Publications by authors named "Ljubica Caldovic"

43 Publications

Fifteen years of urea cycle disorders brain research: Looking back, looking forward.

Anal Biochem 2021 Oct 9:114343. Epub 2021 Oct 9.

Division of Neurogenetics and Neurodevelopmental Pediatrics, Children's National Hospital, Washington D.C., United States. Electronic address:

Urea cycle disorders (UCD) are inherited diseases resulting from deficiency in one of six enzymes or two carriers that are required to remove ammonia from the body. UCD may be associated with neurological damage encompassing a spectrum from asymptomatic/mild to severe encephalopathy, which results in most cases from Hyperammonemia (HA) and elevation of other neurotoxic intermediates of metabolism. Electroencephalography (EEG), Magnetic resonance imaging (MRI) and Proton Magnetic resonance spectroscopy (MRS) are noninvasive measures of brain function and structure that can be used during HA to guide management and provide prognostic information, in addition to being research tools to understand the pathophysiology of UCD associated brain injury. The Urea Cycle Rare disorders Consortium (UCDC) has been invested in research to understand the immediate and downstream effects of hyperammonemia (HA) on brain using electroencephalogram (EEG) and multimodal brain MRI to establish early patterns of brain injury and to track recovery and prognosis. This review highlights the evolving knowledge about the impact of UCD and HA in particular on neurological injury and recovery and use of EEG and MRI to study and evaluate prognostic factors for risk and recovery. It recognizes the work of others and discusses the UCDC's prior work and future research priorities.
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http://dx.doi.org/10.1016/j.ab.2021.114343DOI Listing
October 2021

Noncoding sequence variants define a novel regulatory element in the first intron of the N-acetylglutamate synthase gene.

Hum Mutat 2021 Sep 12. Epub 2021 Sep 12.

Center for Genetic Medicine Research, Children's National Hospital, Washington, District of Columbia, USA.

N-acetylglutamate synthase deficiency is an autosomal recessive urea cycle disorder caused either by decreased expression of the NAGS gene or defective NAGS enzyme resulting in decreased production of N-acetylglutamate (NAG), an allosteric activator of carbamylphosphate synthetase 1 (CPS1). NAGSD is the only urea cycle disorder that can be effectively treated with a single drug, N-carbamylglutamate (NCG), a stable NAG analog, which activates CPS1 to restore ureagenesis. We describe three patients with NAGSD due to four novel noncoding sequence variants in the NAGS regulatory regions. All three patients had hyperammonemia that resolved upon treatment with NCG. Sequence variants NM_153006.2:c.427-222G>A and NM_153006.2:c.427-218A>C reside in the 547 bp-long first intron of NAGS and define a novel NAGS regulatory element that binds retinoic X receptor α. Sequence variants NC_000017.10:g.42078967A>T (NM_153006.2:c.-3065A>T) and NC_000017.10:g.42078934C>T (NM_153006.2:c.-3098C>T) reside in the NAGS enhancer, within known HNF1 and predicted glucocorticoid receptor binding sites, respectively. Reporter gene assays in HepG2 and HuH-7 cells demonstrated that all four substitutions could result in reduced expression of NAGS. These findings show that analyzing noncoding regions of NAGS and other urea cycle genes can reveal molecular causes of disease and identify novel regulators of ureagenesis.
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http://dx.doi.org/10.1002/humu.24281DOI Listing
September 2021

Mitochondrial Enzymes of the Urea Cycle Cluster at the Inner Mitochondrial Membrane.

Front Physiol 2020 21;11:542950. Epub 2021 Jan 21.

Center for Genetic Medicine Research, Children's National Medical Center, Washington, DC, United States.

Mitochondrial enzymes involved in energy transformation are organized into multiprotein complexes that channel the reaction intermediates for efficient ATP production. Three of the mammalian urea cycle enzymes: N-acetylglutamate synthase (NAGS), carbamylphosphate synthetase 1 (CPS1), and ornithine transcarbamylase (OTC) reside in the mitochondria. Urea cycle is required to convert ammonia into urea and protect the brain from ammonia toxicity. Urea cycle intermediates are tightly channeled in and out of mitochondria, indicating that efficient activity of these enzymes relies upon their coordinated interaction with each other, perhaps in a cluster. This view is supported by mutations in surface residues of the urea cycle proteins that impair ureagenesis in the patients, but do not affect protein stability or catalytic activity. We find the NAGS, CPS1, and OTC proteins in liver mitochondria can associate with the inner mitochondrial membrane (IMM) and can be co-immunoprecipitated. Our analysis of vertebrate NAGS proteins, the least abundant of the urea cycle enzymes, identified a protein-protein interaction region present only in the mammalian NAGS protein-"variable segment," which mediates the interaction of NAGS with CPS1. Use of super resolution microscopy showed that NAGS, CPS1 and OTC are organized into clusters in the hepatocyte mitochondria. These results indicate that mitochondrial urea cycle proteins cluster, instead of functioning either independently or in a rigid multienzyme complex.
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http://dx.doi.org/10.3389/fphys.2020.542950DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7860981PMC
January 2021

Clinical and structural insights into potential dominant negative triggers of proximal urea cycle disorders.

Biochimie 2021 Apr 9;183:89-99. Epub 2020 Dec 9.

Division of Metabolism and Children's Research Center, University Children's Hospital Zurich, Zurich, Switzerland. Electronic address:

Despite biochemical and genetic testing being the golden standards for identification of proximal urea cycle disorders (UCDs), genotype-phenotype correlations are often unclear. Co-occurring partial defects affecting more than one gene have not been demonstrated so far in proximal UCDs. Here, we analyzed the mutational spectrum of 557 suspected proximal UCD individuals. We probed oligomerizing forms of NAGS, CPS1 and OTC, and evaluated the surface exposure of residues mutated in heterozygously affected individuals. BN-PAGE and gel-filtration chromatography were employed to discover protein-protein interactions within recombinant enzymes. From a total of 281 confirmed patients, only 15 were identified as "heterozygous-only" candidates (i.e. single defective allele). Within these cases, the only missense variants to potentially qualify as dominant negative triggers were CPS1 p.Gly401Arg and NAGS p.Thr181Ala and p.Tyr512Cys, as assessed by residue oligomerization capacity and surface exposure. However, all three candidates seem to participate in critical intramolecular functions, thus, unlikely to facilitate protein-protein interactions. This interpretation is further supported by BN-PAGE and gel-filtration analyses revealing no multiprotein proximal urea cycle complex formation. Collectively, genetic analysis, structural considerations and in vitro experiments point against a prominent role of dominant negative effects in human proximal UCDs.
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http://dx.doi.org/10.1016/j.biochi.2020.12.003DOI Listing
April 2021

The Urine Microbiome of Healthy Men and Women Differs by Urine Collection Method.

