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Literature summary extracted from

  • Gladyshev, V.N.; Khangulov, S.V.; Stadtman, T.C.
    Properties of the selenium- and molybdenum-containing nicotinic acid hydroxylase from Clostridium barkeri (1996), Biochemistry, 35, 212-223.
    View publication on PubMed

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
1.17.1.5
-
Eubacterium barkeri

General Stability

EC Number General Stability Organism
1.17.1.5 enzyme is most stable at alkaline pH in the presence of glycerol, 20% glycerol and 400 mM KCl stabilize Eubacterium barkeri

Inhibitors

EC Number Inhibitors Comment Organism Structure
1.17.1.5 6-Hydroxynicotinate inhibits effectively Eubacterium barkeri
1.17.1.5 additional information not inhibited by incubation for 1 h at room temperature with 100 mM KCN Eubacterium barkeri
1.17.1.5 Selenophosphate 7 mM, 30 min, anaerobic conditions, reversible complete inactivation, time-dependent Eubacterium barkeri
1.17.1.5 sodium selenide 7 mM, 10 min, anaerobic conditions, reversible complete inactivation, time-dependent Eubacterium barkeri
1.17.1.5 Sulfide 1 mM, 10 min, reversible time-dependent inactivation Eubacterium barkeri

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
1.17.1.5 23000
-
1 * 50000 + 1 * 37000 + 1 * 33000 + 1 * 23000, SDS-PAGE Eubacterium barkeri
1.17.1.5 33000
-
1 * 50000 + 1 * 37000 + 1 * 33000 + 1 * 23000, SDS-PAGE Eubacterium barkeri
1.17.1.5 37000
-
1 * 50000 + 1 * 37000 + 1 * 33000 + 1 * 23000, SDS-PAGE Eubacterium barkeri
1.17.1.5 50000
-
1 * 50000 + 1 * 37000 + 1 * 33000 + 1 * 23000, SDS-PAGE Eubacterium barkeri
1.17.1.5 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

EC Number Organism UniProt Comment Textmining
1.17.1.5 Eubacterium barkeri
-
-
-

Oxidation Stability

EC Number Oxidation Stability Organism
1.17.1.5 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)

EC Number Purification (Comment) Organism
1.17.1.5 112fold purification Eubacterium barkeri

Specific Activity [micromol/min/mg]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
1.17.1.5 18
-
-
Eubacterium barkeri

Storage Stability

EC Number Storage Stability Organism
1.17.1.5 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
1.17.1.5 room temperature, pH8, 9 days, 75% loss of hydroxylase activity Eubacterium barkeri

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.17.1.5 2,3-pyrazinedicarboxylate + H2O + NADP+ 10-20% of the activity with nicotinate Eubacterium barkeri ?
-
?
1.17.1.5 2-pyrazinecarboxylate + H2O + NADP+ equally as effective as nicotinate Eubacterium barkeri ?
-
?
1.17.1.5 3,5-pyridinedicarboxylate + H2O + NADP+ 5-10% of the activity with nicotinate Eubacterium barkeri ?
-
?
1.17.1.5 6-methylnicotinate + H2O + NADP+ 5-10% of the activity with nicotinate Eubacterium barkeri ?
-
?
1.17.1.5 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 Eubacterium barkeri 6-hydroxynicotinate + NADPH
-
?
1.17.1.5 trigonelline + H2O + NADP+ 5-10% of the activity with nicotinate Eubacterium barkeri ?
-
?

Subunits

EC Number Subunits Comment Organism
1.17.1.5 heterotetramer 1 * 50000 + 1 * 37000 + 1 * 33000 + 1 * 23000, SDS-PAGE Eubacterium barkeri
1.17.1.5 heterotetramer 23 kDa protein is less stained and may be a degradation product Eubacterium barkeri

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.17.1.5 additional information
-
assay at room temperature Eubacterium barkeri

pH Stability

EC Number pH Stability pH Stability Maximum Comment Organism
1.17.1.5 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
1.17.1.5 8
-
most stable at alkaline pH Eubacterium barkeri

Cofactor

EC Number Cofactor Comment Organism Structure
1.17.1.5 4Fe-4S-center two [2Fe-2S] clusters, 5-7 atoms Fe per 160 kDa enzyme molecule Eubacterium barkeri
1.17.1.5 FAD 1 FAD molecule per 160 kDa protein protomer Eubacterium barkeri
1.17.1.5 molybdenum cofactor
-
Eubacterium barkeri
1.17.1.5 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
1.17.1.5 additional information NAD+ can not replace NADP+, but NADH can replace NADPH Eubacterium barkeri
1.17.1.5 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 Eubacterium barkeri
1.17.1.5 NADP+
-
Eubacterium barkeri
1.17.1.5 NADPH is able to reduce FAD cofactor, NADH can replace NADPH Eubacterium barkeri
1.17.1.5 NADPH NADPH oxidase activity Eubacterium barkeri