N-acetylglutamate Synthase Research Articles

A Novel Method for Development and Validation of the Degradation Products Analysis of N-Carbamylglutamate with UHPLC by Using Design of Experiment Approach.

Carglumic acid, also known as N-carbamyl-l-glutamic acid, is a medication used in the treatment of a rare genetic disorder called N-acetylglutamate synthase (NAGS) deficiency. To the authors' knowledge, there was no method reported in the literature for the determination of degradation products suitable for quality control analyses of carglumic acid. Thus, the aim of the presented study is to develop an impurity method with a UHPLC/DAD detector configuration compatible with industrial standards from the European Pharmacopeia and the United States Pharmacopeia, making the drug more accessible for developing and underdeveloped countries through its precise evaluation. The method involved the separation of carglumic acid and its degradation products using a reverse-phase C18 column (Waters, BEH 150 mm × 2.1 mm, 1.7 μm) at a flow rate of 0.39 mL/min with a stop time of 10 min. To separate all unknown and known impurities, a gradient elution (phosphate buffer, pH 2.4, and acetonitrile) system was used. The detection was performed at 214 nm. Forced degradation studies were conducted under different stress conditions, including acidic, basic, oxidative, thermal, and photolytic stress. Placket-Burman statistical experimental design was used to demonstrate the robustness of this method, and the suitability of the method was confirmed under the applied conditions. Box-Behnken design was used to provide the optimum resolution between the peaks determined to be critical during the optimization. The developed method was validated according to ICH guidelines for specificity, linearity, accuracy, precision, and robustness. The limit of detection and limit of quantification were 0.7 and 0.15 μg/mL for carglumic acid, respectively.

The Mechanism of Deprotonation of the Amino Group of Glutamate upon Binding to N-Acetylglutamate Synthase

The Mechanism of Deprotonation of the Amino Group of Glutamate upon Binding to N-Acetylglutamate Synthase

MECHANISM OF DEPROTONATION OF THE AMINO GROUP OF GLUTAMATE UPON BINDING TO N-ACETYLGLUTAMATE SYNTHASE

Gcn5-related N-acetyltransferases catalyze the transfer of an acetyl group to a primary amino group of a wide class of substrates. Protonation of the amino group upon binding to the enzyme is necessary to activate the nucleophilic attack on the substrate. The process of glutamate binding to N-acetylglutamate synthase is considered using molecular modeling and quantum chemistry methods. It has been shown that deprotonation of the primary amino group of glutamate occurs upon binding to the active site of the enzyme with the participation of the side chain of the aspartate residue.

Use of pure recombinant human enzymes to assess the disease-causing potential of missense mutations in urea cycle disorders, applied to N-acetylglutamate synthase deficiency.

N-acetylglutamate synthase (NAGS) makes acetylglutamate, the essential activator of the first, regulatory enzyme of the urea cycle, carbamoyl phosphate synthetase 1 (CPS1). NAGS deficiency (NAGSD) and CPS1 deficiency (CPS1D) present identical phenotypes. However, they must be distinguished, because NAGSD is cured by substitutive therapy with the N-acetyl-L-glutamate analogue N-carbamyl-L-glutamate, while curative therapy of CPS1D requires liver transplantation. Since their differentiation is done genetically, it is important to ascertain the disease-causing potential of CPS1 and NAGS genetic variants. With this goal, we previously carried out site-directed mutagenesis studies with pure recombinant human CPS1. We could not do the same with human NAGS (HuNAGS) because of enzyme instability, leading to our prior utilization of a bacterial NAGS as an imperfect surrogate of HuNAGS. We now use genuine HuNAGS, stabilized as a chimera of its conserved domain (cHuNAGS) with the maltose binding protein (MBP), and produced in Escherichia coli. MBP-cHuNAGS linker cleavage allowed assessment of the enzymatic properties and thermal stability of cHuNAGS, either wild-type or hosting each one of 23 nonsynonymous single-base changes found in NAGSD patients. For all but one change, disease causation was accounted by the enzymatic alterations identified, including, depending on the variant, loss of arginine activation, increased Km Glutamate, active site inactivation, decreased thermal stability, and protein misfolding. Our present approach outperforms experimental in vitro use of bacterial NAGS or in silico utilization of prediction servers (including AlphaMissense), illustrating with HuNAGS the value for UCDs of using recombinant enzymes for assessing disease-causation and molecular pathogenesis, and for therapeutic guidance.

