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Purpose: Description of retinal phenotype by structural and functional testing, ornithine plasma levels and mutational data of OAT gene in patients with Gyrate Atrophy (GA)., Methods: Ophthalmologic examination, fundus photography (CFP), autofluorescence (FAF), spectral-domain optical coherence tomography (SD-OCT), Goldmann perimetry (GP), full-field electroretinogram (ffERG) and chromatic perimetry (CP) testing were performed. Ornithine plasma levels were measured. Sanger sequencing mutational analysis of the coding exons and exon-intron junctions of the OAT gene were analyzed., Results: Twelve eyes of seven Mexican patients with GA were included. CFF showed peripheric patches of chorioretinal atrophy; FAF revealed peripheric oval areas of hypoautofluorescence; SD-OCT exhibited outer retinal tubulations in 58%, cystoid macular edema in 50%, epiretinal membrane in 42%, foveoschisis and staphyloma in 17%, and hyperreflective deposits in 100% of the eyes; GP showed constricted visual fields in 100% of the eyes; ffERG revealed preserved photopic response in 17% and preserved scotopic response in 17% of the eyes; CP exposed a deficit in generalized response of rods and cones in 100% of the eyes. Mean ornithine plasma levels were 509.5 µmol/L. One patient with genetic confirmation of GA had normal ornithine plasma levels (48 µmol/L). Molecular findings in OAT gene detected two novel pathogenic variants: c.796 C > T (p.Gln266*) and c.721_722dupCC (p.Asp242ArgfsTer6)., Conclusion: This study provides new information regarding functional and structural diagnosis in patients with GA, expands the understanding of retinal phenotype in patients with GA, reports two novel mutations and presents the first case of GA confirmed by genetic testing with normal ornithine levels., Competing Interests: Declarations. Conflict of interest: The authors declare no competing interests. Consent to participate: Informed consent was obtained from all individual participants included in the study. Consent to publish: The authors affirm that human research participants provided informed consent for publication of the images in this manuscript. Ethical approval: This study was performed in line with the principles of the Declaration of Helsinki., (© 2024. The Author(s).)

Gyrate atrophy of the choroid and retina (GACR) is a rare autosomal recessive disease characterised by elevated plasma ornithine levels due to deficiency of the enzyme ornithine aminotransferase (OAT). The accumulation of this amino acid in plasma leads to the development of patches of chorioretinal atrophy in the peripheral retina extending into the macular area. Patients usually present with night blindness followed by constriction of the visual field and, finally, decreased central vision and blindness. The disease is diagnosed by the presence of the characteristic clinical picture, the presence of hyperornithinaemia in plasma and the detection of mutations in the OAT enzyme gene. There is currently no effective gene therapy and the most common therapeutic intervention mainly involves dietary modifications with arginine restriction. This article aims to summarise the pathogenesis, clinical and diagnostic findings and treatment options in patients with GACR., (Copyright © 2024 Sociedad Española de Oftalmología. Published by Elsevier España, S.L.U. All rights reserved.)

Gyrate atrophy of the choroid and retina (GACR) is caused by pathogenic biallelic variants in the gene encoding ornithine-δ-aminotransferase (OAT), and is characterized by progressive vision loss leading to blindness. OAT is a pyridoxal-5'-phosphate (PLP) dependent enzyme that is mainly involved in ornithine catabolism, and patients with a deficiency develop profound hyperornithinemia. Therapy is aimed at lowering ornithine levels through dietary arginine restriction and, in some cases, through enhancement of OAT activity via supraphysiological dosages of pyridoxine. In this study, we aimed to extend diagnostic practices in GACR by extensively characterizing the consequences of pathogenic variants on the enzymatic function of OAT, both at the level of the enzyme itself as well as the flux through the ornithine degradative pathway. In addition, we developed an in vitro pyridoxine responsiveness assay. We identified 14 different pathogenic variants, of which one variant was present in all patients of Dutch ancestry (p.(Gly353Asp)). In most patients the enzymatic activity of OAT as well as the rate of [ 14 C]-ornithine flux was below the limit of quantification (LOQ). Apart from our positive control, only one patient cell line showed responsiveness to pyridoxine in vitro, which is in line with the reported in vivo pyridoxine responsiveness in this patient. None of the patients harboring the p.(Gly353Asp) substitution were responsive to pyridoxine in vivo or in vitro. In silico analysis and small-scale expression experiments showed that this variant causes a folding defect, leading to increased aggregation properties that could not be rescued by PLP. Using these results, we developed a diagnostic pipeline for new patients suspected of having GACR. Adding OAT enzymatic analyses and in vitro pyridoxine responsiveness to diagnostic practices will not only increase knowledge on the consequences of pathogenic variants in OAT, but will also enable expectation management for therapeutic modalities, thus eventually improving clinical care., Competing Interests: Declaration of competing interest The authors have no conflicts of interests to declare., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

This study aimed to identify whether gyrate atrophy of the choroid and retina (GACR) heterozygous individuals have possible clinical manifestations and to explore the potential pathogenic mechanism. In this retrospective study, we surveyed a two-generation pedigree of an individual diagnosed with GACR. Two family members underwent ophthalmological, hematologic, and genetic tests. An arginine-restricted diet with vitamin B6 supplementation was implemented; clinical assessments were repeated every 3 months during follow-up. The relative OAT mRNA expression was determined using a real-time quantitative polymerase chain reaction. The 19-year-old compound heterozygous daughter ( OAT : c.1186C>T; c.748C>T) had bilateral pathologic myopia, posterior staphyloma, chorioretinal atrophy, macular abnormalities, and elevated hematologic ornithine. The 54-year-old heterozygous mother ( OAT : c.1186C>T) presented with bilateral pathologic myopia, asymmetric posterior staphyloma, retina and choroidal capillary layer atrophy, retinal pigment epithelium abnormalities, and mildly elevated hematologic ornithine. Compared to normal individuals, the daughter and mother had 29% and 46% relative OAT mRNA expression, respectively ( p < 0.001). We believe that this is the first report of a carrier of one OAT variant allele exhibiting a mild phenotype, suggesting that family members should be aware of the possibility of clinical involvement in carriers with some autosomal recessive conditions. Additional data suggest that nonsense-mediated, decay-initiated mRNA degradation may cause GACR., Competing Interests: The authors declare no conflicts of interest.

