DHOase is a zinc-dependent metalloenzyme, analysis of the catalytic zinc environment, structure, overview. Cys181 is observed bonded to the zinc atom of the catalytic site, with residues 181-187 visible and tethered by the interaction of loop A with other regions of the protein. On one end, the 1200 bar zinc coordination is like in the monoclinic-ap form with the Cys181 recruited again by the zinc atom, instead of the water molecule; the Cys-181 sulfur atom rotates and becomes more and more closely connected to the zinc when pressure increases
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
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
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)
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)
in noncovalent association with aspartate transcarbamoylase, possible model for the mammalian polypeptide chain CPSase/ATCase/DHOase during pyrimidine biosynthesis
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
the replacement of the zinc ligand Cys181 with glycine does not restore the latent catalytic activity suggesting that it plays a minor role in stabilizing loop A
dihydroorotase (DHO) is an amidohydrolase that catalyzes the reversible condensation of carbamoyl aspartate to form dihydroorotate in de novo pyrimidine biosynthesis
dihydroorotase (DHOase) catalyses the third reaction of the de novo pyrimidine biosynthetic pathway, the reversible condensation of carbamylaspartate into dihydroorotate
in the hyperthermophilic bacterium Aquifex aeolicus, aspartate transcarbamylase (ATCase, EC 2.1.3.2) and dihydroorotase (DHOase) are noncovalently associated. Upon dissociation, ATCase keeps its activity entirely while DHOase is totally inactivated. High pressure fully restores the activity of this isolated DHOase. Under high-hydrostatic pressure, at 600 bar, and to a greater extent at 1200 bar, the orthorhombic form of DHOase displays a structure, which includes the Cys-181 bridge of the C2-ap form and some additional residues of the missing loops that become ordered and visible in the electron density
in the hyperthermophilic bacterium Aquifex aeolicus, aspartate transcarbamylase (ATCase, EC 2.1.3.2) and dihydroorotase (DHOase) are noncovalently associated. Upon dissociation, ATCase keeps its activity entirely while DHOase is totally inactivated. High pressure fully restores the activity of this isolated DHOase. Under high-hydrostatic pressure, at 600 bar, and to a greater extent at 1200 bar, the orthorhombic form of DHOase displays a structure, which includes the Cys-181 bridge of the C2-ap form and some additional residues of the missing loops that become ordered and visible in the electron density
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
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
enzymes DHO-ATC complex structure, analysis of the quaternary structural organization and interactions between the subunits in the Aquifex aeolicus complex, overview
enzymes DHO-ATC complex structure, analysis of the quaternary structural organization and interactions between the subunits in the Aquifex aeolicus complex, overview
recombinant protein lacks catalytic activity, activity is acquired by forming a complex with aspartate transcarbamoylase, complex may be a heterohexamer or dodecamer
hanging drop method, monoclinic structure has a novel cysteine ligand to the zinc that blocks the active site and functions as a cysteine switch, active site residues are located in disordered loops, which may function as a disorder-to-order entropy switch
site-directed mutagenesis, the mutant does not restore the activity of DHOase that has been isolated from aspartate transcarbamylase (ATCase, EC 2.1.3.2)
site-directed mutagenesis, the mutant partially restores the activity of DHOase that has been isolated from aspartate transcarbamylase (ATCase, EC 2.1.3.2)
site-directed mutagenesis, the mutant partially restores the activity of DHOase that has been isolated from aspartate transcarbamylase (ATCase, EC 2.1.3.2)
in noncovalent association with aspartate transcarbamoylase, possible model for mammalian polypeptide chain CPSase/ATCase/DHOase during pyrimidine biosynthesis
in noncovalent association with aspartate transcarbamoylase, possible model for mammalian polypeptide chain CPSase/ATCase/DHOase during pyrimidine biosynthesis
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)
Dihydroorotase from the hyperthermophile Aquifex aeolicus is activated by stoichiometric association with aspartate transcarbamoylase and forms a one-pot reactor for pyrimidine biosynthesis