Int Neurourol J 2020 Mar 31;24(1):41-51. Epub 2020 Mar 31.

Division of Urology, Children's National Medical Center, George Washington University School of Medicine, Washington DC, USA.

Purpose: Compared to the microbiome of other body sites, the urinary microbiome remains poorly understood. Although noninvasive voided urine specimens are convenient, contamination by urethral microbiota may confound understanding of the bladder microbiome. Herein we compared the voiding- versus catheterization-associated urine microbiome of healthy men and women.

Methods: An asymptomatic, healthy cohort of 6 women and 14 men underwent midstream urine collection, followed by sterile catheterization of the bladder after bladder refilling. Urine samples underwent urine dipstick testing and conventional microscopy and urine cultures. Samples also underwent Illumina MiSeq-based 16S ribosomal RNA gene amplification and sequencing.

Results: All organisms identified by urine culture were also identified by 16S amplification; however, next-generation sequencing (NGS) also detected bacteria not identified by cultivation. Lactobacillus and Streptococcus were the most abundant species. Abundances of the 9 predominant bacterial genera differed between the urethra and bladder. Voided and catheterized microbiomes share all dominant (>1%) genera and Operational Taxonomic Units but in similar or different proportions. Hence, urethra and bladder microbiomes do not differ in taxonomic composition, but rather in taxonomic structure. Women had higher abundance of Lactobacillus and Prevotella than men.

Conclusion: Our findings lend credence to the hypothesis that Lactobacilli are important members of the healthy urine microbiome. Our data also suggest that the microbiomes of the urethra and bladder differ from one another. In conclusion, urine collection method results in different 16S-based NGS data, likely due to the sensitivity of NGS methods enabling detection of urethral bacteria present in voided but not catheterized urine specimens.
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http://dx.doi.org/10.5213/inj.1938244.122DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7136448PMC
March 2020

AMP-activated protein kinase signaling regulated expression of urea cycle enzymes in response to changes in dietary protein intake.

J Inherit Metab Dis 2019 11 1;42(6):1088-1096. Epub 2019 Aug 1.

Center for Genetic Medicine Research, Children's National Medical Center, Washington, District of Columbia.

Abundance of urea cycle enzymes in the liver is regulated by dietary protein intake. Although urea cycle enzyme levels rise in response to a high-protein (HP) diet, signaling networks that sense dietary protein intake and trigger changes in expression of urea cycle genes have not been identified. The aim of this study was to identify signaling pathway(s) that respond to changes in protein intake and regulate expression of urea cycle genes in mice and human hepatocytes. Mice were adapted to either HP or low-protein diets followed by isolation of liver protein and mRNA and integrated analysis of the proteomic and transcriptomic data. HP diet led to increased expression of mRNA and enzymes in amino acid degradation pathways and decreased expression of mRNA and enzymes in carbohydrate and fat metabolism, which implicated adenosine monophosphate-activated protein kinase (AMPK) as a possible regulator. Primary human hepatocytes, treated with 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) an activator of AMPK, were used to test whether AMPK regulates expression of urea cycle genes. The abundance of carbamoylphosphate synthetase 1 and ornithine transcarbamylase mRNA increased in hepatocytes treated with AICAR, which supports a role for AMPK signaling in regulation of the urea cycle. Because AMPK is either a target of drugs used to treat type-2 diabetes, these drugs might increase the expression of urea cycle enzymes in patients with partial urea cycle disorders, which could be the basis of a new therapeutic approach.
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http://dx.doi.org/10.1002/jimd.12133DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7385982PMC
November 2019

A single intravesical instillation of GG is safe in children and adults with neuropathic bladder: A phase Ia clinical trial.

J Spinal Cord Med 2021 01 17;44(1):62-69. Epub 2019 May 17.

Paralysis Rehabilitation and Recovery Program, Spinal Cord Injury Research, MedStar National Rehabilitation Hospital, Washington, DC, USA.

Manipulation of the microbiome is an emerging approach to promote health. We conducted a Phase Ia safety study of a single bladder instillation of probiotics in asymptomatic patients with neuropathic bladder to determine the tolerability and safety of a single instillation. Phase Ia safety study. Outpatient rehabilitation clinic at a rehabilitation hospital (adults) and urology clinic at a free-standing children's hospital (children). Ten patients with neuropathic bladder were included: five children with spina bifida and five adults with spinal cord injury. A single GG (Culturelle, 20 billion live organisms) instillation. After the instillation, participants self-monitored symptoms using the Urinary Symptoms Questionnaire for People with Neuropathic Bladder using Intermittent Catheterization daily for one week. Repeat urinalysis, urine culture, and 16S bacterial rRNA-based microbiome analyses were performed 7-10 days after instillation. Probiotic instillation was well-tolerated. One child had upper respiratory tract symptoms during the trial, and two had transient cloudy urine. No adults reported any symptoms following instillation. did not grow on culture post-instillation. There were differences in beta diversity of the urine microbiome in children vs. adults with neuropathic bladder (P < 0.0156). was present in the pre-instillation urinary microbiomes all of the adults and 4 out of 5 of the pediatric subjects, and identified in 4 out of 5 of both the adult and pediatric subjects' post-instillation urinary microbiomes. Intravesical instillation of Culturelle probiotic may be safe and well-tolerated in patients with neuropathic bladder.
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http://dx.doi.org/10.1080/10790268.2019.1616456DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7919893PMC
January 2021

N-Acetylglutamate Synthase Deficiency Due to a Recurrent Sequence Variant in the N-acetylglutamate Synthase Enhancer Region.

Sci Rep 2018 10 18;8(1):15436. Epub 2018 Oct 18.

Children's Research Institute, Children's National Medical Center, Washington, DC, 20010, USA.

