Persistent CAD activity in memory CD8+ T cells supports rRNA synthesis and ribosomal biogenesis required at rechallenge.

Memory CD8+ T cells are characterized by their ability to persist long after the initial antigen encounter and their capacity to generate a rapid recall response. Recent studies have identified a role for metabolic reprogramming and mitochondrial function in promoting the longevity of memory T cells. However, detailed mechanisms involved in promoting their rapid recall response are incompletely understood. Here, we identify a role for the initial and continued activation of the trifunctional rate-limiting enzyme of the de novo pyrimidine synthesis pathway CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase) as critical in promoting the rapid recall response of previously activated CD8+ T cells. We found that CAD was rapidly phosphorylated upon naïve T cell activation in an mTORC1-dependent manner, yet remained phosphorylated long after initial activation. Previously activated CD8+ T cells displayed continued de novo pyrimidine synthesis in the absence of mitogenic signals, and interfering with this pathway diminished the speed and magnitude of cytokine production upon rechallenge. Inhibition of CAD did not affect cytokine transcript levels but diminished available pre-rRNA (ribosomal RNA), the polycistronic rRNA precursor whose synthesis is the rate-limiting step in ribosomal biogenesis. CAD inhibition additionally decreased levels of detectable ribosomal proteins in previously activated CD8+ T cells. Conversely, overexpression of CAD improved both the cytokine response and proliferation of memory T cells. Overall, our studies reveal a critical role for CAD-induced pyrimidine synthesis and ribosomal biogenesis in promoting the rapid recall response characteristic of memory T cells.

CAD gene and early infantile epileptic encephalopathy-50; three Iranian deceased patients and a novel mutation: case report.

BACKGROUND: Early infantile epileptic encephalopathy is a severe form of epilepsy that is genetically extremely heterogeneous and characterized by seizures or spasms at the beginning of infancy. Homozygous or compound heterozygous mutation in the CAD gene cause early infantile epileptic encephalopathy-50 (EIEE50). This case report describes the clinical and molecular features of three patients affected with early infantile epileptic encephalopathy. CASE PRESENTATION: In this report, we describe the clinical features of two deceased daughters and one recently deceased son affected with seizure, muscular hypotonia, and developmental delay. After genetic counseling, blood samples were obtained from the parents, and whole-exome sequencing was performed. Genomic DNA was extracted from whole blood, and mutation analysis was performed using PCR and sequencing methods for the CAD gene. Genetic analysis using the whole-exome sequencing method has detected a novel likely pathogenic mutation on CAD gene, c.2995G > A (p.Val999Met), in heterozygous states in asymptomatic parents and homozygous state in affected newborn son. This mutation has not been reported in the literature for its pathogenicity. CONCLUSIONS: The asymptomatic parents are carriers for the likely pathogenic variant in the CAD gene, and the recently deceased newborn son had the same mutation in a homozygous state. Given that, multiple lines of in silico computational analysis support the detrimental impact of the variant on the gene, and this variant is absent in population databases. Pathogenic mutations in the CAD gene are related to autosomal recessive EIEE50 with similar signs and symptoms to our patients. Ultimately, it is confirmed that this mutation is causative in our patients.

Uridine-responsive epileptic encephalopathy due to inherited variants in CAD: A Tale of Two Siblings.

We report two siblings with intractable epilepsy, developmental regression, and progressive cerebellar atrophy due to biallelic variants in the gene CAD. For the affected girl, uridine started at age 5 resulted in dramatic improvements in seizure control and development, cessation of cerebellar atrophy, and resolution of hematological abnormalities. Her older brother had a more severe course and only modest response to uridine started at 14 years old. Treatment of this progressive condition via uridine supplementation provides an example of precision diagnosis and treatment using clear outcome measures and biomarkers to monitor efficacy.

Structural insights on binding mechanism of CAD complexes (CPSase, ATCase and DHOase).

