BRENDA - Enzyme Database
show all sequences of 1.17.1.5

Properties of the selenium- and molybdenum-containing nicotinic acid hydroxylase from Clostridium barkeri

Gladyshev, V.N.; Khangulov, S.V.; Stadtman, T.C.; Biochemistry 35, 212-223 (1996)

Data extracted from this reference:

Crystallization (Commentary)
Crystallization
Organism
-
Eubacterium barkeri
General Stability
General Stability
Organism
enzyme is most stable at alkaline pH in the presence of glycerol, 20% glycerol and 400 mM KCl stabilize
Eubacterium barkeri
Inhibitors
Inhibitors
Commentary
Organism
Structure
6-Hydroxynicotinate
inhibits effectively
Eubacterium barkeri
additional information
not inhibited by incubation for 1 h at room temperature with 100 mM KCN
Eubacterium barkeri
Selenophosphate
7 mM, 30 min, anaerobic conditions, reversible complete inactivation, time-dependent
Eubacterium barkeri
sodium selenide
7 mM, 10 min, anaerobic conditions, reversible complete inactivation, time-dependent
Eubacterium barkeri
Sulfide
1 mM, 10 min, reversible time-dependent inactivation
Eubacterium barkeri
Molecular Weight [Da]
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
23000
-
1 * 50000 + 1 * 37000 + 1 * 33000 + 1 * 23000, SDS-PAGE
Eubacterium barkeri
33000
-
1 * 50000 + 1 * 37000 + 1 * 33000 + 1 * 23000, SDS-PAGE
Eubacterium barkeri
37000
-
1 * 50000 + 1 * 37000 + 1 * 33000 + 1 * 23000, SDS-PAGE
Eubacterium barkeri
50000
-
1 * 50000 + 1 * 37000 + 1 * 33000 + 1 * 23000, SDS-PAGE
Eubacterium barkeri
160000
-
major form, occurence of additional enzyme forms of 400 kDa and 120 kDa with same subunit composition, gel filtration and native PAGE
Eubacterium barkeri
Organism
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
Eubacterium barkeri
-
-
-
Oxidation Stability
Oxidation Stability
Organism
exposure of substrate-reduced enzyme to air results in a complete loss of activity, enzyme before reduction is much less sensitive to oxygen inactivation
Eubacterium barkeri
Purification (Commentary)
Commentary
Organism
112fold purification
Eubacterium barkeri
Specific Activity [micromol/min/mg]
Specific Activity Minimum [µmol/min/mg]
Specific Activity Maximum [µmol/min/mg]
Commentary
Organism
18
-
-
Eubacterium barkeri
Storage Stability
Storage Stability
Organism
room temperature, 50 mM Tris-HCl buffer, pH 8.2, after 1 day 40% loss of hydroxylase activity, after 7 days 62% loss of hydroxylase activity, NADPH oxidase and diaphorase activity of enzyme are more stable
Eubacterium barkeri
room temperature, pH8, 9 days, 75% loss of hydroxylase activity
Eubacterium barkeri
Substrates and Products (Substrate)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
2,3-pyrazinedicarboxylate + H2O + NADP+
10-20% of the activity with nicotinate
644535
Eubacterium barkeri
?
-
-
-
?
2-pyrazinecarboxylate + H2O + NADP+
equally as effective as nicotinate
644535
Eubacterium barkeri
?
-
-
-
?
3,5-pyridinedicarboxylate + H2O + NADP+
5-10% of the activity with nicotinate
644535
Eubacterium barkeri
?
-
-
-
?
6-methylnicotinate + H2O + NADP+
5-10% of the activity with nicotinate
644535
Eubacterium barkeri
?
-
-
-
?
nicotinate + H2O + NADP+
high substrate specificity toward electron donor substrates, unsubstituted nitrogen and a carboxyl group at position 3 are absolutely required for substrate hydroxylation and unsubstituted carbon-5 is important for oxidation at carbon-6 of substrate
644535
Eubacterium barkeri
6-hydroxynicotinate + NADPH
-
644535
Eubacterium barkeri
?
trigonelline + H2O + NADP+
5-10% of the activity with nicotinate
644535
Eubacterium barkeri
?