N-acetylglutamate synthase deficiency (NAGSD, MIM #237310) is an autosomal recessive disorder of the urea cycle that results from absent or decreased production of N-acetylglutamate (NAG) due to either decreased NAGS gene expression or defective NAGS enzyme. NAG is essential for the activity of carbamylphosphate synthetase 1 (CPS1), the first and rate-limiting enzyme of the urea cycle. NAGSD is the only urea cycle disorder that can be treated with a single drug, N-carbamylglutamate (NCG), which can activate CPS1 and completely restore ureagenesis in patients with NAGSD. We describe a novel sequence variant NM_153006.2:c.-3026C > T in the NAGS enhancer that was found in three patients from two families with NAGSD; two patients had hyperammonemia that resolved upon treatment with NCG, while the third patient increased dietary protein intake after initiation of NCG therapy. Two patients were homozygous for the variant while the third patient had the c.-3026C > T variant and a partial uniparental disomy that encompassed the NAGS gene on chromosome 17. The c.-3026C > T sequence variant affects a base pair that is highly conserved in vertebrates; the variant is predicted to be deleterious by several bioinformatics tools. Functional assays in cultured HepG2 cells demonstrated that the c.-3026C > T substitution could result in reduced expression of the NAGS gene. These findings underscore the importance of analyzing NAGS gene regulatory regions when looking for molecular causes of NAGSD.
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http://dx.doi.org/10.1038/s41598-018-33457-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6194121PMC
October 2018

Identification of in the urine of an individual with spinal cord injury and augmentation cystoplasty using 16S sequencing: copathogen or innocent bystander?

Spinal Cord Ser Cases 2018 21;4:85. Epub 2018 Sep 21.

7Division of Urology, Children's National Health System, Washington, DC USA.

Introduction: People with neuropathic bladder (NB) secondary to spinal cord injury (SCI) are at risk for multiple genitourinary complications, the most frequent of which is urinary tract infection (UTI). Despite the high frequency with which UTI occurs, our understanding of the role of urinary microbes in health and disease is limited. In this paper, we present the first prospective case study integrating symptom reporting, urinalysis, urine cultivation, and 16S ribosomal ribonucleic acid (rRNA) sequencing of the urine microbiome.

Case Presentation: A 55-year-old male with NB secondary to SCI contributed 12 urine samples over an 8-month period during asymptomatic, symptomatic, and postantibiotic periods. All bacteria identified on culture were present on 16S rRNA sequencing, however, 16S rRNA sequencing revealed the presence of bacteria not isolated on culture. In particular, was present in three samples during both asymptomatic and symptomatic periods. White blood cells of ≥5-10/high power field and leukocyte esterase ≥2 on urinalysis was associated with the presence of symptoms.

Discussion: In this patient, there was a predominance of pathogenic bacteria and a lack of putative probiotic bacteria during both symptomatic and asymptomatic states. Urinalysis-defined inflammatory markers were present to a greater extent during symptomatic periods compared to the asymptomatic state, which may underscore a role for urinalysis or other inflammatory markers in differentiating asymptomatic bacteriuria from UTI in patients with NB. The finding of potentially pathogenic bacteria identified by sequencing but not cultivation, suggests a need for greater understanding of the relationships amongst bacterial species in the bacteriuric neuropathic bladder.
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http://dx.doi.org/10.1038/s41394-018-0115-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6155001PMC
September 2018

Sources and Fates of Carbamyl Phosphate: A Labile Energy-Rich Molecule with Multiple Facets.

Biology (Basel) 2018 Jun 12;7(2). Epub 2018 Jun 12.

Center for Genetic Medicine Research, Children's National Medical Center, Washington, DC 20010, USA.

Carbamyl phosphate (CP) is well-known as an essential intermediate of pyrimidine and arginine/urea biosynthesis. Chemically, CP can be easily synthesized from dihydrogen phosphate and cyanate. Enzymatically, CP can be synthesized using three different classes of enzymes: (1) ATP-grasp fold protein based carbamyl phosphate synthetase (CPS); (2) Amino-acid kinase fold carbamate kinase (CK)-like CPS (anabolic CK or aCK); and (3) Catabolic transcarbamylase. The first class of CPS can be further divided into three different types of CPS as CPS I, CPS II, and CPS III depending on the usage of ammonium or glutamine as its nitrogen source, and whether -acetyl-glutamate is its essential co-factor. CP can donate its carbamyl group to the amino nitrogen of many important molecules including the most well-known ornithine and aspartate in the arginine/urea and pyrimidine biosynthetic pathways. CP can also donate its carbamyl group to the hydroxyl oxygen of a variety of molecules, particularly in many antibiotic biosynthetic pathways. Transfer of the carbamyl group to the nitrogen group is catalyzed by the anabolic transcarbamylase using a direct attack mechanism, while transfer of the carbamyl group to the oxygen group is catalyzed by a different class of enzymes, CmcH/NodU CTase, using a different mechanism involving a three-step reaction, decomposition of CP to carbamate and phosphate, transfer of the carbamyl group from carbamate to ATP to form carbamyladenylate and pyrophosphate, and transfer of the carbamyl group from carbamyladenylate to the oxygen group of the substrate. CP is also involved in transferring its phosphate group to ADP to generate ATP in the fermentation of many microorganisms. The reaction is catalyzed by carbamate kinase, which may be termed as catabolic CK (cCK) in order to distinguish it from CP generating CK. CP is a thermally labile molecule, easily decomposed into phosphate and cyanate, or phosphate and carbamate depending on the pH of the solution, or the presence of enzyme. Biological systems have developed several mechanisms including channeling between enzymes, increased affinity of CP to enzymes, and keeping CP in a specific conformation to protect CP from decomposition. CP is highly important for our health as both a lack of, or decreased, CP production and CP accumulation results in many disease conditions.
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http://dx.doi.org/10.3390/biology7020034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6022934PMC
June 2018

Disease-causing mutations in the promoter and enhancer of the ornithine transcarbamylase gene.

Hum Mutat 2018 04 16;39(4):527-536. Epub 2018 Jan 16.

Center for Genetic Medicine Research, Children's National Health System, Washington, District of Columbia.