The efficacy of Carbamylglutamate impacts the nutritional management of patients with N-Acetylglutamate synthase deficiency

BackgroundThe autosomal recessive disorder N-acetylglutamate synthase (NAGS) deficiency is the rarest defect of the urea cycle, with an incidence of less than one in 2,000,000 live births. Hyperammonemic crises can be avoided in individuals with NAGS deficiency by the administration of carbamylglutamate (also known as carglumic acid), which activates carbamoyl phosphatase synthetase 1 (CPS1). The aim of this case series was to introduce additional cases of NAGS deficiency to the literature as well as to assess the role of nutrition management in conjunction with carbamylglutamate therapy across new and existing cases.MethodsWe conducted retrospective chart reviews of seven cases of NAGS deficiency in the US and Canada, focusing on presentation, diagnosis, medication management, nutrition management, and outcomes.ResultsFive new and two previously published cases were included. Presenting symptoms were consistent with previous reports. Diagnostic confirmation via molecular testing varied in protocol across cases, with consecutive single gene tests leading to long delays in diagnosis in some cases. All patients responded well to carbamylglutamate therapy, as indicated by normalization of plasma ammonia and citrulline, as well as urine orotic acid in patients with abnormal levels at baseline. Although protein restriction was not prescribed in any cases after carbamylglutamate initiation, two patients continued to self-restrict protein intake. One patient experienced two episodes of hyperammonemia that resulted in poor long-term outcomes. Both episodes occurred after a disruption in access to carbamylglutamate, once due to insurance prior authorization requirements and language barriers and once due to seizure activity limiting the family’s ability to administer carbamylglutamate.ConclusionsFollow-up of patients with NAGS deficiency should include plans for illness and for disruption of carbamylglutamate access, including nutrition management strategies such as protein restriction. Carbamylglutamate can help patients with NAGS deficiency to liberalize their diets, but the maximum safe level of protein intake to prevent hyperammonemia is not yet known. Patients using this medication should still monitor their diet closely and be prepared for any disruptions in medication access, which might require immediate dietary adjustments or medical intervention to prevent hyperammonemia.

Datamining approaches for examining the low prevalence of N-acetylglutamate synthase deficiency and understanding transcriptional regulation of urea cycle genes.

Ammonia, which is toxic to the brain, is converted into non-toxic urea, through a pathway of six enzymatically catalyzed steps known as the urea cycle. In this pathway, N-acetylglutamate synthase (NAGS, EC 2.3.1.1) catalyzes the formation of N-acetylglutamate (NAG) from glutamate and acetyl coenzyme A. NAGS deficiency (NAGSD) is the rarest of the urea cycle disorders, yet is unique in that ureagenesis can be restored with the drug N-carbamylglutamate (NCG). We investigated whether the rarity of NAGSD could be due to low sequence variation in the NAGS genomic region, high NAGS tolerance for amino acid replacements, and alternative sources of NAG and NCG in the body. We also evaluated whether the small genomic footprint of the NAGS catalytic domain might play a role. The small number of patients diagnosed with NAGSD could result from the absence of specific disease biomarkers and/or short NAGS catalytic domain. We screened for sequence variants in NAGS regulatory regions in patients suspected of having NAGSD and found a novel NAGS regulatory element in the first intron of the NAGS gene. We applied the same datamining approach to identify regulatory elements in the remaining urea cycle genes. In addition to the known promoters and enhancers of each gene, we identified several novel regulatory elements in their upstream regions and first introns. The identification of cis-regulatory elements of urea cycle genes and their associated transcription factors holds promise for uncovering shared mechanisms governing urea cycle gene expression and potentially leading to new treatments for urea cycle disorders.

