Literature summary for 1.3.1.14 extracted from

  • Argyrou, A.; Washabaugh, M.W.; Pickart, C.M.
    Dihydroorotate dehydrogenase from Clostridium oroticum is a class 1B enzyme and utilizes a concerted mechanism of catalysis (2000), Biochemistry, 39, 10373-10384.
    View publication on PubMed

Activating Compound

Activating Compound Comment Organism Structure
glutathione activates Faecalicatena orotica
dithiothreitol activates Faecalicatena orotica
2-mercaptoethanol activates Faecalicatena orotica
Mercaptoacetic acid activates Faecalicatena orotica
cysteine L-cysteine activates strongly, D-cysteine is also an activator, 1.8 mM L-cysteine is required at pH 8 for half-maximal activity, 10 mM for near-maximal activity Faecalicatena orotica
additional information thiol activation involves reduction of side chain of a cysteine residue corresponding to Cys-130, which functions as a general base Faecalicatena orotica
additional information not activated by L-serine or L-alanine Faecalicatena orotica

Cloned(Commentary)

Cloned (Comment) Organism
genes encoding the two alpha and beta polypeptides, expression in Escherichia coli Bacillus subtilis
genes encoding the two alpha and beta polypeptides, expression in Escherichia coli Lactococcus lactis

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.09
-
L-dihydroorotate pH 8, 25°C Faecalicatena orotica
0.13
-
NAD+ pH 8, 25°C Faecalicatena orotica

Localization

Localization Comment Organism GeneOntology No. Textmining
cytosol
-
Bacillus subtilis
-
-
cytosol
-
Lactococcus lactis
-
-
cytosol
-
Enterococcus faecalis
-
-
cytosol
-
Faecalicatena orotica
-
-

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
120000
-
analytical gel filtration Faecalicatena orotica
32000
-
alpha2,beta2, 2 * 32000 + 2 * 28000, SDS-PAGE Faecalicatena orotica
28000
-
alpha2,beta2, 2 * 32000 + 2 * 28000, SDS-PAGE Faecalicatena orotica

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
dihydroorotate + NAD+ Lactococcus lactis fourth step and sole redox reaction in the pyrimidine de novo biosynthetic pathway orotate + NADH + H+
-
r
dihydroorotate + NAD+ Enterococcus faecalis fourth step and sole redox reaction in the pyrimidine de novo biosynthetic pathway orotate + NADH + H+
-
r
dihydroorotate + NAD+ Faecalicatena orotica fourth step and sole redox reaction in the pyrimidine de novo biosynthetic pathway orotate + NADH + H+
-
r

Organism

Organism UniProt Comment Textmining
Bacillus subtilis
-
-
-
Faecalicatena orotica
-
identical with Clostridium oroticum
-
Enterococcus faecalis
-
-
-
Lactococcus lactis
-
family IB enzyme, also contains family IA enzyme
-

Purification (Commentary)

Purification (Comment) Organism
-
Faecalicatena orotica

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
additional information
-
-
Faecalicatena orotica

Storage Stability

Storage Stability Organism
-20°C, 0.27 M sodium phosphate, pH 6.3, over 6 months, less than 15% loss of activity Faecalicatena orotica

