Structure and Functional Characterization of Human Aspartate Transcarbamoylase, the Target of the Anti-tumoral Drug PALA

Ruiz‐Ramos, Alba; Velázquez‐Campoy, Adrián; Grande-García, A.; Moreno-Morcillo, María; Ramón‐Maiques, Santiago

doi:10.1016/j.str.2016.05.001

Cited by 33 publications

(67 citation statements)

References 51 publications

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“…The relative orientation of the domains is similar to the open conformation observed in other ATCs crystallized without ligands ( Supplementary Fig. 3) 26,28,34 . In addition, atATC has the CP-loop (aa 156-169) and Asp-loop (aa 309-332) ( Fig.…”

Section: Crystal Structure Of Arabidopsis Atc Bound To Umpsupporting

confidence: 73%

“…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%

“…In addition, atATC has the CP-loop (aa 156-169) and Asp-loop (aa 309-332) ( Fig. 2a,d) that in other ATCs undergo large conformational movements upon substrate binding 16,28 .…”

Section: Crystal Structure Of Arabidopsis Atc Bound To Umpmentioning

confidence: 99%

“…In eukaryotes other than plants, on the other hand, ATC is fused together with CPS into a single multienzymatic protein named CAD that also contains an active DHO (animals) or an inactive DHO-like domain (fungi) 10,[20][21][22][23][24] . This ATC also forms homotrimers, favoring the assembly of CAD into large hexameric particles [25][26][27][28] , and its activity is modulated by the binding of UTP to an allosteric region within CPS 29,30 . In striking contrast, plant ATCs consist of a UMP-inhibitable homotrimer with no associated subunits, meaning that both the catalytic and regulatory sites must reside within the same polypeptide chain 31 .…”

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confidence: 99%

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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: Crystal Structure Of Arabidopsis Atc Bound To Umpsupporting

confidence: 73%

Section: Atatc Only Binds One Molecule Of Pala Per Trimermentioning

confidence: 64%

“…In addition, atATC has the CP-loop (aa 156-169) and Asp-loop (aa 309-332) ( Fig. 2a,d) that in other ATCs undergo large conformational movements upon substrate binding 16,28 .…”

Section: Crystal Structure Of Arabidopsis Atc Bound To Umpmentioning

confidence: 99%

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confidence: 99%

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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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“…To confirm that all four enzymatic activities of CAD were needed for de novo pyrimidine synthesis and cell growth in absence of uridine, we measured the proliferation of CAD-KO cells transfected with GFP-CAD bearing well-known inactivating mutations for each activity (Figure 2g). The transfected inactivated variants in the SYN (p.H627N, p.E682Q) 13,14 , DHO (p.D1686N) 15 and ATC (p.R2024Q) 7,16 domains failed to rescue the growth of CAD-KO cells. In turn, the GLN inactive mutant (p.C252S) 17 showed a partial rescue, with transfected cells doubling every ~2.5 days, suggesting that free-ammonia can, to some extent, contribute to the synthesis of carbamoyl phosphate (Figure 1).…”

Section: Validation Of a Growth Complementation Assay In Cad Knockoutmentioning

confidence: 98%

Cell-based analysis ofCADvariants identifies individuals likely to benefit from uridine therapy

Caño-Ochoa

Abedalthagafi

et al. 2020

Preprint

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Purpose: Pathogenic autosomal recessive variants in CAD, encoding the multienzymatic protein initiating pyrimidine de novo biosynthesis, cause a severe inborn metabolic disorder treatable with a dietary supplement of uridine. This condition is difficult to diagnose given the large size of CAD with over 1000 missense variants and the non-specific clinical presentation. We aimed to develop a reliable and discerning assay to assess the pathogenicity of CAD variants and to select affected individuals that might benefit from uridine therapy.Methods: Using CRISPR/Cas9, we generated a human CAD-knockout cell line that requires uridine supplements for survival. Transient transfection of the knockout cells with recombinant CAD restores growth in absence of uridine. This system determines missense variants that inactivate CAD and do not rescue the growth phenotype. Results:We identified 25 individuals with biallelic variants in CAD and a phenotype consistent with a CAD deficit. We used the CAD-knockout complementation assay to test a total of 34 variants, identifying 16 as deleterious for CAD activity. Combination of these pathogenic variants confirmed 11 subjects with a CAD deficit, for whom we describe the clinical phenotype. Conclusions:We designed a cell-based assay to test the pathogenicity of CAD variants, identifying 11 CAD deficient individuals, who could benefit from uridine therapy. KeywordsCongenital disorder of glycosylation; de novo pyrimidine biosynthesis; carbamoyl phosphate synthetase; aspartate transcarbamoylase; dihydroorotase

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A Biophysical Toolkit for Molecular Interactions

Santiveri

López-Méndez

Huecas

et al. 2017

Encyclopedia of Life Sciences

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Intermolecular interactions inside and around the cell are key to maintain basic cell homeostasis and to sense and react to external stimuli. Moreover, the action of therapeutics and other xenobiotics occurs through physical interactions of these agents with biomolecules present in the cell or tissue, thereby interfering with the ‘native’ interactions of the biomolecules. Biophysical characterisation of molecular interactions is a bottom‐up reductionist approach that implies measuring the physicochemical properties of the interacting species (two biomacromolecules or a biomacromolecule and a small‐molecule ligand) in order to quantify the binding strength (thermodynamics) and association and dissociation speed (kinetics) between them. A large variety of macroscopic experimental techniques are available and constitute a biophysical toolkit to study molecular interactions in vitro . Among others, isothermal titration calorimetry, surface plasmon resonance, analytical ultracentrifugation, nuclear magnetic resonance, microscale thermophoresis, thermal shift and fluorescence‐based methods are most commonly used. Key Concepts The strength (affinity) and speed (kinetics) of interactions between biomolecules can be quantified using biophysical experimental methods. Isothermal titration calorimetry (ITC), fluorescence and nuclear magnetic resonance (NMR) spectroscopies, thermal shift assay (TSA), analytical ultracentrifugation (AUC), microscale thermophoresis (MST) and surface plasmon resonance (SPR) are arguably the most used and versatile techniques to study binding in vitro and constitute a biophysical toolkit. Equilibrium affinity constants can be robustly obtained by titration using ITC, fluorescence, NMR, MST, AUC and SPR. Kinetic information can be obtained from time‐resolved fluorescence spectroscopy and SPR methods. The recently developed kinITC method allows obtaining kinetic information from ITC data. Biophysical techniques can tackle the study of a wide range of systems and great progress has been made in the characterisation of interactions taking place at the cell membrane. Basically, all biophysical methods can be automatised to screen for small‐molecule ligands of proteins and other macromolecules, providing starting points for chemical biology and drug discovery.

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Structure and Functional Characterization of Human Aspartate Transcarbamoylase, the Target of the Anti-tumoral Drug PALA

Cited by 33 publications

References 51 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

Cell-based analysis ofCADvariants identifies individuals likely to benefit from uridine therapy

A Biophysical Toolkit for Molecular Interactions

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