Pyrimidine biosynthetic pathway enzymes constitute an important target for the development of antitumor drugs. To understand the role of binding mechanisms underlying the inborn errors of pyrimidine biosynthetic pathway, structure and function of enzymes have been analyzed. Pyrimidine biosynthetic pathway is initiated by CAD enzymes that harbor the first three enzymatic activities facilitated by Carbamoyl Phosphate Synthetase (CPSase), Aspartate Transcarbamoylase (ATCase) and Dihydroorotase (DHOase). While being an attractive therapeutic target, the lack of data driven us to study the CPSase (CarA and CarB) and its mode of binding to ATCase and DHOase which are the major limitation for its structural optimization. Understanding the binding mode of CPSase, ATCase and DHOase could help to identify the potential interface hotspot residues that favor the mechanism behind it. The mechanistic insight into the CAD complexes were achieved through Molecular modeling, Protein-Protein docking, Alanine scanning and Molecular dynamics (MD) Studies. The hotspot residues present in the CarB region of carboxy phosphate and carbamoyl phosphate synthetic domains are responsible for the assembly of CAD (CPSase-ATCase-DHOase) complexes. Overall analysis suggests that the identified hotspot residues were confirmed by alanine scanning and important for the regulation of pyrimidine biosynthesis. MD simulations analysis provided the prolonged stability of the interacting complexes. The present study reveals the novel hotspot residues such as Glu134, Glu147, Glu154, Asp266, Lys269, Glu274, Asp333, Trp459, Asp526, Asp528, Glu533, Glu544, Glu546, Glu800, Val855, Asp877, Tyr884 and Gln919 which could be targeted for structure-based inhibitor design to potentiate the CAD mediated regulation of aggressive tumors.Communicated by Ramaswamy H. Sarma.

Triacetyluridine treats epileptic encephalopathy from CAD mutations: a case report and review.

Refractory epilepsy and encephalopathy are frequently encountered in patients with inborn errors of metabolism. We report a case of an 8-year-old girl with history of developmental delay, autism and intractable epilepsy that was found to have a pathogenic variant in CAD. We briefly review the biochemical pathway of CAD and the preclinical and clinical studies that suggest uridine supplementation can rescue the CAD deficiency phenotypes. Our case demonstrates a relatively late-onset case of refractory epilepsy with a rapid response to treatment using the uridine pro-drug triacetyluridine (TAU), the FDA-approved treatment for hereditary orotic aciduria.

Combined small molecule and loss-of-function screen uncovers estrogen receptor alpha and CAD as host factors for HDV infection and antiviral targets.

OBJECTIVE: Hepatitis D virus (HDV) is a circular RNA virus coinfecting hepatocytes with hepatitis B virus. Chronic hepatitis D results in severe liver disease and an increased risk of liver cancer. Efficient therapeutic approaches against HDV are absent. DESIGN: Here, we combined an RNAi loss-of-function and small molecule screen to uncover host-dependency factors for HDV infection. RESULTS: Functional screening unravelled the hypoxia-inducible factor (HIF)-signalling and insulin-resistance pathways, RNA polymerase II, glycosaminoglycan biosynthesis and the pyrimidine metabolism as virus-hepatocyte dependency networks. Validation studies in primary human hepatocytes identified the carbamoyl-phosphatesynthetase 2, aspartate transcarbamylase and dihydroorotase (CAD) enzyme and estrogen receptor alpha (encoded by ESR1) as key host factors for HDV life cycle. Mechanistic studies revealed that the two host factors are required for viral replication. Inhibition studies using N-(phosphonoacetyl)-L-aspartic acid and fulvestrant, specific CAD and ESR1 inhibitors, respectively, uncovered their impact as antiviral targets. CONCLUSION: The discovery of HDV host-dependency factors elucidates the pathogenesis of viral disease biology and opens therapeutic strategies for HDV cure.

Characterization of the catalytic flexible loop in the dihydroorotase domain of the human multi-enzymatic protein CAD.

The dihydroorotase (DHOase) domain of the multifunctional protein carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotase (CAD) catalyzes the third step in the de novo biosynthesis of pyrimidine nucleotides in animals. The crystal structure of the DHOase domain of human CAD (huDHOase) revealed that, despite evolutionary divergence, its active site components are highly conserved with those in bacterial DHOases, encoded as monofunctional enzymes. An important element for catalysis, conserved from Escherichia coli to humans, is a flexible loop that closes as a lid over the active site. Here, we combined mutagenic, structural, biochemical, and molecular dynamics analyses to characterize the function of the flexible loop in the activity of CAD's DHOase domain. A huDHOase chimera bearing the E. coli DHOase flexible loop was inactive, suggesting the presence of distinctive elements in the flexible loop of huDHOase that cannot be replaced by the bacterial sequence. We pinpointed Phe-1563, a residue absolutely conserved at the tip of the flexible loop in CAD's DHOase domain, as a critical element for the conformational equilibrium between the two catalytic states of the protein. Substitutions of Phe-1563 with Ala, Leu, or Thr prevented the closure of the flexible loop and inactivated the protein, whereas substitution with Tyr enhanced the interactions of the loop in the closed position and reduced fluctuations and the reaction rate. Our results confirm the importance of the flexible loop in CAD's DHOase domain and explain the key role of Phe-1563 in configuring the active site and in promoting substrate strain and catalysis.