The ornithine transcarbamylase (OTC) gene is on the X chromosome and its product catalyzes the formation of citrulline from ornithine and carbamylphosphate in the urea cycle. About 10%-15% of patients, clinically diagnosed with OTC deficiency (OTCD), lack identifiable mutations in the coding region or splice junctions of the OTC gene on routine molecular testing. We collected DNA from such patients via retrospective review and by prospective enrollment. In nine of 38 subjects (24%), we identified a sequence variant in the OTC regulatory regions. Eight subjects had unique sequence variants in the OTC promoter and one subject had a novel sequence variant in the OTC enhancer. All sequence variants affect positions that are highly conserved in mammalian OTC genes. Functional studies revealed reduced reporter gene expression with all sequence variants. Two sequence variants caused decreased binding of the HNF4 transcription factor to its mutated binding site. Bioinformatic analyses combined with functional assays can be used to identify and authenticate pathogenic sequence variants in regulatory regions of the OTC gene, in other urea cycle disorders or other inborn errors of metabolism.
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http://dx.doi.org/10.1002/humu.23394DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7388160PMC
April 2018

Corrigendum to "GJC2 promoter mutations causing Pelizaeus-Merzbacher-like disease" [Mol. Genet. Metab. 111 (2014) 393-398].

Mol Genet Metab 2016 11;119(3):293

Department of Neurology, Children's National Medical Center, Washington, DC, USA; Department of Pediatrics, Children's National Medical Center, Washington, DC, USA; Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA, USA. Electronic address:

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http://dx.doi.org/10.1016/j.ymgme.2016.06.011DOI Listing
November 2016

Whole exome sequencing in patients with white matter abnormalities.

Ann Neurol 2016 06 9;79(6):1031-1037. Epub 2016 May 9.

School of Medicine and Health Sciences, George Washington University, Washington, DC.

Here we report whole exome sequencing (WES) on a cohort of 71 patients with persistently unresolved white matter abnormalities with a suspected diagnosis of leukodystrophy or genetic leukoencephalopathy. WES analyses were performed on trio, or greater, family groups. Diagnostic pathogenic variants were identified in 35% (25 of 71) of patients. Potentially pathogenic variants were identified in clinically relevant genes in a further 7% (5 of 71) of cases, giving a total yield of clinical diagnoses in 42% of individuals. These findings provide evidence that WES can substantially decrease the number of unresolved white matter cases. Ann Neurol 2016;79:1031-1037.
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http://dx.doi.org/10.1002/ana.24650DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5354169PMC
June 2016

Redefining Healthy Urine: A Cross-Sectional Exploratory Metagenomic Study of People With and Without Bladder Dysfunction.

J Urol 2016 Aug 22;196(2):579-87. Epub 2016 Jan 22.

Division of Urology, Children's National Health System, Washington, D.C.

Purpose: We used the PathoScope platform to perform species level analyses of publicly available, 16S rRNA pyrosequenced, asymptomatic urine data to determine relationships between microbiomes, and clinical and functional phenotypes.

Materials And Methods: We reanalyzed previously reported, cross-sectionally acquired urine samples from 47 asymptomatic subjects, including 23 controls and 24 subjects with neuropathic bladder. Urine was originally collected by the usual method of bladder drainage and analyzed by urinalysis, culture and pyrosequencing. Urinalysis and culture values were stratified as leukocyte esterase (0, or 1 or greater), nitrite (positive or negative), pyuria (fewer than 5, or 5 or greater white blood cells per high power field), cloudy urine (positive or negative) and urine culture bacterial growth (less than 50,000, or 50,000 or greater cfu/ml). PathoScope was used for next generation sequencing alignment, bacterial classification and microbial diversity characterization.

Results: Subjects with neuropathic bladder were significantly more likely to have positive leukocyte esterase and pyuria, cloudy urine and bacterial growth. Of 47 samples 23 showed bacterial growth on culture and in all samples bacteria were identified by pyrosequencing. Nonneuropathic bladder urine microbiomes included greater proportions of Lactobacillus crispatus in females and Staphylococcus haemolyticus in males. The Lactobacillus community differed significantly among females depending on bladder function. Irrespective of gender the subjects with neuropathic bladder had greater proportions of Enterococcus faecalis, Proteus mirabilis and Klebsiella pneumonia. In 4 subjects with neuropathic bladder Actinobaculum sp. was detected by sequencing and by PathoScope but not by cultivation and in all cases it was associated with pyuria.

Conclusions: Using PathoScope plus 16S pyrosequencing we were able to identify unique, phenotype dependent, species level microbes. Novel findings included absent L. crispatus in the urine of females with neuropathic bladder and the presence of Actinobaculum only in subjects with neuropathic bladder.
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http://dx.doi.org/10.1016/j.juro.2016.01.088DOI Listing
August 2016

Magnetic resonance imaging spectrum of succinate dehydrogenase-related infantile leukoencephalopathy.

Ann Neurol 2016 Mar 12;79(3):379-86. Epub 2016 Feb 12.

Department of Child Neurology, VU University Medical Center, Amsterdam, The Netherlands.

Objective: Succinate dehydrogenase-deficient leukoencephalopathy is a complex II-related mitochondrial disorder for which the clinical phenotype, neuroimaging pattern, and genetic findings have not been comprehensively reviewed.

Methods: Nineteen individuals with succinate dehydrogenase deficiency-related leukoencephalopathy were reviewed for neuroradiological, clinical, and genetic findings as part of institutional review board-approved studies at Children's National Health System (Washington, DC) and VU University Medical Center (Amsterdam, the Netherlands).

Results: All individuals had signal abnormalities in the central corticospinal tracts and spinal cord where imaging was available. Other typical findings were involvement of the cerebral hemispheric white matter with sparing of the U fibers, the corpus callosum with sparing of the outer blades, the basis pontis, middle cerebellar peduncles, and cerebellar white matter, and elevated succinate on magnetic resonance spectroscopy (MRS). The thalamus was involved in most studies, with a predilection for the anterior nucleus, pulvinar, and geniculate bodies. Clinically, infantile onset neurological regression with partial recovery and subsequent stabilization was typical. All individuals had mutations in SDHA, SDHB, or SDHAF1, or proven biochemical defect.

Interpretation: Succinate dehydrogenase deficiency is a rare leukoencephalopathy, for which improved recognition by magnetic resonance imaging (MRI) in combination with advanced sequencing technologies allows noninvasive diagnostic confirmation. The MRI pattern is characterized by cerebral hemispheric white matter abnormalities with sparing of the U fibers, corpus callosum involvement with sparing of the outer blades, and involvement of corticospinal tracts, thalami, and spinal cord. In individuals with infantile regression and this pattern of MRI abnormalities, the differential diagnosis should include succinate dehydrogenase deficiency, in particular if MRS shows elevated succinate.
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http://dx.doi.org/10.1002/ana.24572DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5712845PMC
March 2016

Genotype-Phenotype Correlations in Ornithine Transcarbamylase Deficiency: A Mutation Update.