-
-
-
?
Subunits
Subunits
Commentary
Organism
heterotetramer
1 * 50000 + 1 * 37000 + 1 * 33000 + 1 * 23000, SDS-PAGE; 23 kDa protein is less stained and may be a degradation product
Eubacterium barkeri
Temperature Optimum [°C]
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
additional information
-
assay at room temperature
Eubacterium barkeri
pH Stability
pH Stability
pH Stability Maximum
Commentary
Organism
additional information
-
maximal stability during incubation for 14 h at room temperature, after 9 days storage at pH 8, 25% of the initial activity retained
Eubacterium barkeri
8
-
most stable at alkaline pH
Eubacterium barkeri
Cofactor
Cofactor
Commentary
Organism
Structure
4Fe-4S-center
two [2Fe-2S] clusters, 5-7 atoms Fe per 160 kDa enzyme molecule
Eubacterium barkeri
FAD
1 FAD molecule per 160 kDa protein protomer
Eubacterium barkeri
molybdenum cofactor
-
Eubacterium barkeri
molybdopterin
Mo is bound to a dinucleotide form of molybdopterin and is coordinated with selenium, 1 mol Mo per 160 kDa enzyme molecule, molybdenum is directly coordinated to selenium, Se-Mo center is required for enzymic oxidation of nicotinate
Eubacterium barkeri
additional information
enzyme contains labile selenium cofactor which is essential for hydroxylase activity of enzyme, Se is directly coordinated to Mo, up to 1 Se atom per enzyme molecule; NAD+ can not replace NADP+, but NADH can replace NADPH
Eubacterium barkeri
NADP+
-
Eubacterium barkeri
NADPH
is able to reduce FAD cofactor, NADH can replace NADPH; NADPH oxidase activity
Eubacterium barkeri
Cofactor (protein specific)
Cofactor
Commentary
Organism
Structure
4Fe-4S-center
two [2Fe-2S] clusters, 5-7 atoms Fe per 160 kDa enzyme molecule
Eubacterium barkeri
FAD
1 FAD molecule per 160 kDa protein protomer
Eubacterium barkeri
molybdenum cofactor
-
Eubacterium barkeri
molybdopterin
Mo is bound to a dinucleotide form of molybdopterin and is coordinated with selenium, 1 mol Mo per 160 kDa enzyme molecule, molybdenum is directly coordinated to selenium, Se-Mo center is required for enzymic oxidation of nicotinate
Eubacterium barkeri
additional information
enzyme contains labile selenium cofactor which is essential for hydroxylase activity of enzyme, Se is directly coordinated to Mo, up to 1 Se atom per enzyme molecule; NAD+ can not replace NADP+, but NADH can replace NADPH
Eubacterium barkeri
NADP+
-
Eubacterium barkeri
NADPH
is able to reduce FAD cofactor, NADH can replace NADPH; NADPH oxidase activity
Eubacterium barkeri
Crystallization (Commentary) (protein specific)
Crystallization
Organism
-
Eubacterium barkeri
General Stability (protein specific)
General Stability
Organism
enzyme is most stable at alkaline pH in the presence of glycerol, 20% glycerol and 400 mM KCl stabilize
Eubacterium barkeri
Inhibitors (protein specific)
Inhibitors
Commentary
Organism
Structure
6-Hydroxynicotinate
inhibits effectively
Eubacterium barkeri
additional information
not inhibited by incubation for 1 h at room temperature with 100 mM KCN
Eubacterium barkeri
Selenophosphate
7 mM, 30 min, anaerobic conditions, reversible complete inactivation, time-dependent
Eubacterium barkeri
sodium selenide
7 mM, 10 min, anaerobic conditions, reversible complete inactivation, time-dependent
Eubacterium barkeri
Sulfide