Mepiquat chloride increases Cry1Ac protein content by regulating the carbon and amino acid metabolism of Bt cotton under high temperature and drought stress

The effects of mepiquat chloride (DPC) on Cry1Ac protein contents in Bacillus thuringiensis (Bt) cotton boll shells under high temperature and drought stress were investigated to provide a theoretical reference for Bt cotton breeding and high-yield and high-efficiency cotton cultivation. This study was undertaken on the Bt cotton cultivar ‘Sikang 3’ during the 2020 and 2021 growing seasons at Yangzhou University Farm, Yangzhou, China. The potted cotton plants were exposed to high temperature and drought stress, and 20 mg L−1 DPC and water (CK) were sprayed on cotton plants. Seven days after treatment, Cry1Ac protein content, α-ketoglutarate content, pyruvic acid content, glutamate synthase activity, glutamic oxaloacetic transaminase activity, soluble protein content, and amino acid content were measured, and transcriptome sequencing was performed. DESeq was used for differential gene analysis. Under the DPC treatment, in contrast with the water treatment (CK), Cry1Ac protein content increased by 4.7-11.9%. α-Ketoglutarate content, pyruvic acid content, glutamate synthase activity, glutamic oxaloacetic transaminase activity, soluble protein content, and amino acid content all increased. Transcriptome analysis revealed that there were 7542 upregulated genes and 10449 downregulated genes for DPC vs. CK. GO and KEGG analyses showed that differentially expressed genes were mainly involved in biological processes, such as amino acid metabolism and carbon metabolism. Genes coding 6-phosphofructokinase, pyruvate kinase, glutamic pyruvate transaminase, pyruvate dehydrogenase, citrate synthase, isocitrate dehydrogenase, 2-oxoglutarate dehydrogenase, glutamate synthase, 1-pyrroline-5-carboxylate dehydrogenase, glutamic oxaloacetic transaminase, amino-acid N-acetyltransferase, and acetylornithine deacetylase were all significantly upregulated. DPC increased pyruvate, α-ketoglutarate, and oxaloacetate by increasing the operating rate of the glycolytic pathway, the citric acid cycle. It also significantly up-regulated the genes encoding glutamate synthase and pyrrolidine-5-carboxylic acid dehydrogenase, and down-regulated the genes encoding glutamine synthetase as well as up-regulated the genes encoding glutamate oxaloacetate transaminase and N-acetylglutamate synthetase. Therefore, the synthesis ability of aspartic acid, glutamic acid, pyruvate, and arginine was increased by DPC, and then Cry1Ac protein contents were increased by regulating carbon and amino acid metabolism.