J Genet Genomics 2015 May 19;42(5):181-94. Epub 2015 May 19.

Department of Integrative Systems Biology, Center for Genetic Medicine Research, Children's National Health System, Washington DC 20010, USA. Electronic address:

Ornithine transcarbamylase (OTC) deficiency is an X-linked trait that accounts for nearly half of all inherited disorders of the urea cycle. OTC is one of the enzymes common to both the urea cycle and the bacterial arginine biosynthesis pathway; however, the role of OTC has changed over evolution. For animals with a urea cycle, defects in OTC can trigger hyperammonemic episodes that can lead to brain damage and death. This is the fifth mutation update for human OTC with previous updates reported in 1993, 1995, 2002, and 2006. In the 2006 update, 341 mutations were reported. This current update contains 417 disease-causing mutations, and also is the first report of this series to incorporate information about natural variation of the OTC gene in the general population through examination of publicly available genomic data and examination of phenotype/genotype correlations from patients participating in the Urea Cycle Disorders Consortium Longitudinal Study and the first to evaluate the suitability of systematic computational approaches to predict severity of disease associated with different types of OTC mutations.
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http://dx.doi.org/10.1016/j.jgg.2015.04.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4565140PMC
May 2015

Expression pattern and biochemical properties of zebrafish N-acetylglutamate synthase.

PLoS One 2014 22;9(1):e85597. Epub 2014 Jan 22.

Department of Biological Sciences, Rowan University, Glassboro, New Jersey, United States of America.

The urea cycle converts ammonia, a waste product of protein catabolism, into urea. Because fish dispose ammonia directly into water, the role of the urea cycle in fish remains unknown. Six enzymes, N-acetylglutamate synthase (NAGS), carbamylphosphate synthetase III, ornithine transcarbamylase, argininosuccinate synthase, argininosuccinate lyase and arginase 1, and two membrane transporters, ornithine transporter and aralar, comprise the urea cycle. The genes for all six enzymes and both transporters are present in the zebrafish genome. NAGS (EC 2.3.1.1) catalyzes the formation of N-acetylglutamate from glutamate and acetyl coenzyme A and in zebrafish is partially inhibited by L-arginine. NAGS and other urea cycle genes are highly expressed during the first four days of zebrafish development. Sequence alignment of NAGS proteins from six fish species revealed three regions of sequence conservation: the mitochondrial targeting signal (MTS) at the N-terminus, followed by the variable and conserved segments. Removal of the MTS yields mature zebrafish NAGS (zfNAGS-M) while removal of the variable segment from zfNAGS-M results in conserved NAGS (zfNAGS-C). Both zfNAGS-M and zfNAGS-C are tetramers in the absence of L-arginine; addition of L-arginine decreased partition coefficients of both proteins. The zfNAGS-C unfolds over a broader temperature range and has higher specific activity than zfNAGS-M. In the presence of L-arginine the apparent Vmax of zfNAGS-M and zfNAGS-C decreased, their Km(app) for acetyl coenzyme A increased while the Km(app) for glutamate remained unchanged. The expression pattern of NAGS and other urea cycle genes in developing zebrafish suggests that they may have a role in citrulline and/or arginine biosynthesis during the first day of development and in ammonia detoxification thereafter. Biophysical and biochemical properties of zebrafish NAGS suggest that the variable segment may stabilize a tetrameric state of zfNAGS-M and that under physiological conditions zebrafish NAGS catalyzes formation of N-acetylglutamate at the maximal rate.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0085597PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3899043PMC
December 2014

GJC2 promoter mutations causing Pelizaeus-Merzbacher-like disease.

Mol Genet Metab 2014 03 16;111(3):393-398. Epub 2013 Dec 16.

Department of Neurology, Children's National Medical Center, Washington, DC, USA; Department of Pediatrics, Children's National Medical Center, Washington, DC, USA; Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA, USA. Electronic address:

Objective: Pelizaeus-Merzbacher-like disease is a rare hypomyelinating leukodystrophy caused by autosomal recessive mutations in GJC2, encoding a gap junction protein essential for production of a mature myelin sheath. A previously identified GJC2 mutation (c.-167A>G) in the promoter region is hypothesized to disrupt a putative SOX10 binding site; however, the lack of additional mutations in this region and contradictory functional data have limited the interpretation of this variant.

Methods: We describe two independent Pelizaeus-Merzbacher-like disease families with a novel promoter region mutation and updated in vitro functional assays.

Results: A novel GJC2 mutation (c.-170A>G) in the promoter region was identified in Pelizaeus-Merzbacher-like disease patients. In vitro functional assays using human GJC2 promoter constructs demonstrated that this mutation and the previously described c.-167A>G mutation similarly diminished the transcriptional activity driven by SOX10 and the binding affinity for SOX10.

Interpretation: These findings support the role of GJC2 promoter mutations in Pelizaeus-Merzbacher-like disease. GJC2 promoter region mutation screening should be included in the evaluation of patients with unexplained hypomyelinating leukodystrophies.
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http://dx.doi.org/10.1016/j.ymgme.2013.12.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4183365PMC
March 2014

A de novo mutation in the β-tubulin gene TUBB4A results in the leukoencephalopathy hypomyelination with atrophy of the basal ganglia and cerebellum.

Am J Hum Genet 2013 May 11;92(5):767-73. Epub 2013 Apr 11.

Institute for Molecular Bioscience, University of Queensland, St. Lucia, Queensland 4072, Australia.