The gyrate atrophy of the choroid and retina (GACR) is a rare genetic disease for which no definitive cure is available. GACR is due to the deficit of ornithine aminotransferase (hOAT), a pyridoxal 5'-phosphate-dependent enzyme responsible for ornithine catabolism. The hallmark of the disease is plasmatic ornithine accumulation, which damages retinal epithelium leading to progressive vision loss and blindness within the fifth decade. Here, we characterized the biochemical properties of tetrameric and dimeric hOAT and evaluated hOAT loaded in red blood cells (RBCs) as a possible enzyme replacement therapy (ERT) for GACR. Our results show that (i) hOAT has a relatively wide specificity for amino acceptors, with pyruvate being the most suitable candidate for ornithine catabolism within RBCs; (ii) both the tetrameric and dimeric enzyme can be loaded in RBC retaining their activity; and (iii) hOAT displays reduced stability in plasma, but is partly protected from inactivation upon incubation in a mixture mimicking the intracellular erythrocyte environment. Preliminary ex vivo experiments indicate that hOAT-loaded RBCs are able to metabolize extracellular ornithine at a concentration mimicking that found in patients, both in buffer and, although with lower efficiency, in plasma. Overall, our data provide a proof of concept that an RBC-mediated ERT is feasible and can be exploited as a new therapeutic approach in GACR.

Gyrate atrophy of choroid and retina (GACR) is a chorioretinal degeneration caused by pathogenic variants in the gene encoding ornithine aminotransferase (OAT), an enzyme mainly expressed in liver. Affected patients have increased ornithine concentrations in blood and other body fluids and develop progressive constriction of vision fields leading to blindness. Current therapies are unsatisfactory and better treatments are highly needed. In two mouse models of OAT deficiency that recapitulates biochemical and retinal changes of GACR, we investigated the efficacy of an intravenously injected serotype 8 adeno-associated (AAV8) vector expressing OAT under the control of a hepatocyte-specific promoter. Following injections, OAT-deficient mice showed reductions of ornithine concentrations in blood and eye cups compared with control mice injected with a vector expressing green fluorescent protein. AAV-injected mice showed improved electroretinogram response and partial restoration of retinal structure up to one-year post-injection. In summary, hepatic OAT expression by AAV8 vector was effective at correction of hyperornithinemia and improved function and structure of the retina. In conclusion, this study provides proof-of-concept of efficacy of liver-directed AAV-mediated gene therapy of GACR., (© 2023 The Authors. Published under the terms of the CC BY 4.0 license.)

Objective: The sensitivity and specificity of current biomarkers for gastric cancer were insufficient. The aim of the present study was to screen novel biomarkers and determine the diagnostic values of ornithine aminotransferase (OAT) and carbamoyl phosphate synthetase 1 (CPS1) for detecting gastric cancer., Methods: With stable isotope tags, we labelled an initial discovery group of four paired gastric cancer tissue samples and identified with LC-ESI-MS/MS. A validation group of 159 gastric cancer samples and 30 healthy controls were used to validate the candidate targets. GSEA was used to explore the pathways activated in gastric cancer., Results: Four hundred and thirty one proteins were found differentially expressed in gastric cancer tissues. Of these proteins, OAT and CPS1 were found over-expressed in gastric cancer patients, with sensitivity of 70.4% (95% CI: 63.3%-77.6%) and specificity of 80.5% (95% CI: 74.3%-86.7%) for ornithine aminotransferase, and with sensitivity of 68.6% (95% CI: 61.3%-75.8%) and specificity of 73% (95% CI: 66%-79.9%) for carbamoyl phosphate synthetase 1. The co-expression of OAT and CPS1 in gastric cancer tissues has a sensitivity of 81% (95% CI: 73.2%-88.8%) and specificity of 89% (95% CI: 83%-95%). Furthermore, both OAT and CPS1 were overexpressed in patients with local invasion T3 and T4 stages than those in patients with T1 and T2 stages. The co-expression of OAT and CPS1 was strongly correlated with histological grade I 68% (95% CI: 58.7%-77.3%) and TNM stage I/II 52% (95% CI: 42%-62%). The areas under ROC curves were up to 0.758 for the co-expression of OAT and CPS1 in gastric cancer. GSEA results showed that two gene sets and 30 gene sets were activated in OAT high- and CPS1 high-expression patients with gastric cancer, respectively., Conclusions: The present findings indicated a tight correlation between the co-expression of OAT and CPS1 and the histological grade, local invasion, and TNM stages of gastric cancer. Therefore, OAT and CPS1 might be predictors for gastric cancer invasion and potential targets for anticancer drug design for gastric cancer., (© 2022 The Authors. Journal of Clinical Laboratory Analysis published by Wiley Periodicals LLC.)

Gyrate atrophy (GA) of the choroid and retina is a rare autosomal recessive genetic condition characterized by elevation of the plasma level of the amino acid ornithine due to deficiency of the enzyme ornithine ketoacid aminotransferase. Accumulation of ornithine occurs in various body tissues but leads primarily to characteristic ophthalmic manifestations including myopia, cataract, progressive chorioretinal atrophy, and macular changes. Patients usually present with night blindness that starts in the first decade of life followed by visual field constriction and eventually diminution of the central visual acuity and blindness. The condition has been reported worldwide and its differential diagnosis is broad and includes choroideremia and retinitis pigmentosa. Treatment currently depends on life-long dietary modifications including restriction of the amino acid arginine in diet. This article describes in detail the pathogenesis, clinical features, multimodal imaging findings, and treatment options for GA of the choroid and retina and its complications.

The bifunctional enzyme Δ 1 -pyrroline-5-carboxylate synthase (P5CS) is vital to the synthesis of proline and ornithine, playing an essential role in human health and agriculture. Pathogenic mutations in the P5CS gene (ALDH18A1) lead to neurocutaneous syndrome and skin relaxation connective tissue disease in humans, and P5CS deficiency seriously damages the ability to resist adversity in plants. We have recently found that P5CS forms cytoophidia in vivo and filaments in vitro. However, it is difficult to appreciate the function of P5CS filamentation without precise structures. Using cryo-electron microscopy, here we solve the structures of Drosophila full-length P5CS in three states at resolution from 3.1 to 4.3 Å. We observe distinct ligand-binding states and conformational changes for the GK and GPR domains, respectively. Divergent helical filaments are assembled by P5CS tetramers and stabilized by multiple interfaces. Point mutations disturbing those interfaces prevent P5CS filamentation and greatly reduce the enzymatic activity. Our findings reveal that filamentation is crucial for the coordination between the GK and GPR domains, providing a structural basis for the catalytic function of P5CS filaments., Competing Interests: JZ, CG, XZ, CC, BY, TZ, HH, GL, JL No competing interests declared, (© 2022, Zhong et al.)