1 mM, 10 min, reversible time-dependent inactivation
Eubacterium barkeri
Molecular Weight [Da] (protein specific)
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
23000
-
1 * 50000 + 1 * 37000 + 1 * 33000 + 1 * 23000, SDS-PAGE
Eubacterium barkeri
33000
-
1 * 50000 + 1 * 37000 + 1 * 33000 + 1 * 23000, SDS-PAGE
Eubacterium barkeri
37000
-
1 * 50000 + 1 * 37000 + 1 * 33000 + 1 * 23000, SDS-PAGE
Eubacterium barkeri
50000
-
1 * 50000 + 1 * 37000 + 1 * 33000 + 1 * 23000, SDS-PAGE
Eubacterium barkeri
160000
-
major form, occurence of additional enzyme forms of 400 kDa and 120 kDa with same subunit composition, gel filtration and native PAGE
Eubacterium barkeri
Oxidation Stability (protein specific)
Oxidation Stability
Organism
exposure of substrate-reduced enzyme to air results in a complete loss of activity, enzyme before reduction is much less sensitive to oxygen inactivation
Eubacterium barkeri
Purification (Commentary) (protein specific)
Commentary
Organism
112fold purification
Eubacterium barkeri
Specific Activity [micromol/min/mg] (protein specific)
Specific Activity Minimum [µmol/min/mg]
Specific Activity Maximum [µmol/min/mg]
Commentary
Organism
18
-
-
Eubacterium barkeri
Storage Stability (protein specific)
Storage Stability
Organism
room temperature, 50 mM Tris-HCl buffer, pH 8.2, after 1 day 40% loss of hydroxylase activity, after 7 days 62% loss of hydroxylase activity, NADPH oxidase and diaphorase activity of enzyme are more stable
Eubacterium barkeri
room temperature, pH8, 9 days, 75% loss of hydroxylase activity
Eubacterium barkeri
Substrates and Products (Substrate) (protein specific)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
2,3-pyrazinedicarboxylate + H2O + NADP+
10-20% of the activity with nicotinate
644535
Eubacterium barkeri
?
-
-
-
?
2-pyrazinecarboxylate + H2O + NADP+
equally as effective as nicotinate
644535
Eubacterium barkeri
?
-
-
-
?
3,5-pyridinedicarboxylate + H2O + NADP+
5-10% of the activity with nicotinate
644535
Eubacterium barkeri
?
-
-
-
?
6-methylnicotinate + H2O + NADP+
5-10% of the activity with nicotinate
644535
Eubacterium barkeri
?
-
-
-
?
nicotinate + H2O + NADP+
high substrate specificity toward electron donor substrates, unsubstituted nitrogen and a carboxyl group at position 3 are absolutely required for substrate hydroxylation and unsubstituted carbon-5 is important for oxidation at carbon-6 of substrate
644535
Eubacterium barkeri
6-hydroxynicotinate + NADPH
-
644535
Eubacterium barkeri
?
trigonelline + H2O + NADP+
5-10% of the activity with nicotinate
644535
Eubacterium barkeri
?
-
-
-
?
Subunits (protein specific)
Subunits
Commentary
Organism
heterotetramer
1 * 50000 + 1 * 37000 + 1 * 33000 + 1 * 23000, SDS-PAGE; 23 kDa protein is less stained and may be a degradation product
Eubacterium barkeri
Temperature Optimum [°C] (protein specific)
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
additional information
-
assay at room temperature
Eubacterium barkeri
pH Stability (protein specific)
pH Stability
pH Stability Maximum
Commentary
Organism
additional information
-
maximal stability during incubation for 14 h at room temperature, after 9 days storage at pH 8, 25% of the initial activity retained
Eubacterium barkeri
8
-
most stable at alkaline pH
Eubacterium barkeri
Other publictions for EC 1.17.1.5
No.