A novel strategy for l-arginine production in engineered Escherichia coli

Backgroundl-arginine is an important amino acid with applications in diverse industrial and pharmaceutical fields. n-acetylglutamate, synthesized from l-glutamate and acetyl-CoA, is a precursor of the l-arginine biosynthetic branch in microorganisms. The enzyme that produces n-acetylglutamate, n-acetylglutamate synthase, is allosterically inhibited by l-arginine. l-glutamate, as a central metabolite, provides carbon backbone for diverse biological compounds besides l-arginine. When glucose is the sole carbon source, the theoretical maximum carbon yield towards l-arginine is 96.7%, but the experimental highest yield was 51%. The gap of l-arginine yield indicates the regulation complexity of carbon flux and energy during the l-arginine biosynthesis. Besides endogenous biosynthesis, n-acetylglutamate, the key precursor of l-arginine, can be obtained by chemical acylation of l-glutamate with a high yield of 98%. To achieve high-yield production of l-arginine, we demonstrated a novel approach by directly feeding precursor n-acetylglutamate to engineered Escherichia coli.ResultsWe reported a new approach for the high yield of l-arginine production in E. coli. Gene argA encoding n-acetylglutamate synthase was deleted to disable endogenous biosynthesis of n-acetylglutamate. The feasibility of external n-acetylglutamate towards l-arginine was verified via growth assay in argA− strain. To improve l-arginine production, astA encoding arginine n-succinyltransferase, speF encoding ornithine decarboxylase, speB encoding agmatinase, and argR encoding an arginine responsive repressor protein were disrupted. Based on overexpression of argDGI, argCBH operons, encoding enzymes of the l-arginine biosynthetic pathway, ~ 4 g/L l-arginine was produced in shake flask fermentation, resulting in a yield of 0.99 mol l-arginine/mol n-acetylglutamate. This strain was further engineered for the co-production of l-arginine and pyruvate by removing genes adhE, ldhA, poxB, pflB, and aceE, encoding enzymes involved in the conversion and degradation of pyruvate. The resulting strain was shown to produce 4 g/L l-arginine and 11.3 g/L pyruvate in shake flask fermentation.ConclusionsHere, we developed a novel approach to avoid the strict regulation of l-arginine on ArgA and overcome the metabolism complexity in the l-arginine biosynthesis pathway. We achieve a high yield of l-arginine production from n-acetylglutamate in E. coli. Co-production pyruvate and l-arginine was used as an example to increase the utilization of input carbon sources.

#4542 L-ARGININE-NITRIC OXIDE MOLECULAR PATHWAY IN AUTOSOMAL DOMINANT POLYCYSTIC KIDNEY DISEASE

Abstract Background and Aims Autosomal Dominant Polycystic Kidney Disease (ADPKD) is an inherited, complex disease, cystogenesis and phenotypic changes in this disorder being not yet fully understood. Recently, arginine, important for renal cells metabolism, was proved to play an important role in ADPKD physiopathology (1). The inability of cells to recycle or synthesize intracellular arginine through the urea cycle is defined as arginine auxotrophy. Arginine auxotrophy in ADPKD induces cell hyperproliferation, and cysts formation (2). We aimed to explore L-Arginine (Arg) - Nitric Oxide (NO) molecular pathway in ADPKD, a multisystemic, arginine auxotrophe disease. Method We developed a prospective, case control study, in Carol Davila Clinical Hospital of Nephrology, Bucharest, Romania. The study included a group of 62 subjects with ADPKD (mean age -54.3years old, men:women 34:28, with 1A and 1B Mayo ADPKD risk classes) and a group of 37 healthy subjects, similar as sex, mean age. ADPKD diagnosis was based on familial history, clinical exam and CT or MRI scan. We excluded from the study subjects that presented cysts in other organs except the kidney, with eGFR<60mL/min/1.73mp, with history of hematuria, cysts infection, urinary tract infection, renal lithiasis, with unstable blood pressure and hypertensive treatment for less than 6 months; metabolic disorders (carbamoyl phosphate synthase 1 and N-acetyl glutamate synthase deficiencies, lack of ornithine transcarbamilase, hyperargininemia, phenylketonuria). Laboratory tests: serum level of arginine, enzymatic activity of arginase 2 (ARG-2) and nitric oxide inducible synthase (NOS-2), serum levels of stable metabolites of nitric oxide (nitrate, direct nitrite, total nitrite), endogenous inhibitors of nitric oxide synthesis (Asymmetric Dimethylarginine -ADMA, Symmetric Dimethylarginine SDMA). Results In ADPKD patients, the serum levels of arginine and of the stable metabolites of nitric oxide had statistically significant lower levels compared with control group (Table 1). The levels of the principal enzymes that metabolizes arginine – arginase 2 and nitric oxide inducible synthase, respectively the endogenous inhibitors of nitric oxide synthesis were statistically significant higher in ADPKD group when compared with control group (Table 1). The ARG2/Arg, NOS2/Arg, Nitrite/Arg, Nitrate/Arg, ADMA/Arg and SDMA/Arg ratios were statistically significant overexpressed in ADPKD group when compared with control group. Conclusion ADPKD is a metabolic kidney disease, auxotrophic for arginine. The metabolic ADPKD phenotype of renal cells with low risk of progression (1A and 1B Mayo) is defined by the alteration of L-Arginine-NO molecular pathway, the significant reduction of systemic arginine, moderately increased enzymatic activities of ARG2 and NOS2, the reduction of the synthesis and bioactivity of NO. Exploring arginine reprogramming and related molecular L-Arg-NO pathways disturbances could offer more information about ADPKD physiopathology.