Chronic HBV infection is a major cause of hepatocellular carcinoma (HCC) worldwide. The phenotypes of HCC are diverse, in part, due to mutations in distinct oncogenes and/or tumor suppressor genes. These genetic drivers of HCC development have generally been considered as major mediators of tumor heterogeneity. Using the liver-specific Pten-null HBV transgenic mouse model of chronic viral infection, a critical role for liver lobule zone-specific gene expression patterns in determining HCC phenotype and β-catenin-dependent HBV biosynthesis is demonstrated. These observations suggest that the position of the hepatocyte within the liver lobule, and hence its intrinsic gene expression pattern at the time of cellular transformation, make critical contributions to the properties of the resulting liver tumor. These results may explain why therapies targeting pathways modulated by specific identified tumor driver genes display variable treatment efficacy., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Activation of mTORC1 (mechanistic target of rapamycin complex 1) in renal tissue has been reported in chronic kidney disease (CKD)-induced renal fibrosis. However, the molecular mechanisms responsible for activating mTORC1 in CKD pathology are not well understood. The purpose of this study was to identify the uremic toxin involved in mTORC1-induced renal fibrosis. Among the seven protein-bound uremic toxins, only indoxyl sulfate (IS) caused significant activation of mTORC1 in human kidney 2 cells (HK-2 cells). This IS-induced mTORC1 activation was inhibited in the presence of an organic anion transporter inhibitor, a NADPH oxidase inhibitor, and an antioxidant. IS also induced epithelial-mesenchymal transition of tubular epithelial cells (HK-2 cells), differentiation of fibroblasts into myofibroblasts (NRK-49F cells), and inflammatory response of macrophages (THP-1 cells), which are associated with renal fibrosis, and these effects were inhibited in the presence of rapamycin (mTORC1 inhibitor). In in vivo experiments, IS overload was found to activate mTORC1 in the mouse kidney. The administration of AST-120 or rapamycin targeted to IS or mTORC1 ameliorated renal fibrosis in Adenine-induced CKD mice. The findings reported herein indicate that IS activates mTORC1, which then contributes to renal fibrosis. Therapeutic interventions targeting IS and mTORC1 could be effective against renal fibrosis in CKD.

Free proline has multiple functions in plant cells, such as regulating osmotic potential and protecting both proteins and cell membranes. The expression of Δ1-Pyrroline-5-carboxylate synthase (P5CS), a key enzyme in the proline biosynthetic pathway, increases under drought, salt and cold stress conditions, causing plant cells to accumulate large amounts of proline. In this study, we cloned and identified the P5CS gene from Stipa purpurea , which has a full-length of 2196 bp and encodes 731 amino acids. A subcellular localization analysis indicated that SpP5CS localized to the cytoplasm. The ectopic overexpression of SpP5CS in Arabidopsis thaliana resulted in higher proline contents, longer roots, higher survival rates and less membrane damage under drought stress conditions compared with wild-type controls. SpP5CS -overexpressing A. thaliana was more resistant to drought stress than the wild type, whereas the deletion mutant sp5cs was less resistant to drought stress. Thus, SpP5CS may be a potential candidate target gene for increasing plant resistance to drought stress.

Background: Gyrate atrophy is a rare autosomal recessive inherited genetic disease. Progressive deterioration of peripheral night vision and blindness are the foremost clinical manifestations of the disease caused by mutations of ornithine aminotransferase gene., Case: The presented case was an 18-year-old male referred for a progressive reduction of visual acuity, which started when the subject was 7 years old, blurred vision, and hypotonic muscles., Observations: The findings by liquid chromatography with tandem mass spectrometry and high-performance liquid chromatography methods exhibited a high level of ornithine: 248 μmol/L (reference range: 44-206 μmol/L) and 818 μmol/L (reference: 25-123 μmol/L), respectively. After genetic counseling and conducting further investigation, a novel mutation (c.425-1G>A) in ornithine aminotransferase gene was recognized through whole exome sequencing and the mutation was verified using Sanger sequencing method, which is associated with gyrate atrophy phenotype., Conclusion: The exact mechanism of chorioretinal atrophy in hyperornithinemia is not known but the increased ornithine level is the clinical manifestation of gyrate atrophy of choroid and retina, muscle weakness, moderate mental retardation, and low cerebral creatine. Pathogenic variant in the ornithine aminotransferase gene associated with gyrate atrophy, may be beneficial as a biomarker to initial diagnosis and treatment of gyrate atrophy disease.

Key Message: The drought and salt tolerances of wheat were enhanced by ectopic expression of the Arabidopsis ornithine aminotransferase (AtOAT) encoded gene. The OAT was confirmed to play a role in proline biosynthesis in wheat. Proline (Pro) accumulation is a common response to both abiotic and biotic stresses in plants. Ornithine aminotransferase (OAT) is pyridoxal-5-phosphate dependent enzyme involved in plant proline biosynthesis. During stress condition, proline is synthesized via glutamate and ornithine pathways. The OAT is the key enzyme in ornithine pathway. In this study, an OAT gene AtOAT from Arabidopsis was expressed in wheat for its functional characterization under drought, salinity, and heat stress conditions. We found that the expression of AtOAT enhanced the drought and salt stress tolerances of wheat by increasing the proline content and peroxidase activity. In addition, it was confirmed that the expression of AtOAT also played a partial tolerance to heat stress in the transgenic wheat plants. Moreover, quantitative real-time PCR (qRT-PCR) analysis showed that the transformation of AtOAT up-regulated the expression of the proline biosynthesis associated genes TaOAT, TaP5CS, and TaP5CR, and down-regulated that of the proline catabolism related gene TaP5CDH in the transgenic plants under stress conditions. Moreover, the genes involved in ornithine pathway (Orn-OAT-P5C/GSA-P5CR-Pro) were up-regulated along with the up-regulation of those genes involved in glutamate pathway (Glu-P5CS-P5C/GSA-P5CR-Pro). Therefore, we concluded that the expression of AtOAT enhanced wheat abiotic tolerance via modifying the proline biosynthesis by up-regulating the expression of the proline biosynthesis-associated genes and down-regulating that of the proline catabolic gene under stresses condition.

