1 mM	Aspergillus fumigatus
Li+	1 mM, slight inhibition; slight inhibition at 1 mM	Aspergillus fumigatus
Mn2+	1 mM, slight inhibition; slight inhibition at 1 mM	Aspergillus fumigatus
additional information	AfGE activity is not affected by EDTA, indicating that this enzyme is not a metalloenzyme. No significant effect by glycerol, acetic anhydride, ascorbic acid, dimethyl sulfoxide, SDS, and urea at 10 mM; the enzyme is not affected by EDTA, indicating that this enzyme is not a metalloenzyme	Aspergillus fumigatus
Ni2+	1 mM, slight inhibition; about 40% inhibition at 1 mM	Aspergillus fumigatus
PMSF	10 mM	Aspergillus fumigatus

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
15.8	-	3-(4-methoxyphenyl) propyl methyl 4-O-methyl-alpha-D-glucopyranosiduronate	pH 5.0, 30°C	Aspergillus fumigatus
16.4	-	benzyl D-glucuronate	pH 5.0, 30°C	Aspergillus fumigatus
23.7	-	benzyl methyl 4-O-methyl-alpha-D-glucopyranosiduronate	pH 5.0, 30°C	Aspergillus fumigatus
38	-	benzyl methyl alpha-D-glucopyranosiduronate	pH 5.0, 30°C	Aspergillus fumigatus

Localization

Localization	Comment	Organism	GeneOntology No.	Textmining
extracellular	-	Aspergillus fumigatus	-	-

Metals/Ions

Metals/Ions	Comment	Organism
Mg2+	1 mM, increases activity	Aspergillus fumigatus
Mg2+	activates at 1 mM	Aspergillus fumigatus
additional information	the enzyme is not affected by Zn2+ at 1 mM. AfGE activity is not affected by EDTA, indicating that this enzyme is not a metalloenzyme	Aspergillus fumigatus

Molecular Weight [Da]

Molecular Weight [Da]	Molecular Weight Maximum [Da]	Comment	Organism
44000	-	calculated, enzyme with a Myc-His tag added to the C-terminal end of AfGE by the fusion PCR	Aspergillus fumigatus

Organism

Organism	UniProt	Comment	Textmining
Aspergillus fumigatus	Q4WL89	-	-
Aspergillus fumigatus	Q4WL89	i.e. Aspergillus fumigatus	-
Aspergillus fumigatus ATCC MYA-4609	Q4WL89	-	-
Aspergillus fumigatus ATCC MYA-4609	Q4WL89	i.e. Aspergillus fumigatus	-

Posttranslational Modification

Posttranslational Modification	Comment	Organism
glycoprotein	the enzyme has two potential N-glycosylation sites	Aspergillus fumigatus

Purification (Commentary)

Purification (Comment)	Organism
-	Aspergillus fumigatus
recombinant Myc-His-tagged enzyme AfGE from Aspergillus oryzae strain RIB40 by nickel affinity chromatography, and anion exchange chromatography	Aspergillus fumigatus

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
3-(4-methoxyphenyl) propyl methyl 4-O-methyl-alpha-D-glucopyranosiduronate + H2O	-	Aspergillus fumigatus	3-(4-methoxyphenyl)propan-1-ol + methyl 4-O-methyl-alpha-D-glucopyranosiduronic acid	-	?
3-(4-methoxyphenyl) propyl methyl 4-O-methyl-alpha-D-glucopyranosiduronate + H2O	-	Aspergillus fumigatus ATCC MYA-4609	3-(4-methoxyphenyl)propan-1-ol + methyl 4-O-methyl-alpha-D-glucopyranosiduronic acid	-	?
3-(4-methoxyphenyl) propyl methyl 4-O-methyl-alpha-D-glucopyranosiduronate + H2O	the substrate mimics the ester linkage between lignin and hemicellulose	Aspergillus fumigatus	methanol + 3-(4-methoxyphenyl) propyl 4-O-methyl-alpha-D-glucopyranosiduronate	-	?
benzyl D-glucuronate + H2O	-	Aspergillus fumigatus	benzyl alcohol + D-glucuronic acid	-	?
benzyl D-glucuronate + H2O	-	Aspergillus fumigatus ATCC MYA-4609	benzyl alcohol + D-glucuronic acid	-	?
benzyl D-glucuronate + H2O	-	Aspergillus fumigatus	benzyl alcohol + glucuronic acid	-	?
benzyl D-glucuronate + H2O	-	Aspergillus fumigatus ATCC MYA-4609	benzyl alcohol + glucuronic acid	-	?
benzyl methyl 4-O-methyl-alpha-D-glucopyranosiduronate + H2O	-	Aspergillus fumigatus	benzyl alcohol + methyl 4-O-methyl-alpha-D-glucopyranosiduronic acid	-	?
benzyl methyl 4-O-methyl-alpha-D-glucopyranosiduronate + H2O	-	Aspergillus fumigatus ATCC MYA-4609	benzyl alcohol + methyl 4-O-methyl-alpha-D-glucopyranosiduronic acid	-	?
benzyl methyl alpha-D-glucopyranosiduronate + H2O	-	Aspergillus fumigatus	benzyl alcohol + methyl alpha-D-glucopyranosiduronic acid	-	?
additional information	Lys209 plays an important role in the preference for the substrates containing 4-O-methyl group in the glucopyranose ring. The purified recombinant enzyme displays the ability to degrade the synthetic substrates mimicking the ester linkage between hemicellulose and lignin. Self-degradation of the substrates under high alkaline condition	Aspergillus fumigatus	?	-	-
additional information	Lys209 plays an important role in the preference for the substrates containing 4-O-methyl group in the glucopyranose ring. The purified recombinant enzyme displays the ability to degrade the synthetic substrates mimicking the ester linkage between hemicellulose and lignin. Self-degradation of the substrates under high alkaline condition	Aspergillus fumigatus ATCC MYA-4609	?	-	-