Hypomyelination with atrophy of the basal ganglia and cerebellum (H-ABC) is a rare hereditary leukoencephalopathy that was originally identified by MRI pattern analysis, and it has thus far defied all attempts at identifying the causal mutation. Only 22 cases are published in the literature to date. We performed exome sequencing on five family trios, two family quartets, and three single probands, which revealed that all eleven H-ABC-diagnosed individuals carry the same de novo single-nucleotide TUBB4A mutation resulting in nonsynonymous change p.Asp249Asn. Detailed investigation of one of the family quartets with the singular finding of an H-ABC-affected sibling pair revealed maternal mosaicism for the mutation, suggesting that rare de novo mutations that are initially phenotypically neutral in a mosaic individual can be disease causing in the subsequent generation. Modeling of TUBB4A shows that the mutation creates a nonsynonymous change at a highly conserved asparagine that sits at the intradimer interface of α-tubulin and β-tubulin, and this change might affect tubulin dimerization, microtubule polymerization, or microtubule stability. Consistent with H-ABC's clinical presentation, TUBB4A is highly expressed in neurons, and a recent report has shown that an N-terminal alteration is associated with a heritable dystonia. Together, these data demonstrate that a single de novo mutation in TUBB4A results in H-ABC.
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http://dx.doi.org/10.1016/j.ajhg.2013.03.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3644625PMC
May 2013

A novel biochemically salvageable animal model of hyperammonemia devoid of N-acetylglutamate synthase.

Mol Genet Metab 2012 Jun 17;106(2):160-8. Epub 2012 Mar 17.

Research Center for Genetic Medicine, Children's National Medical Center, Washington, DC 20010, USA.

All knockout mouse models of urea cycle disorders die in the neonatal period or shortly thereafter. Since N-acetylglutamate synthase (NAGS) deficiency in humans can be effectively treated with N-carbamyl-l-glutamate (NCG), we sought to develop a mouse model of this disorder that could be rescued by biochemical intervention, reared to adulthood, reproduce, and become a novel animal model for hyperammonemia. Founder NAGS knockout heterozygous mice were obtained from the trans-NIH Knock-Out Mouse Project. Genotyping of the mice was performed by PCR and confirmed by Western blotting of liver and intestine. NCG and L-citrulline (Cit) were used to rescue the NAGS knockout homozygous (Nags(-/-)) pups and the rescued animals were characterized. We observed an 85% survival rate of Nags(-/-) mice when they were given intraperitoneal injections with NCG and Cit during the newborn period until weaning and supplemented subsequently with both compounds in their drinking water. This regimen has allowed for normal development, apparent health, and reproduction. Interruption of this rescue intervention resulted in the development of severe hyperammonemia and death within 48 h. In addition to hyperammonemia, interruption of rescue supplementation was associated with elevated plasma glutamine, glutamate, and lysine, and reduced citrulline, arginine, ornithine and proline levels. We conclude that NAGS deprived mouse model has been developed which can be rescued by NCG and Cit and reared to reproduction and beyond. This biochemically salvageable mouse model recapitulates the clinical phenotype of proximal urea cycle disorders and can be used as a reliable model of induced hyperammonemia by manipulating the administration of the rescue compounds.
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http://dx.doi.org/10.1016/j.ymgme.2012.03.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3356441PMC
June 2012

Transcriptional regulation of N-acetylglutamate synthase.

PLoS One 2012 27;7(2):e29527. Epub 2012 Feb 27.

Center for Genetic Medicine Research, Children's National Medical Center, Washington, DC, United States of America.

The urea cycle converts toxic ammonia to urea within the liver of mammals. At least 6 enzymes are required for ureagenesis, which correlates with dietary protein intake. The transcription of urea cycle genes is, at least in part, regulated by glucocorticoid and glucagon hormone signaling pathways. N-acetylglutamate synthase (NAGS) produces a unique cofactor, N-acetylglutamate (NAG), that is essential for the catalytic function of the first and rate-limiting enzyme of ureagenesis, carbamyl phosphate synthetase 1 (CPS1). However, despite the important role of NAGS in ammonia removal, little is known about the mechanisms of its regulation. We identified two regions of high conservation upstream of the translation start of the NAGS gene. Reporter assays confirmed that these regions represent promoter and enhancer and that the enhancer is tissue specific. Within the promoter, we identified multiple transcription start sites that differed between liver and small intestine. Several transcription factor binding motifs were conserved within the promoter and enhancer regions while a TATA-box motif was absent. DNA-protein pull-down assays and chromatin immunoprecipitation confirmed binding of Sp1 and CREB, but not C/EBP in the promoter and HNF-1 and NF-Y, but not SMAD3 or AP-2 in the enhancer. The functional importance of these motifs was demonstrated by decreased transcription of reporter constructs following mutagenesis of each motif. The presented data strongly suggest that Sp1, CREB, HNF-1, and NF-Y, that are known to be responsive to hormones and diet, regulate NAGS transcription. This provides molecular mechanism of regulation of ureagenesis in response to hormonal and dietary changes.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0029527PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3287996PMC
August 2012

N-carbamylglutamate enhancement of ureagenesis leads to discovery of a novel deleterious mutation in a newly defined enhancer of the NAGS gene and to effective therapy.

Hum Mutat 2011 Oct 9;32(10):1153-60. Epub 2011 Sep 9.

Research Center for Genetic Medicine, Children's National Medical Center, The George Washington University, Washington, DC, USA.

N-acetylglutamate synthase (NAGS) catalyzes the conversion of glutamate and acetyl-CoA to NAG, the essential allosteric activator of carbamyl phosphate synthetase I, the first urea cycle enzyme in mammals. A 17-year-old female with recurrent hyperammonemia attacks, the cause of which remained undiagnosed for 8 years in spite of multiple molecular and biochemical investigations, showed markedly enhanced ureagenesis (measured by isotope incorporation) in response to N-carbamylglutamate (NCG). This led to sequencing of the regulatory regions of the NAGS gene and identification of a deleterious single-base substitution in the upstream enhancer. The homozygous mutation (c.-3064C>A), affecting a highly conserved nucleotide within the hepatic nuclear factor 1 (HNF-1) binding site, was not found in single nucleotide polymorphism databases and in a screen of 1,086 alleles from a diverse population. Functional assays demonstrated that this mutation decreases transcription and binding of HNF-1 to the NAGS gene, while a consensus HNF-1 binding sequence enhances binding to HNF-1 and increases transcription. Oral daily NCG therapy restored ureagenesis in this patient, normalizing her biochemical markers, and allowing discontinuation of alternate pathway therapy and normalization of her diet with no recurrence of hyperammonemia. Inc.
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http://dx.doi.org/10.1002/humu.21553DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3976964PMC
October 2011

Down-regulation of hepatic urea synthesis by oxypurines: xanthine and uric acid inhibit N-acetylglutamate synthase.