Background: Gyrate Atrophy (GA) is a rare autosomal recessive disorder characterized by progressive chorioretinal degeneration. It is caused due to mutations in OAT gene that encodes a defective ornithine-δ-aminotransferase enzyme. We aim to identify the molecular cause of the disease and correlate it with the phenotype. Materials and Methods: Clinical, biochemical and genetic analyses were performed in siblings with GA. Case Description: A 10-year-old girl presented with impaired vision was clinically diagnosed to have peripheral chorioretinal degeneration in both eyes due to GA with vitreous hemorrhage in the right eye. Similar chorioretinal degeneration was observed in the patient's sibling, while parents were normal. Biochemical analysis of plasma by LC-MS/MS showed an elevated ornithine level of 892.8 µmol/L in the patient and 572.3 µmol/L in the sibling. Familial genetic screening by Sanger sequencing revealed a nonsense mutation in exon 11 of the OAT gene (c.1192C>T; p.Arg398Ter) in all the family members with a homozygous mutation in the patient and sibling, and heterozygous mutation in the parents. The patient was under follow-up with an arginine-restricted diet. At the last follow-up, the vitreous hemorrhage of right eye had resolved with an improvement in visual acuity and left eye remained stable with 6/12. Conclusion: Our patient is a rare case of gyrate atrophy presented with vitreous hemorrhage and nonsense OAT gene mutation, inherited in the autosomal recessive pattern. This report highlights the phenotypic variability among the siblings with the same mutation in OAT gene for the first time.

Background : Gyrate atrophy of the choroid and retina (GA) is a rare autosomal recessive disorder characterized by nyctalopia, myopia, sharply demarcated expanding peripheral chorioretinal atrophic lesions, early cataract, progressive visual loss and hyperornithinemia. Only three cases of GA associated with rhegmatogenous retinal detachments (RRD) have been reported. The genotype-phenotype correlation of RRD in GA is limited by lack of genetic information in the previously reported cases. Here we report two young sisters with a characteristic GA phenotype associated with a novel variant in the ornithine aminotransferase gene (OAT ), in whom one developed unilateral RRD at the age of 9 years. Materials and Methods : Retrospective report of two cases including genetic analysis and multimodal retinal imaging. Results : A 9-year-old Saudi girl presented with a funnel-shaped RRD, extensive proliferative vitreoretinopathy, peripheral choroidal detachment and neovascular glaucoma in her right eye. Fundus examination of her left eye showed an attached retina with sharply-demarcated peripheral chorioretinal atrophic patches suggestive of GA. Whole exome sequencing confirmed GA by revealing a homozygous c.980 C > G (p. Pro327Arg) variant in exon 8 of OAT . The RRD was inoperable. The chorioretinal lesions in the left eye enlarged slowly over 3 years of follow up. Examination of the proband's older sister revealed a similar but more advanced GA phenotype in both eyes. Conclusions : A characteristic GA phenotype associated with a novel variant in OAT is reported. This variant might be associated with childhood-onset RRD in the proband.

Pyrroline-5-carboxylate synthase (P5CS) catalyzes the synthesis of pyrroline-5-carboxylate (P5C), a key precursor for the synthesis of proline and ornithine. P5CS malfunction leads to multiple human diseases; however, the molecular mechanism underlying these diseases is unknown. We found that P5CS localizes in mitochondria in rod- and ring-like patterns but diffuses inside the mitochondria upon cellular starvation or exposure to oxidizing agents. Some of the human disease-related mutant forms of P5CS also exhibit diffused distribution. Multimerization (but not the catalytic activity) of P5CS regulates its localization. P5CS mutant cells have a reduced proliferation rate and are sensitive to cellular stresses. Flies lacking P5CS have reduced eclosion rates. Lipid droplets accumulate in the eyes of the newly eclosed P5CS mutant flies, which degenerate with aging. The loss of P5CS in cells leads to abnormal purine metabolism and lipid-droplet accumulation. The reduced lipid-droplet consumption is likely due to decreased expression of the fatty acid transporter, CPT1, and few β-oxidation-related genes following P5CS knockdown. Surprisingly, we found that P5CS is required for mitochondrial respiratory complex organization and that the respiration defects in P5CS knockout cells likely contribute to the metabolic defects in purine synthesis and lipid consumption. This study links amino acid synthesis with mitochondrial respiration and other key metabolic processes, whose imbalance might contribute to P5CS-related disease conditions.

Gyrate Atrophy (GA) of the choroid and retina (MIM# 258870) is an autosomal recessive disorder due to mutations of the OAT gene encoding ornithine-delta-aminotransferase (OAT), associated with progressive retinal deterioration and blindness. The disease has a theoretical global incidence of approximately 1:1,500,000. OAT is mainly involved in ornithine catabolism in adults, thus explaining the hyperornithinemia as hallmark of the disease. Patients are treated with an arginine-restricted diet, to limit ornithine load, or the administration of Vitamin B6, a precursor of the OAT coenzyme pyridoxal phosphate. Although the clinical and genetic aspects of GA are known for many years, the enzymatic phenotype of pathogenic variants and their response to Vitamin B6, as well as the molecular mechanisms explaining retinal damage, are poorly clarified. Herein, we provide an overview of the current knowledge on the biochemical properties of human OAT and on the molecular, cellular, and clinical aspects of GA., (Copyright © 2020 Elsevier B.V. All rights reserved.)

l-Proline takes a significant role in the pharmaceutical and chemical industries as well as graziery. Typical biosynthesis of l-proline is from l-glutamate, involving three enzyme reactions as well as a spontaneous cyclization. Alternatively, l-proline can be also synthesized in l-ornithine and/or l-arginine producing strains by an ornithine aminotransferase (OCD). In this study, a strategy of directed evolution combining rare codon selection and pEvolvR was developed to screen OCD with high catalytic efficiency, improving l-proline production from l-arginine chassis cells. The mutations were generated by CRISPR-assisted DNA polymerases and were screened by growth-coupled rare codon selection system. OCD K205G/M86K/T162A from Pseudomonas putida was identified with 2.85-fold increase in catalytic efficiency for the synthesis of l-proline. Furthermore, we designed and optimized RBS for the BaargI and Ppocd coupling cascade using RedLibs, as well as sRNA inhibition of argF to moderate l-proline biosynthesis in l-arginine overproducing Corynebacterium crenatum . The strain PS6 with best performance reached 15.3 g/L l-proline in the shake flask and showed a titer of 38.4 g/L in a 5 L fermenter with relatively low concentration of residual l-ornithine and/or l-arginine.