Whole-cell modeling of E. coli confirms that in vitro tRNA aminoacylation measurements are insufficient to support cell growth and predicts a positive feedback mechanism regulating arginine biosynthesis.

In Escherichia coli, inconsistencies between in vitro tRNA aminoacylation measurements and in vivo protein synthesis demands were postulated almost 40years ago, but have proven difficult to confirm. Whole-cell modeling can test whether a cell behaves in a physiologically correct manner when parameterized with in vitro measurements by providing a holistic representation of cellular processes in vivo. Here, a mechanistic model of tRNA aminoacylation, codon-based polypeptide elongation, and N-terminal methionine cleavage was incorporated into a developing whole-cell model of E. coli. Subsequent analysis confirmed the insufficiency of aminoacyl-tRNA synthetase kinetic measurements for cellular proteome maintenance, and estimated aminoacyl-tRNA synthetase kcats that were on average 7.6-fold higher. Simulating cell growth with perturbed kcats demonstrated the global impact of these in vitro measurements on cellular phenotypes. For example, an insufficient kcat for HisRS caused protein synthesis to be less robust to the natural variability in aminoacyl-tRNA synthetase expression in single cells. More surprisingly, insufficient ArgRS activity led to catastrophic impacts on arginine biosynthesis due to underexpressed N-acetylglutamate synthase, where translation depends on repeated CGG codons. Overall, the expanded E. coli model deepens understanding of how translation operates in an in vivo context.

Generation of a Yeast Cell Model Potentially Useful to Identify the Mammalian Mitochondrial N-Acetylglutamate Transporter.

The human mitochondrial carrier family (MCF) consists of 53 members. Approximately one-fifth of them are still orphans of a function. Most mitochondrial transporters have been functionally characterized by reconstituting the bacterially expressed protein into liposomes and transport assays with radiolabeled compounds. The efficacy of this experimental approach is constrained to the commercial availability of the radiolabeled substrate to be used in the transport assays. A striking example is that of N-acetylglutamate (NAG), an essential regulator of the carbamoyl synthetase I activity and the entire urea cycle. Mammals cannot modulate mitochondrial NAG synthesis but can regulate the levels of NAG in the matrix by exporting it to the cytosol, where it is degraded. The mitochondrial NAG transporter is still unknown. Here, we report the generation of a yeast cell model suitable for identifying the putative mammalian mitochondrial NAG transporter. In yeast, the arginine biosynthesis starts in the mitochondria from NAG which is converted to ornithine that, once transported into cytosol, is metabolized to arginine. The deletion of ARG8 makes yeast cells unable to grow in the absence of arginine since they cannot synthetize ornithine but can still produce NAG. To make yeast cells dependent on a mitochondrial NAG exporter, we moved most of the yeast mitochondrial biosynthetic pathway to the cytosol by expressing four E. coli enzymes, argB-E, able to convert cytosolic NAG to ornithine. Although argB-E rescued the arginine auxotrophy of arg8∆ strain very poorly, the expression of the bacterial NAG synthase (argA), which would mimic the function of a putative NAG transporter increasing the cytosolic levels of NAG, fully rescued the growth defect of arg8∆ strain in the absence of arginine, demonstrating the potential suitability of the model generated.

