Any feedback?
Please rate this page
(literature.php)
(0/150)

BRENDA support

Literature summary for 3.5.2.3 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
Search for more literature for 3.5.2.3

Activating Compound

Activating Compound Comment Organism Structure
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)

Cloned (Comment) Organism
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

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

Inhibitors

Inhibitors Comment Organism Structure
DHCEDD the peptide corresponds to the sequence 179DHCEDD185, and causes 50% inhibition of the wild-type enzyme Aquifex aeolicus
DHCEDDKLA the peptide corresponds to the sequence 179DHCEDDKLA187, and causes 76% inhibition of the wild-type enzyme Aquifex aeolicus
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]

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

Natural Substrates/ Products (Substrates)

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

Organism

Organism UniProt Comment Textmining
Aquifex aeolicus O66990
-
-

Purification (Commentary)

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

Substrates and Products (Substrate)

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

Subunits

Subunits Comment Organism
? x * 43000, DHO, SDS-PAGE Aquifex aeolicus
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

Synonyms Comment Organism
DHO
-
 Aquifex aeolicus

Temperature Optimum [°C]

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

Turnover Number [1/s]

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

pH Optimum

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

General Information

General Information Comment Organism
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
metabolism dihydroorotase (DHO) is an amidohydrolase that catalyzes the reversible condensation of carbamoyl aspartate to form dihydroorotate in de novo pyrimidine biosynthesis Aquifex aeolicus
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]

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