Chinese hamster ovary (CHO) cells are the superior host cell culture models used for the bioproduction of therapeutic proteins. One of the prerequisites for bioproduction using CHO cell lines is the need to generate stable CHO cell lines with optimal expression output. Antibiotic selection is commonly employed to isolate and select CHO cell lines with stable expression, despite its potential negative impact on cellular metabolism and expression level. Herein, we present a novel proline-based selection system for the isolation of stable CHO cell lines. The system exploits a dysfunctional proline metabolism pathway in CHO cells by using a pyrroline-5-carboxylate synthase gene as a selection marker, enabling selection to be made using proline-free media. The selection system was demonstrated by expressing green fluorescent protein (GFP) and a monoclonal antibody. When GFP was expressed, more than 90% of stable transfectants were enriched within 2 weeks of the selection period. When a monoclonal antibody was expressed, we achieved comparable titers (3.35 ± 0.47 μg/mL) with G418 and Zeocin-based selections (1.65 ± 0.46 and 2.25 ± 0.07 μg/mL, respectively). We further developed a proline-based coselection by using S. cerevisiae PRO1 and PRO2 genes as markers, which enables the generation of 99.5% double-transgenic cells. The proline-based selection expands available selection tools and provides an alternative to antibiotic-based selections in CHO cell line development.

Background: Ornithine aminotransferase (OAT, EC:2.6.1.13), alternatively known as ornithine delta aminotransferase (δOAT), is a pyridoxal phosphate (PLP)-dependent enzyme involved in the conversion of ornithine into glutamyl-5-semi-aldehyde (GSA) and vice versa. Up till now, there has been no study on OAT in wheat despite the success of its isolation from rice, maize, and sorghum. This study focuses on identification and molecular characterization of OAT in wheat., Results: In total, three homeologous OAT genes in wheat genome were found on chromosome group 5, named as TaOAT-5AL, TaOAT-5BL, and TaOAT-5DL. Sequence alignment between gDNA and its corresponding cDNA obtained a total of ten exons and nine introns. A phylogenetic tree was constructed and results indicated that OATs shared highly conserved domains between monocots and eudicots, which was further illustrated by using WebLogo to generate a sequence logo. Further subcellular localization analysis indicated that they functioned in mitochondria. Protein-protein interactions supported their role in proline biosynthesis through interactions with genes, such as delta 1-pyrroline-5-carboxylate synthetase (P5CS) and pyrroline-5-carboxylate reductase (P5CR), involved in the proline metabolic pathway. Promoter analysis exposed the presence of several stress responsive elements, implying their involvement in stress regulation. Expression profiling illustrated that TaOAT was highly induced in the wheat plants exposed to drought or salt stress condition. Upregulated expression of TaOATs was observed in stamens and at the heading stage. A potential role of TaOAT genes during floret development was also revealed. Furthermore, the transgenic plants overexpressing TaOAT showed enhanced tolerance to drought stress by increasing proline accumulation. In addition, salt tolerance of the transgenic plants was also enhanced., Conclusion: TaOATs genes were involved in proline synthesis and nitrogen remobilization because they interacted with genes related to proline biosynthesis enzymes and arginine catabolism. In addition, TaOAT genes had a role in abiotic stress tolerance and a potential role in floret development. The results of this study may propose future research in the improvement of wheat resistance to abiotic stresses.

Compartmentation of enzymes via filamentation has arisen as a mechanism for the regulation of metabolism. In 2010, three groups independently reported that CTP synthase (CTPS) can assemble into a filamentous structure termed the cytoophidium. In searching for CTPS-interacting proteins, here we perform a yeast two-hybrid screening of Drosophila proteins and identify a putative CTPS-interacting protein, △ 1 -pyrroline-5-carboxylate synthase (P5CS). Using the Drosophila follicle cell as the in vivo model, we confirm that P5CS forms cytoophidia, which are associated with CTPS cytoophidia. Overexpression of P5CS increases the length of CTPS cytoophidia. Conversely, filamentation of CTPS affects the morphology of P5CS cytoophidia. Finally, in vitro analyses confirm the filament-forming property of P5CS. Our work links CTPS with P5CS, two enzymes involved in the rate-limiting steps in pyrimidine and proline biosynthesis, respectively., (Copyright © 2020 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Ltd. All rights reserved.)

Among the over 50 gyrate atrophy-causing mutations of ornithine δ-aminotransferase (OAT), the R180T involves an active site residue located at the dimer interface, which in the crystal structure of OAT complexed with 5-fluoromethylornithine engages a salt bridge with the α-carboxylate of the substrate analogue. Starting from the previous finding that no transaminase activity was detected in CHO-K 1 cells expressing the R180T variant, here we try to shed light at the protein level on the structural and/or functional defects of the R180T variant. To this aim, the variant has been cloned, expressed, purified and characterized by a combination of biochemical and structural studies. Although the R180T variant shares a similar overall conformation with the wild-type, its crystal structure solved at 1.8 Ǻ reveals slight structural alterations at the active site and at the dimeric interface. These changes are consistent with the spectroscopic and kinetic results, indicating that the variant, as compared with the wild-type OAT, shows (a) an increased K m value for l-ornithine (l-Orn), (b) an altered pyridoxal 5'-phosphate binding mode and affinity and (c) an increased thermostability. In addition, the R180T mutant exhibits a remarkable loss of catalytic activity and is endowed with the ability to catalyse not only the δ-transamination but also, albeit to a lesser extent, the α-transamination of l-Orn. Overall, these data indicate that the slight structural changes caused by the R180T mutation, preventing a proper collocation of l-Orn at the active site of OAT, are responsible for the notable reduction of the catalytic efficiency. ENZYMES: Ornithine aminotransferase EC 2.6.1.13. DATABASES: 6HX7.pdb., (© 2019 Federation of European Biochemical Societies.)

Background/aim: Gyrate atrophy of the choroid and retina (GACR) is an extremely rare autosomal recessive inherited disorder characterised by progressive vision loss. To identify the disease-causing gene in a consanguineous Chinese pedigree with GACR, we aimed to accurately diagnose patients with GACR through a combination of next-generation sequencing (NGS) genetic diagnosis, clinical imaging and amino acid metabolic analysis., Methods: A consanguineous Chinese pedigree with GACR, including two patients, was recruited and a comprehensive ophthalmological evaluation was performed. DNA was extracted from a proband and her family members, and the sample from the proband was analysed using targeted NGS. Variants ‎detected by NGS were confirmed by Sanger sequencing and subjected to segregation analysis. Tandem mass spectrometry (MS/MS) was subsequently performed for metabolic assessment., Results: We identified a ‎novel, deleterious, homologous ornithine aminotransferase ( OAT ) variant, c.G248A: p.S83N, which contributes to ‎the progression of GACR in patients. Our results showed that the p.S83N autosomal recessive ‎variant of OAT is most likely ‎pathogenic, with changes in protein stability drastically decreasing functionality. MS/MS verified that ornithine levels in patients were significantly elevated., Conclusions: Recruitment of a third-degree first cousin consanguineous marriage family with GACR allowed us to identify a novel pathogenic OAT variant in the Chinese population, broadening the mutation spectrum. Our findings reported the diagnostic value of a combination of NGS, retinal imaging and metabolic analysis of consanguineous marriage pedigrees in low-income/middle-income and low-incidence countries, including China, and may help to guide accurate diagnosis and ‎treatment of this disease., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2019. No commercial re-use. See rights and permissions. Published by BMJ.)