Urea Cycle Dysregulation Generates Clinically Relevant Genomic and Biochemical Signatures.

The urea cycle (UC) is the main pathway by which mammals dispose of waste nitrogen. We find that specific alterations in the expression of most UC enzymes occur in many tumors, leading to a general metabolic hallmark termed "UC dysregulation" (UCD). UCD elicits nitrogen diversion toward carbamoyl-phosphate synthetase2, aspartate transcarbamylase, and dihydrooratase (CAD) activation and enhances pyrimidine synthesis, resulting in detectable changes in nitrogen metabolites in both patient tumors and their bio-fluids. The accompanying excess of pyrimidine versus purine nucleotides results in a genomic signature consisting of transversion mutations at the DNA, RNA, and protein levels. This mutational bias is associated with increased numbers of hydrophobic tumor antigens and a better response to immune checkpoint inhibitors independent of mutational load. Taken together, our findings demonstrate that UCD is a common feature of tumors that profoundly affects carcinogenesis, mutagenesis, and immunotherapy response.

Executioner caspases and CAD are essential for mutagenesis induced by TRAIL or vincristine.

Chemotherapy drugs interfere with cellular processes to generate genotoxic lesions that activate cell death pathways. Sustained DNA damage induced by these drugs can provoke mutations in surviving non-cancerous cells, potentially increasing the risk of therapy-related cancers. Ligation of death receptors by ligands such as TRAIL, and subsequent activation of extrinsic apoptotic pathways, also provokes mutations. In this study, we show that executioner caspase activation of the apoptotic nuclease CAD/DFF40 is essential for TRAIL-induced mutations in surviving cells. As exposure to chemotherapy drugs also activates apoptotic caspases and presumably CAD, we hypothesized that these pathways may also contribute to the mutagenesis induced by conventional chemotherapy drugs, perhaps augmenting the mutations that arise from direct DNA damage provoked by these agents. Interestingly, vincristine-mediated mutations were caspase and CAD dependent. Executioner caspases accounted for some of the mutations caused by the topoisomerase poisons doxorubicin and SN38, but were dispensable for mutagenesis following treatment with cisplatin or temozolomide. These data highlight a non-apoptotic role of caspases in mutagenesis mediated by death receptor agonists, microtubule poisons and topoisomerase inhibitors, and provide further evidence for a potential carcinogenic consequence of sublethal apoptotic signaling stimulated by anticancer therapies.

Structural Insight into the Core of CAD, the Multifunctional Protein Leading De Novo Pyrimidine Biosynthesis.

CAD, the multifunctional protein initiating and controlling de novo biosynthesis of pyrimidines in animals, self-assembles into ∼1.5 MDa hexamers. The structures of the dihydroorotase (DHO) and aspartate transcarbamoylase (ATC) domains of human CAD have been previously determined, but we lack information on how these domains associate and interact with the rest of CAD forming a multienzymatic unit. Here, we prove that a construct covering human DHO and ATC oligomerizes as a dimer of trimers and that this arrangement is conserved in CAD-like from fungi, which holds an inactive DHO-like domain. The crystal structures of the ATC trimer and DHO-like dimer from the fungus Chaetomium thermophilum confirm the similarity with the human CAD homologs. These results demonstrate that, despite being inactive, the fungal DHO-like domain has a conserved structural function. We propose a model that sets the DHO and ATC complex as the central element in the architecture of CAD.

Tracking a CAD-ALK gene rearrangement in urine and blood of a colorectal cancer patient treated with an ALK inhibitor.