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
(S)-dihydroorotate + NAD+ equilibrium favours direction of orotate reduction Faecalicatena orotica orotate + NADH + H+
-
?
(S)-dihydroorotate + NAD+ equilibrium favours direction of orotate reduction Faecalicatena orotica orotate + NADH + H+
-
r
(S)-dihydroorotate + NAD+ mechanism of dehydrogenation, mechanism of electron transfer: L-dihydroorotate transfers a pair of electrons to FMN via iron-sulfur cluster via FAD to NAD+ Lactococcus lactis orotate + NADH + H+
-
?
(S)-dihydroorotate + NAD+ mechanism of dehydrogenation, mechanism of electron transfer: L-dihydroorotate transfers a pair of electrons to FMN via iron-sulfur cluster via FAD to NAD+ Lactococcus lactis orotate + NADH + H+
-
r
(S)-dihydroorotate + NAD+ NAD+ as ultimate electron acceptor Bacillus subtilis orotate + NADH + H+
-
?
(S)-dihydroorotate + NAD+ NAD+ as ultimate electron acceptor Lactococcus lactis orotate + NADH + H+
-
?
(S)-dihydroorotate + NAD+ NAD+ as ultimate electron acceptor Lactococcus lactis orotate + NADH + H+
-
r
(S)-dihydroorotate + NAD+ NAD+ as ultimate electron acceptor Enterococcus faecalis orotate + NADH + H+
-
?
(S)-dihydroorotate + NAD+ NAD+ as ultimate electron acceptor Faecalicatena orotica orotate + NADH + H+
-
?
(S)-dihydroorotate + NAD+ NAD+ as ultimate electron acceptor Faecalicatena orotica orotate + NADH + H+
-
r
dihydroorotate + NAD+ fourth step and sole redox reaction in the pyrimidine de novo biosynthetic pathway Lactococcus lactis orotate + NADH + H+
-
r
dihydroorotate + NAD+ fourth step and sole redox reaction in the pyrimidine de novo biosynthetic pathway Enterococcus faecalis orotate + NADH + H+
-
r
dihydroorotate + NAD+ fourth step and sole redox reaction in the pyrimidine de novo biosynthetic pathway Faecalicatena orotica orotate + NADH + H+
-
r

Subunits

Subunits Comment Organism
heterotetramer alpha2,beta2 Bacillus subtilis
heterotetramer alpha2,beta2, 2 * 32000 + 2 * 28000, SDS-PAGE Faecalicatena orotica
heterotetramer alpha2,beta2, family IB enzyme Lactococcus lactis
heterotetramer alpha subunit is named PyrDB, beta subunit is named PyrK Bacillus subtilis
heterotetramer alpha subunit is named PyrDB, beta subunit is named PyrK Lactococcus lactis

Synonyms

Synonyms Comment Organism
More family IB dihydroorotate dehydrogenase Lactococcus lactis
More family IB dihydroorotate dehydrogenase Faecalicatena orotica

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
25
-
assay at Faecalicatena orotica

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
22
-
L-dihydroorotate formation of orotate, in absence of NAD+ or NADH, pH 8, 10 mM cysteine Faecalicatena orotica
45
-
L-dihydroorotate in presence of NAD+ Faecalicatena orotica
3.97
-
Orotate formation of L-dihydroorotate, in absence of NAD+ or NADH, pH 8, 10 mM cysteine Faecalicatena orotica

pH Range

pH Minimum pH Maximum Comment Organism
6.4 8.9 below and above the turnover number decreases Faecalicatena orotica

Cofactor

Cofactor Comment Organism Structure
FAD FAD is located on the beta subunit Lactococcus lactis
FAD two molecules FAD per heterotetramer Bacillus subtilis
FAD two molecules FAD per heterotetramer Lactococcus lactis
FAD two molecules FAD per heterotetramer Enterococcus faecalis
FAD two molecules FAD per heterotetramer Faecalicatena orotica
FAD flavoprotein, ratio FAD to FMN 1:1 Faecalicatena orotica
FMN flavoprotein, ratio FAD to FMN 1:1 Faecalicatena orotica
FMN two molecules of FMN per heterotetramer Bacillus subtilis
FMN two molecules of FMN per heterotetramer Lactococcus lactis
FMN two molecules of FMN per heterotetramer Enterococcus faecalis
FMN two molecules of FMN per heterotetramer Faecalicatena orotica
FMN FMN is located on the alpha subunit Lactococcus lactis
4Fe-4S-center iron-sulfur cluster resides on the beta subunit Lactococcus lactis
4Fe-4S-center cofactor content: 4 non-heme iron and 4 labile sulfur atoms per heterotetramer Bacillus subtilis
4Fe-4S-center cofactor content: 4 non-heme iron and 4 labile sulfur atoms per heterotetramer Lactococcus lactis
4Fe-4S-center cofactor content: 4 non-heme iron and 4 labile sulfur atoms per heterotetramer Enterococcus faecalis
4Fe-4S-center cofactor content: 4 non-heme iron and 4 labile sulfur atoms per heterotetramer Faecalicatena orotica