The non-proteinogenic amino acid ornithine links several stress response pathways. From a previous study we know that ornithine accumulates in response to low CO2. To investigate ornithine accumulation in plants, we shifted plants to either low CO2 or low light. Both conditions increased carbon limitation, but only low CO2 also increased the rate of photorespiration. Changes in metabolite profiles of light- and CO2-limited plants were quite similar. Several amino acids that are known markers of senescence accumulated strongly under both conditions. However, urea cycle intermediates respond differently between the two treatments. While the levels of both ornithine and citrulline were much higher in plants shifted to 100 ppm CO2 compared to those kept in 400 ppm CO2, their metabolite abundance did not significantly change in response to a light limitation. Furthermore, both ornithine and citrulline accumulation is independent from sugar starvation. Exogenous supplied sugar did not significantly change the accumulation of the two metabolites in low CO2-stressed plants, while the accumulation of other amino acids was reduced by about 50%. Gene expression measurements showed a reduction of the entire arginine biosynthetic pathway in response to low CO2. Genes in both proline biosynthesis and degradation were induced. Hence, proline did not accumulate in response to low CO2 like observed for many other stresses. We propose that excess of nitrogen re-fixed during photorespiration can be alternatively stored in ornithine and citrulline under low CO2 conditions. Furthermore, ornithine is converted to pyrroline-5-carboxylate by the action of δOAT., Competing Interests: The authors have declared that no competing interests exist.

Plant tolerance to biotic and abiotic stresses is complicated by interactions between different stresses. Maintaining crop yield under abiotic stresses is the most daunting challenge for breeding resilient crop varieties. In response to environmental stresses, plants produce several metabolites, such as proline (Pro), polyamines (PAs), asparagine, serine, carbohydrates including glucose and fructose, and pools of antioxidant reactive oxygen species. Among these metabolites, Pro has long been known to accumulate in cells and to be closely related to drought, salt, and pathogen resistance. Pyrroline-5-carboxylate (P5C) is a common intermediate of Pro synthesis and metabolism that is produced by ornithine aminotransferase (OAT), an enzyme that functions in an alternative Pro metabolic pathway in the mitochondria under stress conditions. OAT is highly conserved and, to date, has been found in all prokaryotic and eukaryotic organisms. In addition, ornithine (Orn) and arginine (Arg) are both precursors of PAs, which confer plant resistance to drought and salt stresses. OAT is localized in the cytosol in prokaryotes and fungi, while OAT is localized in the mitochondria in higher plants. We have comprehensively reviewed the research on Orn, Arg, and Pro metabolism in plants, as all these compounds allow plants to tolerate different kinds of stresses.

Nitrogen is one of the most important nutrient element that is essential for plant growth and development. Many genes have been reported to contribute to nitrogen absorption and transportation. However, genes involved in nitrogen reutilization are seldom reported. Ornithine δ-aminotransferase (δOAT) is the enzyme connecting arginine cycling and proline cycling. Here, we found that OsOAT, the homologue of δOAT in rice, is essential for nitrogen reutilization through mediating arginase activity. In the Osoat mutant, metabolic abnormality induced by nitrogen deficiency in floret causes malformed glumes, incapable glume opening and anther indehiscence. These defects in the mutant affect the pollination process and lead to a low seed setting rate as well as abnormal seed shape. Intriguingly, urea can rescue the phenotypes of the Osoat mutant. Therefore, OsOAT is crucial for nitrogen reutilization and plays a critical role in floret development and seed setting in rice., (© 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.)

Gyrate atrophy (GA) is a rare recessive disorder characterized by progressive blindness, chorioretinal degeneration and systemic hyperornithinemia. GA is caused by point mutations in the gene encoding ornithine δ-aminotransferase (OAT), a tetrameric pyridoxal 5'-phosphate-dependent enzyme catalysing the transamination of l-ornithine and α-ketoglutarate to glutamic-γ-semialdehyde and l-glutamate in mitochondria. More than 50 OAT variants have been identified, but their molecular and cellular properties are mostly unknown. A subset of patients is responsive to pyridoxine administration, although the mechanisms underlying responsiveness have not been clarified. Herein, we studied the effects of the V332M mutation identified in pyridoxine-responsive patients. The Val332-to-Met substitution does not significantly affect the spectroscopic and kinetic properties of OAT, but during catalysis it makes the protein prone to convert into the apo-form, which undergoes unfolding and aggregation under physiological conditions. By using the CRISPR/Cas9 technology we generated a new cellular model of GA based on HEK293 cells knock-out for the OAT gene (HEK-OAT&#95;KO). When overexpressed in HEK-OAT&#95;KO cells, the V332M variant is present in an inactive apodimeric form, but partly shifts to the catalytically-competent holotetrameric form in the presence of exogenous PLP, thus explaining the responsiveness of these patients to pyridoxine administration. Overall, our data represent the first integrated molecular and cellular analysis of the effects of a pathogenic mutation in OAT. In addition, we validated a novel cellular model for the disease that could prove instrumental to define the molecular defect of other GA-causing variants, as well as their responsiveness to pyridoxine and other putative drugs., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Gyrate atrophy (GA) of the choroid and retina is a rare autosomal recessive disorder that occurs due to deficiency of the mitochondrial enzyme ornithine aminotransferase (OAT). Hyperornithinemia causes degeneration of the retina with symptoms like myopia, reduced night vision and progressive vision loss. Our patient is a 10-year-old girl with impaired vision and strabismus. As part of the metabolic work-up, plasma amino acid analysis revealed significantly increased concentration of ornithine (1039 μmol/L; reference interval 20 - 155 μmol/L). Molecular genetic analysis revealed homozygous mutation in exon 7 of the OAT gene that has not been reported previously (c.868&#95;870delCTT p.(Leu290del)). This in frame deletion was predicted to be deleterious by in silico software analysis. Our patient was treated with pyridoxine (vitamin B 6 in a dose of 2 x 100 mg/day), low-protein diet (0.6 g/kg/day) and L-lysine supplementation which resulted in a significant reduction in plasma ornithine concentrations to 53% of the initial concentration and the ophthalmologic findings showed significant improvement. We conclude that low protein diet and lysine supplementation can lead to long-term reduction in plasma ornithine concentrations and, if started at an early age, notably slow the progression of retinal function loss in patients with GA. The effect of therapy can be reliably monitored by periodical measurement of plasma ornithine concentration. To our knowledge, this is the first report of OAT deficiency in Croatia., Competing Interests: Potential conflict of interest: None declared.