BACKGROUND: Monitoring response and resistance to kinase inhibitors is essential to precision cancer medicine, and is usually investigated by molecular profiling of a tissue biopsy obtained at progression. However, tumor heterogeneity and tissue sampling bias limit the effectiveness of this strategy. In addition, tissue biopsies are not always feasible and are associated with risks due to the invasiveness of the procedure. To overcome these limitations, blood-based liquid biopsy analysis has proven effective to non-invasively follow tumor clonal evolution. PATIENTS AND METHODS: We exploited urine cell-free, trans-renal DNA (tr-DNA) and matched plasma circulating tumor DNA (ctDNA) to monitor a metastatic colorectal cancer patient carrying a CAD-ALK translocation during treatment with an ALK inhibitor. RESULTS: Using a custom next generation sequencing panel we identified the genomic CAD-ALK rearrangement and a TP53 mutation in plasma ctDNA. Sensitive assays were developed to detect both alterations in urine tr-DNA. The dynamics of the CAD-ALK rearrangement in plasma and urine were concordant and paralleled the patient's clinical course. Detection of the CAD-ALK gene fusion in urine tr-DNA anticipated radiological confirmation of disease progression. Analysis of plasma ctDNA identified ALK kinase mutations that emerged during treatment with the ALK inhibitor entrectinib. CONCLUSION: We find that urine-based genetic testing allows tracing of tumor-specific oncogenic rearrangements. This strategy could be effectively applied to non-invasively monitor tumor evolution during therapy. The same approach could be exploited to monitor minimal residual disease after surgery with curative intent in patients whose tumors carry gene fusions. The latter could be implemented without the need of patient hospitalization since urine tr-DNA can be self-collected, is stable over time and can be shipped at specified time-points to central labs for testing.

CAD mutations and uridine-responsive epileptic encephalopathy.

Unexplained global developmental delay and epilepsy in childhood pose a major socioeconomic burden. Progress in defining the molecular bases does not often translate into effective treatment. Notable exceptions include certain inborn errors of metabolism amenable to dietary intervention. CAD encodes a multifunctional enzyme involved in de novo pyrimidine biosynthesis. Alternatively, pyrimidines can be recycled from uridine. Exome sequencing in three families identified biallelic CAD mutations in four children with global developmental delay, epileptic encephalopathy, and anaemia with anisopoikilocytosis. Two died aged 4 and 5 years after a neurodegenerative disease course. Supplementation of the two surviving children with oral uridine led to immediate cessation of seizures in both. A 4-year-old female, previously in a minimally conscious state, began to communicate and walk with assistance after 9 weeks of treatment. A 3-year-old female likewise showed developmental progress. Blood smears normalized and anaemia resolved. We establish CAD as a gene confidently implicated in this neurometabolic disorder, characterized by co-occurrence of global developmental delay, dyserythropoietic anaemia and seizures. While the natural disease course can be lethal in early childhood, our findings support the efficacy of uridine supplementation, rendering CAD deficiency a treatable neurometabolic disorder and therefore a potential condition for future (genetic) newborn screening.

Community Sampling and Integrative Taxonomy Reveal New Species and Host Specificity in the Army Ant-Associated Beetle Genus Tetradonia (Coleoptera, Staphylinidae, Aleocharinae).

Army ant colonies host a diverse community of arthropod symbionts. Among the best-studied symbiont communities are those of Neotropical army ants of the genus Eciton. It is clear, however, that even in these comparatively well studied systems, a large proportion of symbiont biodiversity remains unknown. Even more striking is our lack of knowledge regarding the nature and specificity of these host-symbiont interactions. Here we surveyed the diversity and host specificity of rove beetles of the genus Tetradonia Wasmann, 1894 (Staphylinidae: Aleocharinae). Systematic community sampling of 58 colonies of the six local Eciton species at La Selva Biological Station, Costa Rica, combined with an integrative taxonomic approach, allowed us to uncover species diversity, host specificity, and co-occurrence patterns of symbionts in unprecedented detail. We used an integrative taxonomic approach combining morphological and genetic analyses, to delineate species boundaries. Mitochondrial DNA barcodes were analyzed for 362 Tetradonia specimens, and additional nuclear markers for a subset of 88 specimens. All analyses supported the presence of five Tetradonia species, including two species new to science. Host specificity is highly variable across species, ranging from generalists such as T. laticeps, which parasitizes all six local Eciton species, to specialists such as T. lizonae, which primarily parasitizes a single species, E. hamatum. Here we provide a dichotomous key along with diagnostic molecular characters for identification of Tetradonia species at La Selva Biological Station. By reliably assessing biodiversity and providing tools for species identification, we hope to set the baseline for future studies of the ecological and evolutionary dynamics in these species-rich host-symbiont networks.