Construction of an Escherichia coli Strain Capable of Utilizing Steamed Rice as the Sole Carbon and Energy Source by Extracellular Expression of Amylase and Its Use for the Production of Putrescine

The amyE gene encoding α-amylase from Bacillus subtilis 168 was fused to several genes, the products of which are membrane proteins to express AmyE extracellularly. Genes of CapA, a subunit of the capsular poly-γ-glutamate synthetase of Bacillus subtilis subsp. natto; YiaTR232, the first 232 residues of YiaT, an outer membrane protein of Escherichia coli K-12; and PgsA, cytidine 5′-diphosphate-diacylglycerol-glycerol-3-phosphate 3-phosphatidyltransferase of B. subtilis subsp. natto, were used. The Escherichia coli strain harboring the plasmid carrying the pgsA-amyE fusion expressed a very high level of amylase not only on the extracellular surface of the cells but also in the medium and the intracellular space and solubilized steamed rice in two days. The N-terminal amino acid sequence of the amylase purified from the culture medium (Ser-Ala-Glu-Thr-Ala) indicated that it was cleaved at the signal peptide cleavage site of AmyE. The strain SH2204, transformed with pKN11 carrying speA (arginine decarboxylase gene), speB (agmatinase gene) and argAATG Y19C (N-acetylglutamate synthase gene) and pMAN63 carrying pgsA-amyE produced 1.2 mM putrescine from 5 mM arginine by adding 0.5 mM IPTG in eight days. Discarding steamed rice is not only a food loss, but also a waste of renewable, biogenerated resources due to the burning of it with petroleum, increasing carbon dioxide in the atmosphere. The E. coli strain developed in this study can help solve this problem because it can produce an important chemical using steamed rice as the sole carbon and energy source.

NAGS, CPS1, and SLC25A13 (Citrin) at the Crossroads of Arginine and Pyrimidines Metabolism in Tumor Cells.

Urea cycle enzymes and transporters collectively convert ammonia into urea in the liver. Aberrant overexpression of carbamylphosphate synthetase 1 (CPS1) and SLC25A13 (citrin) genes has been associated with faster proliferation of tumor cells due to metabolic reprogramming that increases the activity of the CAD complex and pyrimidine biosynthesis. N-acetylglutamate (NAG), produced by NAG synthase (NAGS), is an essential activator of CPS1. Although NAGS is expressed in lung cancer derived cell lines, expression of the NAGS gene and its product was not evaluated in tumors with aberrant expression of CPS1 and citrin. We used data mining approaches to identify tumor types that exhibit aberrant overexpression of NAGS, CPS1, and citrin genes, and evaluated factors that may contribute to increased expression of the three genes and their products in tumors. Median expression of NAGS, CPS1, and citrin mRNA was higher in glioblastoma multiforme (GBM), glioma, and stomach adenocarcinoma (STAD) samples compared to the matched normal tissue. Median expression of CPS1 and citrin mRNA was higher in the lung adenocarcinoma (LUAD) sample while expression of NAGS mRNA did not differ. High NAGS expression was associated with an unfavorable outcome in patients with glioblastoma and GBM. Low NAGS expression was associated with an unfavorable outcome in patients with LUAD. Patterns of DNase hypersensitive sites and histone modifications in the upstream regulatory regions of NAGS, CPS1, and citrin genes were similar in liver tissue, lung tissue, and A549 lung adenocarcinoma cells despite different expression levels of the three genes in the liver and lung. Citrin gene copy numbers correlated with its mRNA expression in glioblastoma, GBM, LUAD, and STAD samples. There was little overlap between NAGS, CPS1, and citrin sequence variants found in patients with respective deficiencies, tumor samples, and individuals without known rare genetic diseases. The correlation between NAGS, CPS1, and citrin mRNA expression in the individual glioblastoma, GBM, LUAD, and STAD samples was very weak. These results suggest that the increased cytoplasmic supply of either carbamylphosphate, produced by CPS1, or aspartate may be sufficient to promote tumorigenesis, as well as the need for an alternative explanation of CPS1 activity in the absence of NAGS expression and NAG.