J Biol Chem 2011 Jun 3;286(25):22055-68. Epub 2011 May 3.

Division of Child Development, Rehabilitation Medicine, and Metabolic Disease, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania 19104, USA.

We previously reported that isobutylmethylxanthine (IBMX), a derivative of oxypurine, inhibits citrulline synthesis by an as yet unknown mechanism. Here, we demonstrate that IBMX and other oxypurines containing a 2,6-dione group interfere with the binding of glutamate to the active site of N-acetylglutamate synthetase (NAGS), thereby decreasing synthesis of N-acetylglutamate, the obligatory activator of carbamoyl phosphate synthase-1 (CPS1). The result is reduction of citrulline and urea synthesis. Experiments were performed with (15)N-labeled substrates, purified hepatic CPS1, and recombinant mouse NAGS as well as isolated mitochondria. We also used isolated hepatocytes to examine the action of various oxypurines on ureagenesis and to assess the ameliorating affect of N-carbamylglutamate and/or l-arginine on NAGS inhibition. Among various oxypurines tested, only IBMX, xanthine, or uric acid significantly increased the apparent K(m) for glutamate and decreased velocity of NAGS, with little effect on CPS1. The inhibition of NAGS is time- and dose-dependent and leads to decreased formation of the CPS1-N-acetylglutamate complex and consequent inhibition of citrulline and urea synthesis. However, such inhibition was reversed by supplementation with N-carbamylglutamate. The data demonstrate that xanthine and uric acid, both physiologically occurring oxypurines, inhibit the hepatic synthesis of N-acetylglutamate. An important and novel concept emerging from this study is that xanthine and/or uric acid may have a role in the regulation of ureagenesis and, thus, nitrogen homeostasis in normal and disease states.
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http://dx.doi.org/10.1074/jbc.M110.209023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3121350PMC
June 2011

N-acetylglutamate synthase deficiency: an insight into the genetics, epidemiology, pathophysiology, and treatment.

Appl Clin Genet 2011 24;4:127-35. Epub 2011 Aug 24.

Center for Genetic Medicine Research, Children's Research institute, Children's National Medical Center, Washington DC, USA.

The conversion of ammonia into urea by the human liver requires the coordinated function of the 6 enzymes and 2 transporters of the urea cycle. The initial and rate-limiting enzyme of the urea cycle, carbamylphosphate synthetase 1 (CPS1), requires an allosteric activator, N-acetylglutamate (NAG). The formation of this unique cofactor from glutamate and acetyl Coenzyme-A is catalyzed by N-acetylglutamate synthase (NAGS). An absence of NAG as a consequence of NAGS deficiency may compromise flux through CPS1 and result in hyperammonemia. The NAGS gene encodes a 528-amino acid protein, consisting of a C-terminal catalytic domain, a variable segment, and an N-terminal mitochondrial targeting signal. Only 22 mutations in the NAGS gene have been reported to date, mostly in the catalytic domain. NAGS is primarily expressed in the liver and intestine. However, it is also surprisingly expressed in testis, stomach and spleen, and during early embryonic development at levels not concordant with the expression of other urea cycle enzymes, CPS1, or ornithine transcarbamylase. The purpose of NAGS expression in these tissues, and its significance to NAGS deficiency is as yet unknown. Inherited NAGS deficiency is the rarest of the urea cycle disorders, and we review the currently reported 34 cases. Treatment of NAGS deficiency with N-carbamyglutamate, a stable analog of NAG, can restore deficient urea cycle function and normalize blood ammonia in affected patients.
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http://dx.doi.org/10.2147/TACG.S12702DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3681184PMC
June 2013

N-acetylglutamate synthase: structure, function and defects.

Mol Genet Metab 2010 26;100 Suppl 1:S13-9. Epub 2010 Feb 26.

The Children's Research Institute, Children's National Medical Center, The George Washington University, Washington, DC 20010, USA.

N-acetylglutamate (NAG) is a unique enzyme cofactor, essential for liver ureagenesis in mammals while it is the first committed substrate for de novo arginine biosynthesis in microorganisms and plants. The enzyme that produces NAG from glutamate and CoA, NAG synthase (NAGS), is allosterically inhibited by arginine in microorganisms and plants and activated in mammals. This transition of the allosteric effect occurred when tetrapods moved from sea to land. The first mammalian NAGS gene (from mouse) was cloned in 2002 and revealed significant differences from the NAGS ortholog in microorganisms. Almost all NAGS genes possess a C-terminus transferase domain in which the catalytic activity resides and an N-terminus kinase domain where arginine binds. The three-dimensional structure of NAGS shows two distinctly folded domains. The kinase domain binds arginine while the acetyltransferase domain contains the catalytic site. NAGS deficiency in humans leads to hyperammonemia and can be primary, due to mutations in the NAGS gene or secondary due to other mitochondrial aberrations that interfere with the normal function of the same enzyme. For either condition, N-carbamylglutamate (NCG), a stable functional analog of NAG, was found to either restore or improve the deficient urea-cycle function.
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http://dx.doi.org/10.1016/j.ymgme.2010.02.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2876818PMC
July 2010

Mechanism of allosteric inhibition of N-acetyl-L-glutamate synthase by L-arginine.

J Biol Chem 2009 Feb 18;284(8):4873-80. Epub 2008 Dec 18.

Research Center for Genetic Medicine, Children's National Medical Center, The George Washington University, Washington, D. C. 20010, USA.

N-Acetylglutamate synthase (NAGS) catalyzes the first committed step in l-arginine biosynthesis in plants and micro-organisms and is subject to feedback inhibition by l-arginine. This study compares the crystal structures of NAGS from Neisseria gonorrhoeae (ngNAGS) in the inactive T-state with l-arginine bound and in the active R-state complexed with CoA and l-glutamate. Under all of the conditions examined, the enzyme consists of two stacked trimers. Each monomer has two domains: an amino acid kinase (AAK) domain with an AAK-like fold but lacking kinase activity and an N-acetyltransferase (NAT) domain homologous to other GCN5-related transferases. Binding of l-arginine to the AAK domain induces a global conformational change that increases the diameter of the hexamer by approximately 10 A and decreases its height by approximately 20A(.) AAK dimers move 5A outward along their 2-fold axes, and their tilt relative to the plane of the hexamer decreases by approximately 4 degrees . The NAT domains rotate approximately 109 degrees relative to AAK domains enabling new interdomain interactions. Interactions between AAK and NAT domains on different subunits also change. Local motions of several loops at the l-arginine-binding site enable the protein to close around the bound ligand, whereas several loops at the NAT active site become disordered, markedly reducing enzymatic specific activity.
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http://dx.doi.org/10.1074/jbc.M805348200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2643497PMC
February 2009

Inversion of allosteric effect of arginine on N-acetylglutamate synthase, a molecular marker for evolution of tetrapods.

