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Literature summary extracted from

  • Kahler, A.E.; Nielsen, F.S.; Switzer, R.L.
    Biochemical characterization of the heteromeric Bacillus subtilis dihydroorotate dehydrogenase and its isolated subunits (1999), Arch. Biochem. Biophys., 371, 191-201.
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.3.1.14
-
 Bacillus subtilis
1.3.1.14 expression in Escherichia coliBL21 DE3, expression of pyrDI alone and coexpression with pyrDII Bacillus subtilis

Protein Variants

EC Number Protein Variants Comment Organism
1.3.1.14 C220A pyrDII mutant, conserved, essential for activity Bacillus subtilis
1.3.1.14 C225A pyrDII mutant conserved, essential for activity Bacillus subtilis
1.3.1.14 C228A pyrDII mutant conserved, essential for activity Bacillus subtilis
1.3.1.14 C230A pyrDII mutant nonconserved Bacillus subtilis
1.3.1.14 C243A pyrDII mutant conserved, essential for activity Bacillus subtilis

Inhibitors

EC Number Inhibitors Comment Organism Structure
1.3.1.14 Orotate
-
 Bacillus subtilis

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.3.1.14 0.027
-
 S-dihydroorotate holoenzyme with 2,6-dichlorophenolindophenol Bacillus subtilis
1.3.1.14 0.0317
-
 S-dihydroorotate holoenzyme with menadione Bacillus subtilis
1.3.1.14 0.5 1 S-dihydroorotate PyrDI Bacillus subtilis

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
1.3.1.14 28000
-
 2 * 33000 + 2 * 28000 Bacillus subtilis
1.3.1.14 28099
-
 heterotetramer 2 * 33094 and 2 * 28099 predicted from seqeuence, 2 * 38000 and 2 * 31000, SDS-PAGE Bacillus subtilis
1.3.1.14 31000
-
 heterotetramer 2 * 33094 and 2 * 28099 predicted from seqeuence, 2 * 38000 and 2 * 31000, SDS-PAGE Bacillus subtilis
1.3.1.14 33000
-
 2 * 33000 + 2 * 28000 Bacillus subtilis
1.3.1.14 33094
-
 heterotetramer 2 * 33094 and 2 * 28099 predicted from seqeuence, 2 * 38000 and 2 * 31000, SDS-PAGE Bacillus subtilis
1.3.1.14 38000
-
 heterotetramer 2 * 33094 and 2 * 28099 predicted from seqeuence, 2 * 38000 and 2 * 31000, SDS-PAGE Bacillus subtilis
1.3.1.14 85000
-
 native molecular mass of PyrDI Bacillus subtilis
1.3.1.14 114000
-
 gel filtration Bacillus subtilis
1.3.1.14 114000
-
 molecular mass of the holoenzyme, calculated mass 122400 Da Bacillus subtilis

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.3.1.14 (S)-dihydroorotate + acceptor Bacillus subtilis fourth step in UMP-biosynthesis orotate + reduced acceptor
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
1.3.1.14 Bacillus subtilis
-
-
-
1.3.1.14 Mus musculus
-
 Ehrlich ascites tumor cells
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.3.1.14 anion-exchange, gel filtration Bacillus subtilis

Reaction

EC Number Reaction Comment Organism Reaction ID
1.3.1.14 (S)-dihydroorotate + NAD+ = orotate + NADH + H+ PyrDI contains the dihydroorotate-binding site, but PyrDII is required for full activity in vivo. Holoenzyme joins a NAD+-reductase activity Bacillus subtilis
a

Renatured (Commentary)

EC Number Renatured (Comment) Organism
1.3.1.14 activity can be recovered by mixing the purified subunits Bacillus subtilis
1.3.1.14 PyrDII-containing inclusion bodies are denatured and refolded through dialysis into buffer Bacillus subtilis

Storage Stability

EC Number Storage Stability Organism
1.3.1.14 4°C, holoenzyme, stable for many days Bacillus subtilis
1.3.1.14 on ice at room temperature, PyrDI, gradually loses activity over a period of hours Bacillus subtilis
1.3.1.14 room temperature, holoenzyme, stable for many hours Bacillus subtilis

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.3.1.14 (S)-dihydroorotate + acceptor different specific activities with potassium ferricyanide, O2, fumarate and NAD+ as electron acceptors for PyrDI and the holoenzyme Bacillus subtilis orotate + reduced acceptor
-
 ?
1.3.1.14 (S)-dihydroorotate + acceptor fourth step in UMP-biosynthesis Bacillus subtilis orotate + reduced acceptor
-
 ?
1.3.1.14 dihydroorotate + acceptor acceptor: NAD+ Bacillus subtilis orotate + reduced acceptor
-
 ?
1.3.1.14 dihydroorotate + acceptor acceptor: menadione Bacillus subtilis orotate + reduced acceptor
-
 ?
1.3.1.14 dihydroorotate + acceptor activity measurement in permeabilized Ehrlich ascites tumor cells, acceptor: nitroblue tetrazolium Mus musculus orotate + reduced acceptor
-
 ?

Subunits

EC Number Subunits Comment Organism
1.3.1.14 tetramer heterotetramer 2 * 33094 and 2 * 28099 predicted from seqeuence, 2 * 38000 and 2 * 31000, SDS-PAGE Bacillus subtilis
1.3.1.14 tetramer 2 * 33000 + 2 * 28000 Bacillus subtilis

Cofactor

EC Number Cofactor Comment Organism Structure
1.3.1.14 FAD 1 mol PyrDII binds 1 mol FAD and 1 mol [2Fe-2S] Bacillus subtilis
1.3.1.14 FAD 1 mol/mol of subunit PyrDII Bacillus subtilis
1.3.1.14 FMN PyrDI is an FMN-containing iron-sulfur flavoprotein, 1 FMN molecule per PyrDI molecule Bacillus subtilis
1.3.1.14 NAD+ for holoenzyme Bacillus subtilis

Ki Value [mM]

EC Number Ki Value [mM] Ki Value maximum [mM] Inhibitor Comment Organism Structure
1.3.1.14 0.0206
-
 Orotate holoenzyme Bacillus subtilis
1.3.1.14 1
-
 Orotate PyrDI Bacillus subtilis