Urea cycle disorders and indications for liver transplantation.

Urea cycle disorders (UCD) are inborn errors of metabolism caused by deficiency of enzymes required to convert nitrogen from ammonia into urea. Current paradigms of treatment focus on dietary manipulations, ammonia scavenger drugs, and liver transplantation. The aim of this study was to describe the characteristics and indication of liver transplantation in UCD in a tertiary hospital. We performed a retrospective study of children with UCD seen in the period 2000-2021. Data was collected on clinical onset, hyperammonemia severity, evolution and liver transplantation. There were 33 patients in the study period, whose diagnosis were: ornithine transcarbamylase (OTC, n = 20, 10 females), argininosuccinate synthetase (ASS, n = 6), carbamylphosphate synthetase 1 (CPS1, n = 4), argininosuccinate lyase (ASL, n = 2) and N-acetylglutamate synthetase (NAGS, n = 1) deficiency. Thirty one were detected because of clinical symptoms (45% with neonatal onset). The other 2 were diagnosed being presymptomatic, by neonatal/family screening. Neonatal forms (n = 14) were more severe, all of them presented during the first week of life as severe hyperammonemia (mean peak 1,152 µmol/L). Seven patients died (6 at debut) and all survivors received transplantation. There was no mortality among the late forms. Of the 27 patients who did not die in the neonatal period, 16 (59%) received liver transplantationwith 100% survival, normal protein tolerance and usual need of citrulline supplementation. The transplant's metabolic success was accompanied by neurologic sequelae in 69%, but there was no progression of brain damage. Decision of continuous medical treatment in 11 patients appeared to be related with preserved neurodevelopment and fewer metabolic crises.

Novel pathogenic variant (c.2947C > T) of the carbamoyl phosphate synthetase 1 gene in neonatal-onset deficiency.

Carbamoyl phosphate synthetase 1 deficiency (CPS1D) is a rare autosomal recessive urea cycle disorder characterized by hyperammonaemia. The biochemical measurement of the intermediate metabolites is helpful for CPS1D diagnosis; it however cannot distinguish CPS1D from N-acetylglutamate synthetase deficiency. Therefore, next-generation sequencing (NGS) is often essential for the accurate diagnosis of CPS1D. NGS was performed to identify candidate gene variants of CPS1D in a Asian neonatal patient presented with poor feeding, reduced activity, tachypnea, lethargy, and convulsions. The potential pathogenicity of the identified variants was predicted by various types of bioinformatical analyses, including evolution conservation, domain and 3D structure simulations. Compound heterozygosity of CPS1D were identified. One was in exon 24 with a novel heterozygous missense variant c.2947C > T (p.P983S), and another was previously reported in exon 20 with c.2548C > T (p.R850C). Both variants were predicted to be deleterious. Conservation analysis and structural modeling showed that the two substituted amino acids were highly evolutionarily conserved, resulting in potential decreases of the binding pocket stability and the partial loss of enzyme activity. In this study, two pathogenic missense variants were identified with NGS, expanding the variants pectrum of the CPS1 gene. The variants and related structural knowledge of CPS enzyme demonstrate the applicability for the accurate diagnosis of CPS1D.

