The mononuclear metal center of type-I dihydroorotase from aquifex aeolicus

Edwards, Brian F.P.; Fernando, Ravin; Martin, Philip D.; Grimley, Edward; Cordes, Melissa; Vaishnav, Asmita; Brunzelle, J.S.; Evans, Hedeel Guy; Evans, David R.

doi:10.1186/1471-2091-14-36

Cited by 13 publications

(12 citation statements)

References 46 publications

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“…The substoichiometric binding of PALA is remarkable, since other ATC structures proved the binding of three molecules of PALA per trimer 28,[38][39][40][41] and the interactions with the transitionstate analog are virtually identical to those observed in atATC ( Fig. 3d and Supplementary Fig.…”

Section: Atatc Only Binds One Molecule Of Pala Per Trimermentioning

confidence: 64%

UMP inhibition and sequential firing in aspartate transcarbamoylase open ways to regulate plant growth

Bellin

Caño-Ochoa

Velázquez‐Campoy

et al. 2020

Preprint

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Pyrimidine nucleotides are essential to plant development. We proved that Arabidopsis growth can be inhibited or enhanced by down- or upregulating aspartate transcarbamoylase (ATC), the first committed enzyme for de novo biosynthesis of pyrimidines in plants. To understand the unique mechanism of feedback inhibition of this enzyme by uridine 5-monophosphate (UMP), we determined the crystal structure of the Arabidopsis ATC trimer free and bound to UMP, demonstrating that the nucleotide binds and blocks the active site. The regulatory mechanism relies on a loop exclusively conserved in plants, and a single-point mutation (F161A) turns ATC insensitive to UMP. Moreover, the structures in complex with a transition-state analog or with carbamoyl phosphate proved a mechanism in plant ATCs for sequential firing of the active sites. The disclosure of the unique regulatory and catalytic properties suggests new strategies to modulate ATC activity and to control de novo pyrimidine synthesis and plant growth.

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Section: Atatc Only Binds One Molecule Of Pala Per Trimermentioning

confidence: 64%

UMP inhibition and sequential firing in aspartate transcarbamoylase open ways to regulate plant growth

Bellin

Caño-Ochoa

Velázquez‐Campoy

et al. 2020

Preprint

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“…Site-directed Mutagenesis-Site-directed mutagenesis was carried out by PCR as described previously (31) using Pfu Turbo polymerase from Stratagene (ThermoFisher) and the plasmid encoding the DHO subunit as a template. The residues in loop A suspected of anchoring the loop at the entry to the active site were replaced by valine or glycine using forward and reverse oligonucleotides from Invitrogen (ThermoFisher Scientific).…”

Section: Methodsmentioning

confidence: 99%

Activation of Latent Dihydroorotase from Aquifex aeolicus by Pressure

Hervé

Evans

Fernado

et al. 2017

Journal of Biological Chemistry

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Elevated hydrostatic pressure was used to probe conformational changes of Aquifex aeolicus dihydroorotase (DHO), which catalyzes the third step in de novo pyrimidine biosynthesis. The isolated protein, a 45-kDa monomer, lacks catalytic activity but becomes active upon formation of a dodecameric complex with aspartate transcarbamoylase (ATC). X-ray crystallographic studies of the isolated DHO and of the complex showed that association induces several major conformational changes in the DHO structure. 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 induced irreversible dissociation of the obligate ATC trimer, and as a consequence the DHO is also inactivated. However, 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 interpretation that the loop is relocated by pressure was validated by site-directed mutagenesis and by inhibition by small peptides that mimic the loop residues.

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“…The structure of M. profunda was determined in the T-state unliganded form.The only ATCase structure corresponding to a functional catalytic trimer in vivo is that of Bacillus subtilis [ 7 , 49 ]. Two structures of a prokaryotic ATCase from Auifex aeolicus that form a stable dodecameric holoenzyme with DHOase, were determined [ 50 , 51 ]. Only one eukaryotic ATCase structure, of Trypanosoma cruzi , has been determined (PDB code: 4IV5).…”

Section: Structures Deposited In the Protein Data Bank (Pdb)mentioning

confidence: 99%

From Genome to Structure and Back Again: A Family Portrait of the Transcarbamylases

Shi

Allewell

Tuchman

2015

IJMS

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Enzymes in the transcarbamylase family catalyze the transfer of a carbamyl group from carbamyl phosphate (CP) to an amino group of a second substrate. The two best-characterized members, aspartate transcarbamylase (ATCase) and ornithine transcarbamylase (OTCase), are present in most organisms from bacteria to humans. Recently, structures of four new transcarbamylase members, N-acetyl-l-ornithine transcarbamylase (AOTCase), N-succinyl-l-ornithine transcarbamylase (SOTCase), ygeW encoded transcarbamylase (YTCase) and putrescine transcarbamylase (PTCase) have also been determined. Crystal structures of these enzymes have shown that they have a common overall fold with a trimer as their basic biological unit. The monomer structures share a common CP binding site in their N-terminal domain, but have different second substrate binding sites in their C-terminal domain. The discovery of three new transcarbamylases, l-2,3-diaminopropionate transcarbamylase (DPTCase), l-2,4-diaminobutyrate transcarbamylase (DBTCase) and ureidoglycine transcarbamylase (UGTCase), demonstrates that our knowledge and understanding of the spectrum of the transcarbamylase family is still incomplete. In this review, we summarize studies on the structures and function of transcarbamylases demonstrating how structural information helps to define biological function and how small structural differences govern enzyme specificity. Such information is important for correctly annotating transcarbamylase sequences in the genome databases and for identifying new members of the transcarbamylase family.

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The mononuclear metal center of type-I dihydroorotase from aquifex aeolicus

Cited by 13 publications

References 46 publications

UMP inhibition and sequential firing in aspartate transcarbamoylase open ways to regulate plant growth

UMP inhibition and sequential firing in aspartate transcarbamoylase open ways to regulate plant growth

Activation of Latent Dihydroorotase from Aquifex aeolicus by Pressure

From Genome to Structure and Back Again: A Family Portrait of the Transcarbamylases

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