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Literature summary extracted from

  • Herve, G.; Evans, H.G.; Fernado, R.; Patel, C.; Hachem, F.; Evans, D.R.
    Activation of latent dihydroorotase from Aquifex aeolicus by pressure (2017), J. Biol. Chem., 292, 629-637 .
    View publication on PubMedView publication on EuropePMC

Activating Compound

EC Number Activating Compound Comment Organism Structure
3.5.2.3 additional information reversible activation of latent dihydroorotase from Aquifex aeolicus by moderate hydrostatic pressure. Moderate hydrostatic pressure applied to the isolated DHO subunit mimics the complex formation and reversibly activates the isolated subunit in the absence of ATC, suggesting that the loop has been displaced from the active site. This effect of pressure is explained by the negative volume change associated with the disruption of ionic interactions and exposure of ionized amino acids to the solvent (electrostriction). The isolated DHO protein, a 45-kDa monomer, lacks catalytic activity but becomes active upon formation of a dodecameric complex with aspartate transcarbamoylase (ATC, EC 2.1.3.2) Aquifex aeolicus

Cloned(Commentary)

EC Number Cloned (Comment) Organism
3.5.2.3 gene pyrC, recombinant expression of wild-type and mutant DHO enzymes in Escherichia coli strain BL21(DE3), coexpression with pyrB encoding with aspartate transcarbamoylase (ATC, EC 2.1.3.2) Aquifex aeolicus

Protein Variants

EC Number Protein Variants Comment Organism
3.5.2.3 C181A site-directed mutagenesis, the mutant shows reduced activity and altered kinetics compared to wild-type Aquifex aeolicus
3.5.2.3 E179V site-directed mutagenesis, the mutant shows reduced activity and altered kinetics compared to wild-type Aquifex aeolicus
3.5.2.3 E183V site-directed mutagenesis, the mutant shows reduced activity and altered kinetics compared to wild-type Aquifex aeolicus

Inhibitors

EC Number Inhibitors Comment Organism Structure
3.5.2.3 DHCEDD the peptide corresponds to the sequence 179DHCEDD185, and causes 50% inhibition of the wild-type enzyme Aquifex aeolicus
3.5.2.3 DHCEDDKLA the peptide corresponds to the sequence 179DHCEDDKLA187, and causes 76% inhibition of the wild-type enzyme Aquifex aeolicus
3.5.2.3 additional information pressure induces irreversible dissociation of the obligate ATC trimer, and as a consequence the DHO is also inactivated. Inhibition of DHO by small peptides that mimic the loop residues Aquifex aeolicus

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
3.5.2.3 0.24
-
 N-carbamoyl-L-aspartate pH 8.0, 74°C, recombinant DHO-ATC complex with mutant DHO E179V Aquifex aeolicus
3.5.2.3 0.47
-
 N-carbamoyl-L-aspartate pH 8.0, 74°C, recombinant DHO-ATC complex with mutant DHO E183V Aquifex aeolicus
3.5.2.3 2.38
-
 N-carbamoyl-L-aspartate pH 8.0, 74°C, recombinant wild-type DHO-ATC complex Aquifex aeolicus
3.5.2.3 3.89
-
 N-carbamoyl-L-aspartate pH 8.0, 74°C, recombinant DHO-ATC complex with mutant DHO C181A Aquifex aeolicus
3.5.2.3 6.2
-
 N-carbamoyl-L-aspartate pH 8.0, 74°C, recombinant isolated DHO mutant DHO E179V Aquifex aeolicus
3.5.2.3 24
-
 N-carbamoyl-L-aspartate pH 8.0, 74°C, recombinant isolated DHO mutant DHO E183V Aquifex aeolicus

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
3.5.2.3 (S)-dihydroorotate + H2O Aquifex aeolicus
-
 N-carbamoyl-L-aspartate
-
 r
3.5.2.3 N-carbamoyl-L-aspartate Aquifex aeolicus
-
 (S)-dihydroorotate + H2O
-
 r

Organism

EC Number Organism UniProt Comment Textmining
3.5.2.3 Aquifex aeolicus O66990
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
3.5.2.3 recombinant isolated DHO and DDHO-ATC complex from Escherichia coli strain BL21(DE3) by nickel affinity chromatography Aquifex aeolicus