Purpose: Gyrate atrophy (GA) is a rare chorioretinal degeneration that results in the deterioration of night and peripheral vision, eventually leading to blindness. The disorder is caused by mutations in the gene encoding ornithine aminotransferase (OAT), causing increased levels of plasma ornithine. Treatment revolves around lowering plasma ornithine levels, with vitamin B6 supplementation being the preferred treatment. Nevertheless, most patients do not respond to this therapy. Here, we report a rare case of vitamin B6-responsive GA caused by a novel mutation in OAT and characterize the presentation with multimodal imaging., Methods: This is a single-patient case report with a clinical diagnosis based on history, multimodal retinal imaging, laboratory findings, and DNA sequencing analysis. We include a 3D structure prediction of the novel mutant protein., Results: DNA sequencing analysis demonstrated that there is a homozygous, novel variant c.473A>C: p.Y158S in OAT. Upon undergoing two weeks of vitamin B6 supplementation, the patient exhibited a 28.5% reduction in plasma ornithine levels. In a follow-up visit two years later, plasma ornithine levels were reduced by 24.1% from the levels at initial presentation and disease progression was retarded based on clinical findings., Conclusion: One novel homozygous missense mutation in OAT was identified and considered to be pathogenic in a patient with GA. The response for the vitamin B6 supplementation was positive, which is rare in all the GA cases reported in the literature. Our data suggests that further studies regarding the relationship between genotype and responsiveness to vitamin B6 should be conducted.

The invasive pest Drosophila suzukii has evolved morphological and behavioural adaptations to lay eggs under the skin of fresh fruits. This results in severe damage to a wide range of small fruits. Drosophila suzukii females typically lay few eggs per fruit, preferring healthy fruits. Hence, larvae are exposed to a reduced amount of nitrogenous waste. Differently, the innocuous Drosophila melanogaster lays eggs on fermented fruits already infested by conspecifics, with larvae developing in a crowded environment with the accumulation of nitrogenous waste such as ammonia and urea. These compounds derive from nitrogen metabolism, protein degradation, and amino acids catabolism and are relatively toxic at high concentrations in an organism. The observed differences in oviposition site and larval ecological niche suggest that these species might differ in behavioural and physiological mechanisms used to cope with nitrogenous waste. We investigated how different concentrations of ammonia and urea affect oviposition and larval development in both species. Females and larvae of D. suzukii showed greater susceptibility to high concentrations of both compounds, with a dramatic decrease in the number of eggs laid and egg viability. Moreover, we tested the chemotactic response of third instar larvae to high concentrations of the compounds. Interestingly, ammonia resulted in a repulsive behaviour in respect of the control and urea groups. To better understand the pathways underlying these differences, we evaluated the effect on ornithine aminotransferase and glutathione-S-transferase, two enzymes involved in nitrogen metabolism and stress response that are expressed during larval development. Both ammonia and urea significantly reduced the expression of these enzymes in D. suzukii compared to D. melanogaster. This shows how the ecological shift of D. suzukii to fresh fruit is accompanied by less efficient detoxifying and excretory mechanisms, with important implications for evolutionary biology and applied research. Our data suggest that the ecological shift of D. suzukii to fresh fruit as oviposition substrate is accompanied by a reduced tolerance to metabolic toxins during larval development., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Aminotransferases are pyridoxal 5'-phosphate-dependent enzymes that catalyze reversible transamination reactions between amino acids and α-keto acids, and are important for the cellular metabolism of nitrogen. Many bacterial and eukaryotic ω-aminotransferases that use l-ornithine (Orn), l-lysine (Lys), or γ-aminobutyrate (GABA) have been identified and characterized, but the corresponding enzymes from archaea are unknown. Here, we examined the activity and function of TK2101, a gene annotated as a GABA aminotransferase, from the hyperthermophilic archaeon Thermococcus kodakarensis We overexpressed the TK2101 gene in T. kodakarensis and purified and characterized the recombinant protein and found that it displays only low levels of GABA aminotransferase activity. Instead, we observed a relatively high ω-aminotransferase activity with l-Orn and l-Lys as amino donors. The most preferred amino acceptor was 2-oxoglutarate. To examine the physiological role of TK2101, we created a TK2101 gene-disruption strain (ΔTK2101), which was auxotrophic for proline. Growth comparison with the parent strain KU216 and the biochemical characteristics of the protein strongly suggested that TK2101 encodes an Orn aminotransferase involved in the biosynthesis of l-Pro. Phylogenetic comparisons of the TK2101 sequence with related sequences retrieved from the databases revealed the presence of several distinct protein groups, some of which having no experimentally studied member. We conclude that TK2101 is part of a novel group of Orn aminotransferases that are widely distributed at least in the genus Thermococcus , but perhaps also throughout the Archaea., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background: Chronic obstructive pulmonary disease (COPD) is a heterogeneous disease of the lungs that is currently the fourth leading cause of death worldwide. Genetic factors account for only a small amount of COPD risk, but epigenetic mechanisms, including DNA methylation, have the potential to mediate the interactions between an individual's genetics and environmental exposure. DNA methylation is highly cell type-specific, and individual cell type studies of DNA methylation in COPD are sparse. Fibroblasts are present within the airway and parenchyma of the lung and contribute to the aberrant deposition of extracellular matrix in COPD. No assessment or comparison of genome-wide DNA methylation profiles in the airway and parenchymal fibroblasts from individuals with and without COPD has been undertaken. These data provide valuable insight into the molecular mechanisms contributing to COPD and the differing pathologies of small airways disease and emphysema in COPD., Methods: Genome-wide DNA methylation was evaluated at over 485,000 CpG sites using the Illumina Infinium HumanMethylation450 BeadChip array in the airway (non-COPD n  = 8, COPD n  = 7) and parenchymal fibroblasts (non-COPD n  = 17, COPD n  = 29) isolated from individuals with and without COPD. Targeted gene expression was assessed by qPCR in matched RNA samples., Results: Differentially methylated DNA regions were identified between cells isolated from individuals with and without COPD in both airway and parenchymal fibroblasts. Only in parenchymal fibroblasts was differential DNA methylation associated with differential gene expression. A second analysis of differential DNA methylation variability identified 359 individual differentially variable CpG sites in parenchymal fibroblasts. No differentially variable CpG sites were identified in the airway fibroblasts. Five differentially variable-methylated CpG sites, associated with three genes, were subsequently assessed for gene expression differences. Two genes (OAT and GRIK2) displayed significantly increased gene expression in cells isolated from individuals with COPD., Conclusions: Differential and variable DNA methylation was associated with COPD status in the parenchymal fibroblasts but not airway fibroblasts. Aberrant DNA methylation was associated with altered gene expression imparting biological function to DNA methylation changes. Changes in DNA methylation are therefore implicated in the molecular mechanisms underlying COPD pathogenesis and may represent novel therapeutic targets., Competing Interests: The tissue was obtained and cells extracted with the approval of each of the research ethics boards for each of the academic institutions involved: Newcastle University (NRES Committee: Newcastle and North Tyneside 1 Ref:11/NE/0291); McMaster University (Hamilton Integrated Research Ethics Board Ref:00-1839); University of British Columbia (Providence Health Care Research Ethics Board Ref:H13-02173); University of Nottingham (East Midlands Research Ethics Committee Ref: 08/H0407/1); and University Medical Center Groningen. This study was conducted according to the national ethical and professional guidelines on the use human body material (“Code of conduct; Dutch federation of biomedical scientific societies”; https://www.federa.org/codes-conduct) and the Research Code of the University Medical Center Groningen (https://www.umcg.nl/EN/Research/Researchers/General/ResearchCode/Paginas/default.aspx).Not applicableThe authors declare that they have no competing interests.Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Purpose: This study reports the presentation of two families with gyrate atrophy (GA). The aim of this study was to characterize the potential effect of therapeutic regimens on macular edema., Methods: Two unrelated patients with GA were studied for the potential effect of low protein diet (≤ 0.8 g/kg/d), and oral administration of pyridoxine (500 mg/day), on serum ornithine levels, best corrected visual acuity (BCVA), slit-lamp, OCT, and auto-fluorescence findings. Blood samples for DNA, mRNA, and exons of the OAT gene were screened for mutations and splicing effect when relevant., Results: At presentation, both patients manifested typical ophthalmic features of GA including cystoid macular edema (CME). One patient also exhibited optic nerve head hamartoma. Following treatment ornithine levels have lessened, BCVA improved, and central macular thickness (CMT) markedly decreased in all four studied eyes. The molecular pathologic features included a novel splice site mutation (c.900+1G>A)., Conclusions: We have identified a novel mutation and two formerly described mutations in patients with GA. Of them, one patient comprised an unusual phenotype including bilateral astrocytic hamartomas. We have recognized for the first time improvement in CME following treatment with low protein intake and pyridoxine supplement. This finding may have significance in the understanding of treatment options for macular edema regardless of underlying etiology.