Subunits

Subunits	Comment	Organism
?	x * 44000, recombinant deglycosylated Myc-His-tagged enzyme AfGE, SDS-PAGE	Aspergillus fumigatus

Synonyms

Synonyms	Comment	Organism
AfGE	-	Aspergillus fumigatus
Afu6g14390	-	Aspergillus fumigatus
Cip2	-	Aspergillus fumigatus
glucuronoyl esterase	-	Aspergillus fumigatus

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
40	-	-	Aspergillus fumigatus
40	-	recombinant enzyme	Aspergillus fumigatus

Temperature Range [°C]

Temperature Minimum [°C]	Temperature Maximum [°C]	Comment	Organism
30	70	30°C: about 80% of maximal activity, 70°C: about 60% of maximal activity	Aspergillus fumigatus
30	65	over 50% of maximal activity within this range, profile overview	Aspergillus fumigatus

Temperature Stability [°C]

Temperature Stability Minimum [°C]	Temperature Stability Maximum [°C]	Comment	Organism
30	50	30 min, pH 7.0, stable	Aspergillus fumigatus
30	50	purified recombinant enzyme, pH 7.0, 30 min, completely stable at	Aspergillus fumigatus
60	-	30 min, pH 7.0, about 30% loss of activity	Aspergillus fumigatus
70	-	30 min, pH 7.0, about 70% loss of activity	Aspergillus fumigatus
70	-	purified recombinant enzyme, pH 7.0, 30 min, loss of 60% activity	Aspergillus fumigatus

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
23.8	-	benzyl D-glucuronate	pH 5.0, 30°C	Aspergillus fumigatus
42.7	-	benzyl methyl alpha-D-glucopyranosiduronate	pH 5.0, 30°C	Aspergillus fumigatus
66.4	-	benzyl methyl 4-O-methyl-alpha-D-glucopyranosiduronate	pH 5.0, 30°C	Aspergillus fumigatus
67.1	-	3-(4-methoxyphenyl) propyl methyl 4-O-methyl-alpha-D-glucopyranosiduronate	pH 5.0, 30°C	Aspergillus fumigatus

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
5	-	-	Aspergillus fumigatus
5	-	recombinant enzyme	Aspergillus fumigatus

pH Range

pH Minimum	pH Maximum	Comment	Organism
4	8	over 60% of maximal activity within this range, profile overview	Aspergillus fumigatus

pH Stability

pH Stability	pH Stability Maximum	Comment	Organism
3	9	purified recombinant enzyme, 30°C, 30 min, over 40% of maximal activity remaining within this range, 80% at pH 5.0	Aspergillus fumigatus
4	-	30°C, 30 min, activity is significantly lost after pre-incubation in pHs lower than 4	Aspergillus fumigatus
5	7	30°C, 30 min, high stability	Aspergillus fumigatus
8	-	30°C, 30 min, activity is significantly lost after pre-incubation in pHs higher than 8	Aspergillus fumigatus

pI Value

Organism	Comment	pI Value Maximum	pI Value
Aspergillus fumigatus	sequence calculation	-	6.1

General Information

General Information	Comment	Organism
evolution	glucuronoyl esterases (GEs) belong to the family 15 of carbohydrate esterases (CE15). Sequence comparison with enzyme Cip2 (UniProt ID G0RV93) from Hypocrea jecorina strain QM6a	Aspergillus fumigatus
additional information	role of conserved Lys209 residue in the preference for 4-O-methyl glucuronoyl esters, AfGE docking study and structure homology molecular modeling using the crystal structure of StGE2 S213A mutant docked with methyl 4-O-methyl-beta-D-glucopyranuronate (PDB ID 4G4J) as the template, overview	Aspergillus fumigatus
physiological function	cellulose in plant cell walls is mainly covered by hemicellulose and lignin, and thus efficient removal of these components is thought to be a key step in the optimal utilization of lignocellulose. The carbohydrate esterase (CE) 15 family of glucuronoyl esterases (GEs), which cleave the linkages between the free carboxyl group of D-glucuronic acid in hemicellulose and the benzyl groups in lignin residues, can contribute to this process	Aspergillus fumigatus

kcat/KM [mM/s]

kcat/KM Value [1/mMs^-1]	kcat/KM Value Maximum [1/mMs^-1]	Substrate	Comment	Organism
1.12	-	benzyl methyl alpha-D-glucopyranosiduronate	pH 5.0, 30°C	Aspergillus fumigatus
1.45	-	benzyl D-glucuronate	pH 5.0, 30°C	Aspergillus fumigatus
2.8	-	benzyl methyl 4-O-methyl-alpha-D-glucopyranosiduronate	pH 5.0, 30°C	Aspergillus fumigatus
4.24	-	3-(4-methoxyphenyl) propyl methyl 4-O-methyl-alpha-D-glucopyranosiduronate	pH 5.0, 30°C	Aspergillus fumigatus