1st author
Pub Med
title
organims
journal
volume
pages
year
Activating Compound
Application
Cloned(Commentary)
Crystallization (Commentary)
Engineering
General Stability
Inhibitors
KM Value [mM]
Localization
Metals/Ions
Molecular Weight [Da]
Natural Substrates/ Products (Substrates)
Organic Solvent Stability
Organism
Oxidation Stability
Posttranslational Modification
Purification (Commentary)
Reaction
Renatured (Commentary)
Source Tissue
Specific Activity [micromol/min/mg]
Storage Stability
Substrates and Products (Substrate)
Subunits
Temperature Optimum [°C]
Temperature Range [°C]
Temperature Stability [°C]
Turnover Number [1/s]
pH Optimum
pH Range
pH Stability
Cofactor
Ki Value [mM]
pI Value
IC50 Value
Activating Compound (protein specific)
Application (protein specific)
Cloned(Commentary) (protein specific)
Cofactor (protein specific)
Crystallization (Commentary) (protein specific)
Engineering (protein specific)
General Stability (protein specific)
IC50 Value (protein specific)
Inhibitors (protein specific)
Ki Value [mM] (protein specific)
KM Value [mM] (protein specific)
Localization (protein specific)
Metals/Ions (protein specific)
Molecular Weight [Da] (protein specific)
Natural Substrates/ Products (Substrates) (protein specific)
Organic Solvent Stability (protein specific)
Oxidation Stability (protein specific)
Posttranslational Modification (protein specific)
Purification (Commentary) (protein specific)
Renatured (Commentary) (protein specific)
Source Tissue (protein specific)
Specific Activity [micromol/min/mg] (protein specific)
Storage Stability (protein specific)
Substrates and Products (Substrate) (protein specific)
Subunits (protein specific)
Temperature Optimum [°C] (protein specific)
Temperature Range [°C] (protein specific)
Temperature Stability [°C] (protein specific)
Turnover Number [1/s] (protein specific)
pH Optimum (protein specific)
pH Range (protein specific)
pH Stability (protein specific)
pI Value (protein specific)
Expression
General Information
General Information (protein specific)
Expression (protein specific)
KCat/KM [mM/s]
KCat/KM [mM/s] (protein specific)
745661
Yuan
Identification and characteri ...
Pusillimonas sp., Pusillimonas sp. T2 / CCTCC M2014272
Lett. Appl. Microbiol.
66
321-328
2018
2
-
1
-
-
-
-
1
-
-
-
2
-
5
-
-
-
-
-
1
-
-
8
-
1
-
-
-
1
-
-
3
-
-
-
2
-
1
3
-
-
-
-
-
-
1
-
-
-
2
-
-
-
-
-
1
-
-
8
-
1
-
-
-
1
-
-
-
-
1
1
-
-
-
714382
Yang
Cloning, expression and functi ...
Comamonas testosteroni, Comamonas testosteroni JA1
Biodegradation
21
593-602
2010
-
-
1
-
1
-
-
-
-
1
3
12
-
7
-
-
-
-
-
-
-
-
12
1
1
-
-
-
1
-
-
1
-
-
-
-
-
3
3
-
1
-
-
-
-
-
-
3
3
12
-
-
-
-
-
-
-
-
12
3
3
-
-
-
3
-
-
-
-
1
3
-
-
-
696503
Yang
Cloning, heterologous expressi ...
Pseudomonas putida, Pseudomonas putida KT 2240
Biodegradation
20
541-549
2009
-
-
1
-
-
-
-
-
-
-
1
-
-
7
-
-
-
-
-
-
1
-
4
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
1
-
4
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
700995
Wagener
The Mo-Se active site of nicot ...
Eubacterium barkeri
Proc. Natl. Acad. Sci. USA
106
11055-11060
2009
-
-
-
1
-
-
-
-
-
-
3
-
-
2
-
-
1
-
-
-
-
-
1
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
3
-
-
-
-
1
-
-
-
-
1
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
658869
Petersen
-
Biocatalysis - preparation and ...
Achromobacter xylosoxidans, Alcaligenes faecalis
Green Chem.
2
99-106
1999
-
-
-
-
-
-
-
-
-
-
-
-
-
2
-
-
-
-
-
-
-
-
2
-
2
-
-
-
2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
2
-
2
-
-
-
2
-
-
-
-
-
-
-
-
-
657616
Wieser
-
Bioconversion of 2-cyanopyrazi ...