Clinical Characteristics of Pediatric Patients with Urea Cycle Disorders

Objective: Urea cycle disorders (UCDs) are inherited deficiencies of the enzymes or transport molecules involved in the cellular excretion of excess ammonia produced during protein metabolism. The aim of this study was to evaluate the clinical characteristics and long-term outcome of pediatric patients with UCDs. Material and Methods: Our research was conducted between September 2020-March 2021 in Dr. Sami Ulus Maternity and Child Health Training and Research Hospital. Clinical characteristics in 16 patients with UCDs [carbamoyl phosphate synthetase I deficiency (n=1), N-acetylglutamate synthase deficiency(n=1), argininosuccinate lyase deficiency (n=4), argininosuccinate synthetase deficiency (n=4), arginase deficiency (n=2), ornithine transcarbamylase deficiency (n=2), hyperammonemia hyperornithinemia homocitrullinuria syndrome (n=2)] were defined. The term “neonatal-onset” UCD was used if symptoms occurred within 28 days of life, and “late-onset” if symptoms started after the neonatal period. Results: Eight patients presented with acute metabolic crisis during newborn period. Core clinical phenotype in neonatal-onset UCDs included sepsis-like findings, whereas epilepsy and mental retardation was predominant in lateonset UCDs. For patients with neonatal-onset UCDs, hyperammonemia was more severe at the initial period. Conclusion: Despite evolving treatment opportunities, still high mortality rates were found in neonatal-onset UCD. UCDs should be suspected in pediatric patients with hyperammonemia and metabolic investigations should be performed immediately to enlighten diagnosis. Neonatal-onset UCD usually present with symptoms of acute hyperammonemia, while moresubtle neurological manifestations are frequent initial findings in the late onset UCD.

Improvement of putrescine production through the arginine decarboxylase pathway in Escherichia coli K-12

In the bio-based polymer industry, putrescine is in the spotlight for use as a material. We constructed strains of Escherichia coli to assess its putrescine production capabilities through the arginine decarboxylase pathway in batch fermentation. N-Acetylglutamate (ArgA) synthase is subjected to feedback inhibition by arginine. Therefore, the 19th amino acid residue, Tyr, of argA was substituted with Cys to desensitize the feedback inhibition of arginine, resulting in improved putrescine production. The inefficient initiation codon GTG of argA was substituted with the effective ATG codon, but its replacement did not affect putrescine production. The essential genes for the putrescine production pathway, speA and speB, were cloned into the same plasmid with argAATG Y19C to form an operon. These genes were introduced under different promoters; lacIp, lacIqp, lacIq1p, and T5p. Among these, the T5 promoter demonstrated the best putrescine production. In addition, disruption of the puuA gene encoding enzyme of the first step of putrescine degradation pathway increased the putrescine production. Of note, putrescine production was not affected by the disruption of patA, which encodes putrescine aminotransferase, the initial enzyme of another putrescine utilization pathway. We also report that the strain KT160, which has a genomic mutation of YifEQ100TAG, had the greatest putrescine production. At 48 h of batch fermentation, strain KT160 grown in terrific broth with 0.01 mM IPTG produced 19.8 mM of putrescine.

Identification of diphenyl furan derivatives via high throughput and computational studies as ArgA inhibitors of Mycobacterium tuberculosis

Microbial amino acid biosynthetic pathways are underexploited for the development of anti-bacterial agents. N-acetyl glutamate synthase (ArgA) catalyses the first committed step in L-arginine biosynthesis and is essential for M. tuberculosis growth. Here, we have purified and optimized assay conditions for the acetylation of l-glutamine by ArgA. Using the optimized conditions, high throughput screening was performed to identify ArgA inhibitors. We identified 2,5-Bis (2-chloro-4-guanidinophenyl) furan, a dicationic diaryl furan derivatives, as ArgA inhibitor, with a MIC99 values of 1.56 μM against M. tuberculosis. The diaryl furan derivative displayed bactericidal killing against both M. bovis BCG and M. tuberculosis. Inhibition of ArgA by the lead compound resulted in transcriptional reprogramming and accumulation of reactive oxygen species. The lead compound and its derivatives showed micromolar binding with ArgA as observed in surface plasmon resonance and tryptophan quenching experiments. Computational and dynamic analysis revealed that these scaffolds share similar binding site residues with L-arginine, however, with slight variations in their interaction pattern. Partial restoration of growth upon supplementation of liquid cultures with either L-arginine or N-acetyl cysteine suggests a multi-target killing mechanism for the lead compound. Taken together, we have identified small molecule inhibitors against ArgA enzyme from M. tuberculosis.

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

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.

Hepatic Manifestations of Urea Cycle Disorders.

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