[Effect of key-gene modification on uridine biosynthesis in Bacillus subtilis].

OBJECTIVE: We studied several crucial factors influencing the uridine biosynthesis in Bacillus subtilis, including mutations of phosphoribosylpyrophosphate synthetase (PRPP synthetase) (prs) and carbamyl phosphate synthetase (pyrAA/pyrAB), and overexpression of heterologous 5'-nucleotidase (sdt1). METHODS: According to the inferred allosteric sites, we introduced point mutation into coding sequences of prs and pyrAB. The mutated prs gene was integratedly expressed in the xylR locus of the chromosome and the pyrAB gene was modified in-situ. The sdt1 gene was overexpressed in the saB locus of the chromosome. The effect of the genetic modification on uridine biosynthesis was characterized by the analysis of uridine, cytidine and uracil in the fermentation broth. RESULTS: The mutations of Asn120Ser, Leu135Ile, Glu52Gly or Val312Ala on PRPP synthase resulted in an increase of uridine production by 67% and 96%, respectively. The mutations of Ser948Phe, Thr977Ala and Lys993Ile on carbamyl phosphate synthase resulted in a 182% increase of uridine yield to 6.97 g/L. The overexpression of heterologous 5'-nucleotidase resulted in a 17% increase of uridine yield to 8.16 g/L. CONCLUSION: The activity and regulation mechanism of PRPP synthase and carbamyl phosphate synthase was an important factor to limit the excessive synthesis of uridine. Asn120Ser and Leu135Ile mutations of PRPP synthase and Ser948Phe, Thr977Ala and Lys993Ile mutations of carbamyl phosphate synthase will facilitate the biosynthesis of uridine. The additional Glu52Gly and Val312Ala mutations of PRPP synthase were beneficial for uridine biosynthesis. The reaction from UMP to uridine also limited the biosynthesis of uridine in B. subtilis.

Behavior and population structure of Anopheles darlingi in Colombia.

Anopheles darlingi is a widely distributed and important malaria vector in Colombia. Biogeographical and ecological heterogeneity across the Colombian distribution led to the hypothesis of behavioral and genetic differentiation among A. darlingi populations. A total of 2017 A. darlingi specimens were collected during 222 h of sampling. This vector was the most abundant anopheline species in most of the localities sampled. Subdivision between samples collected west and east of the Andes was indicated by 1) mitochondrial COI and nuclear CAD sequences from NW-W and CE-S populations (COI ΦST=0.48761-0.81974, CAD FST=0.11319-0.21321), 2) a COI haplotype network, and 3) SAMOVA. Endo- and exophagy were detected in populations west of the Andes, whereas exophagy was evident in PTG, a locality east of the Andes. Isolation by resistance was significant for COI and explained 26% of the genetic differentiation. We suggest that at a macrogeographic scale, the Andes influence the differentiation of A. darlingi in Colombia and may drive divergence, and, at a microgeographic scale, ecological differences have a significant impact on structure. These data could constitute a baseline for the design of effective vector interventions, locality-specific for the east and similar for panmictic populations west of the Andes.

Novel CAD-ALK gene rearrangement is drugable by entrectinib in colorectal cancer.

BACKGROUND: Activated anaplastic lymphoma kinase (ALK) gene fusions are recurrent events in a small fraction of colorectal cancers (CRCs), although these events have not yet been exploited as in other malignancies. METHODS: We detected ALK protein expression by immunohistochemistry and gene rearrangements by fluorescence in situ hybridisation in the ALKA-372-001 phase I study of the pan-Trk, ROS1, and ALK inhibitor entrectinib. One out of 487 CRCs showed ALK positivity with a peculiar pattern that prompted further characterisation by targeted sequencing using anchored multiplex PCR. RESULTS: A novel ALK fusion with the carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD) gene (CAD-ALK fusion gene) was identified. It resulted from inversion within chromosome 2 and the fusion of exons 1-35 of CAD with exons 20-29 of ALK. After failure of previous standard therapies, treatment of this patient with the ALK inhibitor entrectinib resulted in a durable objective tumour response. CONCLUSIONS: We describe the novel CAD-ALK rearrangement as an oncogene and provide the first evidence of its drugability as a new molecular target in CRC.