BMC Biochem 2008 Sep 18;9:24. Epub 2008 Sep 18.

Research Center for Genetic Medicine, Children's National Medical Center, 111 Michigan Ave NW, Washington, DC 20010, USA.

Background: The efficient conversion of ammonia, a potent neurotoxin, into non-toxic metabolites was an essential adaptation that allowed animals to move from the aquatic to terrestrial biosphere. The urea cycle converts ammonia into urea in mammals, amphibians, turtles, snails, worms and many aquatic animals and requires N-acetylglutamate (NAG), an essential allosteric activator of carbamylphosphate synthetase I (CPSI) in mammals and amphibians, and carbamylphosphate synthetase III (CPSIII) in fish and invertebrates. NAG-dependent CPSI and CPSIII catalyze the formation of carbamylphosphate in the first and rate limiting step of ureagenesis. NAG is produced enzymatically by N-acetylglutamate synthase (NAGS), which is also found in bacteria and plants as the first enzyme of arginine biosynthesis. Arginine is an allosteric inhibitor of microbial and plant NAGS, and allosteric activator of mammalian NAGS.

Results: Information from mutagenesis studies of E. coli and P. aeruginosa NAGS was combined with structural information from the related bacterial N-acetylglutamate kinases to identify four residues in mammalian NAGS that interact with arginine. Substitutions of these four residues were engineered in mouse NAGS and into the vertebrate-like N-acetylglutamate synthase-kinase (NAGS-K) of Xanthomonas campestris, which is inhibited by arginine. All mutations resulted in arginine losing the ability to activate mouse NAGS, and inhibit X. campestris NAGS-K. To examine at what point in evolution inversion of arginine effect on NAGS occur, we cloned NAGS from fish and frogs and examined the arginine response of their corresponding proteins. Fish NAGS were partially inhibited by arginine and frog NAGS were activated by arginine.

Conclusion: Difference in arginine effect on bacterial and mammalian NAGS most likely stems from the difference in the type of conformational change triggered by arginine binding to these proteins. The change from arginine inhibition of NAGS to activation was gradual, from complete inhibition of bacterial NAGS, to partial inhibition of fish NAGS, to activation of frog and mammalian NAGS. This change also coincided with the conquest of land by amphibians and mammals.
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http://dx.doi.org/10.1186/1471-2091-9-24DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2566978PMC
September 2008

N-carbamylglutamate markedly enhances ureagenesis in N-acetylglutamate deficiency and propionic acidemia as measured by isotopic incorporation and blood biomarkers.

Pediatr Res 2008 Aug;64(2):213-7

Children's Research Institute, Children's National Medical Center, George Washington University, Washington, DC 20052, USA.

N-acetylglutamate (NAG) is an endogenous essential cofactor for conversion of ammonia to urea in the liver. Deficiency of NAG causes hyperammonemia and occurs because of inherited deficiency of its producing enzyme, NAG synthase (NAGS), or interference with its function by short fatty acid derivatives. N-carbamylglutamate (NCG) can ameliorate hyperammonemia from NAGS deficiency and propionic and methylmalonic acidemia. We developed a stable isotope (13)C tracer method to measure ureagenesis and to evaluate the effect of NCG in humans. Seventeen healthy adults were investigated for the incorporation of (13)C label into urea. [(13)C]urea appeared in the blood within minutes, reaching maximum by 100 min, whereas breath (13)CO(2) reached a maximum by 60 min. A patient with NAGS deficiency showed very little urea labeling before treatment with NCG and normal labeling thereafter. Correspondingly, plasma levels of ammonia and glutamine decreased markedly and urea tripled after NCG treatment. Similarly, in a patient with propionic acidemia, NCG treatment resulted in a marked increase in urea labeling and decrease in glutamine, alanine, and glycine. These results provide a reliable method for measuring the effect of NCG on nitrogen metabolism and strongly suggest that NCG could be an effective treatment for inherited and secondary NAGS deficiency.
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http://dx.doi.org/10.1203/PDR.0b013e318179454bDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2640836PMC
August 2008

The crystal structure of N-acetyl-L-glutamate synthase from Neisseria gonorrhoeae provides insights into mechanisms of catalysis and regulation.

J Biol Chem 2008 Mar 9;283(11):7176-84. Epub 2008 Jan 9.

Children's Research Institute, Children's National Medical Center, The George Washington University, Washington, DC 20010, USA.

The crystal structures of N-acetylglutamate synthase (NAGS) in the arginine biosynthetic pathway of Neisseria gonorrhoeae complexed with acetyl-CoA and with CoA plus N-acetylglutamate have been determined at 2.5- and 2.6-A resolution, respectively. The monomer consists of two separately folded domains, an amino acid kinase (AAK) domain and an N-acetyltransferase (NAT) domain connected through a 10-A linker. The monomers assemble into a hexameric ring that consists of a trimer of dimers with 32-point symmetry, inner and outer ring diameters of 20 and 100A, respectively, and a height of 110A(.) Each AAK domain interacts with the cognate domains of two adjacent monomers across two 2-fold symmetry axes and with the NAT domain from a second monomer of the adjacent dimer in the ring. The catalytic sites are located within the NAT domains. Three active site residues, Arg316, Arg425, and Ser427, anchor N-acetylglutamate in a position at the active site to form hydrogen bond interactions to the main chain nitrogen atoms of Cys356 and Leu314, and hydrophobic interactions to the side chains of Leu313 and Leu314. The mode of binding of acetyl-CoA and CoA is similar to other NAT family proteins. The AAK domain, although catalytically inactive, appears to bind arginine. This is the first reported crystal structure of any NAGS, and it provides insights into the catalytic function and arginine regulation of NAGS enzymes.
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http://dx.doi.org/10.1074/jbc.M707678200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4099063PMC
March 2008
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