Agrobacterium sp.
Appl. Microbiol. Biotechnol.
48
174-180
1997
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
1
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
644535
Gladyshev
Properties of the selenium- an ...
Eubacterium barkeri
Biochemistry
35
212-223
1996
-
-
-
1
-
1
5
-
-
-
5
-
-
1
1
-
1
-
-
-
1
2
6
1
1
-
-
-
-
-
2
7
-
-
-
-
-
-
7
1
-
1
-
5
-
-
-
-
5
-
-
1
-
1
-
-
1
2
6
1
1
-
-
-
-
-
2
-
-
-
-
-
-
-
644528
Gladyshev
Nicotinic acid hydroxylase fro ...
Eubacterium barkeri
Proc. Natl. Acad. Sci. USA
91
232-236
1994
-
-
-
-
-
-
-
-
-
-
5
-
-
1
1
-
1
-
-
-
3
-
1
1
-
-
-
-
-
-
-
7
-
-
-
-
-
-
7
-
-
-
-
-
-
-
-
-
5
-
-
1
-
1
-
-
3
-
1
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
644529
Hurh
-
Purification and characterizat ...
Delftia acidovorans, Pseudomonas fluorescens, Pseudomonas fluorescens TN5, Serratia marcescens, Serratia marcescens IFO 12648
J. Ferment. Bioeng.
78
19-26
1994
-
-
-
-
-
1
10
5
1
-
2
4
-
6
-
-
1
-
-
-
5
-
16
1
2
-
1
-
1
1
1
2
-
-
-
-
-
-
2
-
-
1
-
10
-
5
1
-
2
4
-
-
-
1
-
-
5
-
16
1
2
-
1
-
1
1
1
-
-
-
-
-
-
-
644533
Nagel
-
Purification and characterizat ...
Bacillus niacini
Arch. Microbiol.
154
605-613
1990
-
-
-
-
-
1
2
1
1
-
4
1
-
1
-
-
1
-
-
-
1
1
4
1
1
-
-
-
1
-
-
4
-
-
-
-
-
-
4
-
-
1
-
2
-
1
1
-
4
1
-
-
-
1
-
-
1
1
4
1
1
-
-
-
1
-
-
-
-
-
-
-
-
-
644532
Nagel
-
Molybdenum-dependent degradati ...
Bacillus niacini, Bacillus niacini DSM 2923
FEMS Microbiol. Lett.
59
147-152
1989
-
-
-
-
1
-
1
-
-
1
-
2
-
2
-
-
-
-
-
-
1
-
4
-
1
-
-
-
-
-
-
1
-
-
-
-
-
-
1
-
1
-
-
1
-
-
-
1
-
2
-
-
-
-
-
-
1
-
4
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
395166
Kruger
-
Evidence for the presence of b ...
Bacillus niacini
FEMS Microbiol. Lett.
48
225-227
1987
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
1
-
-
-
1
-
-
-
-
-
-
-
-
-
-
3
-
-
-
-
-
-
3
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
644530
Hirschberg
Oxidation of nicotinic acid by ...
Bacillus sp. (in: Bacteria)
J. Bacteriol.
108
751-756
1971
-
-
-
-
-
-
4
1
1
-
1
1
-
1
-
-
1
-
1
-
1
-
2
-
1
-
-
-
-
-
-
2
-
-
-
-
-
-
2
-
-
-
-
4
-
1
1
-
1
1
-
-
-
1
1
-
1
-
2
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
644534
Holcenberg
Nicotinic acid metabolism. III ...
Clostridium sp.
J. Biol. Chem.
244
1194-1203
1969
4
-
-
-
-
-
-
2
1
-
1
1
-
1
-
-
1
-
-
-
1
-
5
-
-
-
1
-
5
-
-
5
-
-
-
4
-
-
5
-
-
-
-
-
-
2
1
-
1
1
-
-
-
1
-
-
1
-
5
-
-
-
1
-
5
-
-
-
-
-
-
-
-
-