Creation of a putative third metal binding site in type II dihydroorotases significantly enhances enzyme activity.

Dihydroorotase (DHOase) is the third enzyme in the de novo biosynthesis pathway of pyrimidine nucleotides. DHOase is divided into two types (I and II). Type II DHOase generally contains a binuclear metal center in its active site. Recently, the crystal structure of DHOase domain in human CAD protein (huDHOase) has revealed three metal ions in the protein's active site. However, whether type II DHOase can have the critical third metal ion, as observed in huDHOase, remains unknown. In the present study, the putative third metal binding site in type II enzymes, such as the prokaryotic Salmonella enterica serovar Typhimurium LT2 DHOase (StDHOase) and the eukaryotic Saccharomyces cerevisiae DHOase (ScDHOase), was created and identified. StDHOase T198E and ScDHOase T208E mutants had higher activities compared with their wild-type enzymes. The need for a higher DHOase stability and activity may drive creation of the third metal ion binding site in huDHOase, which can be achieved by mutating a highly conserved position T in type II dihydroorotases to E, similar to that in huDHOase.

Disruption of Transcriptional Coactivator Sub1 Leads to Genome-Wide Re-distribution of Clustered Mutations Induced by APOBEC in Active Yeast Genes.

Mutations in genomes of species are frequently distributed non-randomly, resulting in mutation clusters, including recently discovered kataegis in tumors. DNA editing deaminases play the prominent role in the etiology of these mutations. To gain insight into the enigmatic mechanisms of localized hypermutagenesis that lead to cluster formation, we analyzed the mutational single nucleotide variations (SNV) data obtained by whole-genome sequencing of drug-resistant mutants induced in yeast diploids by AID/APOBEC deaminase and base analog 6-HAP. Deaminase from sea lamprey, PmCDA1, induced robust clusters, while 6-HAP induced a few weak ones. We found that PmCDA1, AID, and APOBEC1 deaminases preferentially mutate the beginning of the actively transcribed genes. Inactivation of transcription initiation factor Sub1 strongly reduced deaminase-induced can1 mutation frequency, but, surprisingly, did not decrease the total SNV load in genomes. However, the SNVs in the genomes of the sub1 clones were re-distributed, and the effect of mutation clustering in the regions of transcription initiation was even more pronounced. At the same time, the mutation density in the protein-coding regions was reduced, resulting in the decrease of phenotypically detected mutants. We propose that the induction of clustered mutations by deaminases involves: a) the exposure of ssDNA strands during transcription and loss of protection of ssDNA due to the depletion of ssDNA-binding proteins, such as Sub1, and b) attainment of conditions favorable for APOBEC action in subpopulation of cells, leading to enzymatic deamination within the currently expressed genes. This model is applicable to both the initial and the later stages of oncogenic transformation and explains variations in the distribution of mutations and kataegis events in different tumor cells.

Biallelic mutations in CAD, impair de novo pyrimidine biosynthesis and decrease glycosylation precursors.

In mitochondria, carbamoyl-phosphate synthetase 1 activity produces carbamoyl phosphate for urea synthesis, and deficiency results in hyperammonemia. Cytoplasmic carbamoyl-phosphate synthetase 2, however, is part of a tri-functional enzyme encoded by CAD; no human disease has been attributed to this gene. The tri-functional enzyme contains carbamoyl-phosphate synthetase 2 (CPS2), aspartate transcarbamylase (ATCase) and dihydroorotase (DHOase) activities, which comprise the first three of six reactions required for de novo pyrimidine biosynthesis. Here we characterize an individual who is compound heterozygous for mutations in different domains of CAD. One mutation, c.1843-1G>A, results in an in-frame deletion of exon 13. The other, c.6071G>A, causes a missense mutation (p.Arg2024Gln) in a highly conserved residue that is essential for carbamoyl-phosphate binding. Metabolic flux studies showed impaired aspartate incorporation into RNA and DNA through the de novo synthesis pathway. In addition, CTP, UTP and nearly all UDP-activated sugars that serve as donors for glycosylation were decreased. Uridine supplementation rescued these abnormalities, suggesting a potential therapy for this new glycosylation disorder.