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
3.5.2.3 (S)-dihydroorotate + H2O
-
 Aquifex aeolicus N-carbamoyl-L-aspartate
-
 r
3.5.2.3 N-carbamoyl-L-aspartate
-
 Aquifex aeolicus (S)-dihydroorotate + H2O
-
 r

Subunits

EC Number Subunits Comment Organism
3.5.2.3 ? x * 43000, DHO, SDS-PAGE Aquifex aeolicus
3.5.2.3 More enzymes DHO-ATC complex structure, analysis of the quaternary structural organization and interactions between the subunits in the Aquifex aeolicus complex, overview Aquifex aeolicus

Synonyms

EC Number Synonyms Comment Organism
3.5.2.3 DHO
-
 Aquifex aeolicus

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
3.5.2.3 25 74 assay at Aquifex aeolicus

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
3.5.2.3 3
-
 N-carbamoyl-L-aspartate pH 8.0, 74°C, recombinant isolated DHO mutant DHO E179V Aquifex aeolicus
3.5.2.3 6.7
-
 N-carbamoyl-L-aspartate pH 8.0, 74°C, recombinant isolated DHO mutant DHO E183V Aquifex aeolicus
3.5.2.3 9.2
-
 N-carbamoyl-L-aspartate pH 8.0, 74°C, recombinant DHO-ATC complex with mutant DHO E179V Aquifex aeolicus
3.5.2.3 9.7
-
 N-carbamoyl-L-aspartate pH 8.0, 74°C, recombinant DHO-ATC complex with mutant DHO E183V Aquifex aeolicus
3.5.2.3 15.5
-
 N-carbamoyl-L-aspartate pH 8.0, 74°C, recombinant DHO-ATC complex with mutant DHO C181A Aquifex aeolicus
3.5.2.3 62.3
-
 N-carbamoyl-L-aspartate pH 8.0, 74°C, recombinant wild-type DHO-ATC complex Aquifex aeolicus

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
3.5.2.3 8
-
 assay at Aquifex aeolicus

General Information

EC Number General Information Comment Organism
3.5.2.3 evolution dihydroorotase (DHO) is an amidohydrolase that catalyzes the reversible condensation of carbamoyl aspartate to form dihydroorotate in de novo pyrimidine biosynthesis in virtually all organisms. Although the same reaction is catalyzed by all DHOs, the structure, oligomeric organization, and metal content of this family of enzymes is diverse Aquifex aeolicus
3.5.2.3 metabolism dihydroorotase (DHO) is an amidohydrolase that catalyzes the reversible condensation of carbamoyl aspartate to form dihydroorotate in de novo pyrimidine biosynthesis Aquifex aeolicus
3.5.2.3 additional information the isolated DHO protein, a 45-kDa monomer, lacks catalytic activity but becomes active upon formation of a dodecameric complex with aspartate transcarbamoylase (ATC, EC 2.1.3.2). In the isolated DHO, a flexible loop occludes the active site blocking the access of substrates. The loop is mostly disordered but is tethered to the active site region by several electrostatic and hydrogen bonds. This loop becomes ordered and is displaced from the active site upon formation of DHO-ATC complex. The application of pressure to the complex causes its time-dependent dissociation and the loss of both DHO and ATC activities. Pressure induces irreversible dissociation of the obligate ATC trimer, and as a consequence the DHO is also inactivated Aquifex aeolicus

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
3.5.2.3 0.28
-
 N-carbamoyl-L-aspartate pH 8.0, 74°C, recombinant isolated DHO mutant DHO E183V Aquifex aeolicus
3.5.2.3 0.48
-
 N-carbamoyl-L-aspartate pH 8.0, 74°C, recombinant isolated DHO mutant DHO E179V Aquifex aeolicus
3.5.2.3 3.98
-
 N-carbamoyl-L-aspartate pH 8.0, 74°C, recombinant DHO-ATC complex with mutant DHO C181A Aquifex aeolicus
3.5.2.3 20.6
-
 N-carbamoyl-L-aspartate pH 8.0, 74°C, recombinant DHO-ATC complex with mutant DHO E183V Aquifex aeolicus
3.5.2.3 26.2
-
 N-carbamoyl-L-aspartate pH 8.0, 74°C, recombinant wild-type DHO-ATC complex Aquifex aeolicus
3.5.2.3 38.3
-
 N-carbamoyl-L-aspartate pH 8.0, 74°C, recombinant DHO-ATC complex with mutant DHO E179V Aquifex aeolicus