Potent mechanism-based inactivators can be rationally designed against pyridoxal 5'-phosphate (PLP)-dependent drug targets, such as ornithine aminotransferase (OAT) or γ-aminobutyric acid aminotransferase (GABA-AT). An important challenge, however, is the lack of selectivity toward other PLP-dependent, off-target enzymes, because of similarities in mechanisms of all PLP-dependent aminotransferase reactions. On the basis of complex crystal structures, we investigate the inactivation mechanism of OAT, a hepatocellular carcinoma target, by (1R,3S,4S)-3-amino-4-fluorocyclopentane-1-carboxylic acid (FCP), a known inactivator of GABA-AT. A crystal structure of OAT and FCP showed the formation of a ternary adduct. This adduct can be rationalized as occurring via an enamine mechanism of inactivation, similar to that reported for GABA-AT. However, the crystal structure of an off-target, PLP-dependent enzyme, aspartate aminotransferase (Asp-AT), in complex with FCP, along with the results of attempted inhibition assays, suggests that FCP is not an inactivator of Asp-AT, but rather an alternate substrate. Turnover of FCP by Asp-AT is also supported by high-resolution mass spectrometry. Amid existing difficulties in achieving selectivity of inactivation among a large number of PLP-dependent enzymes, the obtained results provide evidence that a desirable selectivity could be achieved, taking advantage of subtle structural and mechanistic differences between a drug-target enzyme and an off-target enzyme, despite their largely similar substrate binding sites and catalytic mechanisms.

Human ornithine δ-aminotransferase (hOAT) (EC 2.6.1.13) is a mitochondrial pyridoxal 5'-phosphate (PLP)-dependent aminotransferase whose deficit is associated with gyrate atrophy, a rare autosomal recessive disorder causing progressive blindness and chorioretinal degeneration. Here, both the apo- and holo-form of recombinant hOAT were characterized by means of spectroscopic, kinetic, chromatographic and computational techniques. The results indicate that apo and holo-hOAT (a) show a similar tertiary structure, even if apo displays a more pronounced exposure of hydrophobic patches, (b) exhibit a tetrameric structure with a tetramer-dimer equilibrium dissociation constant about fivefold higher for the apoform with respect to the holoform, and (c) have apparent T m values of 46 and 67 °C, respectively. Moreover, unlike holo-hOAT, apo-hOAT is prone to unfolding and aggregation under physiological conditions. We also identified Arg217 as an important hot-spot at the dimer-dimer interface of hOAT and demonstrated that the artificial dimeric variant R217A exhibits spectroscopic properties, T m values and catalytic features similar to those of the tetrameric species. This finding indicates that the catalytic unit of hOAT is the dimer. However, under physiological conditions the apo-tetramer is slightly less prone to unfolding and aggregation than the apo-dimer. The possible implications of the data for the intracellular stability and regulation of hOAT are discussed.

Toxoplasma gondii is a protozoan parasite of medical and veterinary relevance responsible for toxoplasmosis in humans. As an efficacious vaccine remains a challenge, chemotherapy is still the most effective way to combat the disease. In search of novel druggable targets, we performed a thorough characterization of the putative pyridoxal 5'-phosphate (PLP)-dependent enzyme ornithine aminotransferase from T. gondii ME49 (TgOAT). We overexpressed the protein in Escherichia coli and analysed its molecular and kinetic properties by UV-visible absorbance, fluorescence and CD spectroscopy, in addition to kinetic studies of both the steady state and pre-steady state. TgOAT is largely similar to OATs from other species regarding its general transamination mechanism and spectral properties of PLP; however, it does not show a specific ornithine aminotransferase activity like its human homologue, but exhibits both N-acetylornithine and γ-aminobutyric acid (GABA) transaminase activity in vitro , suggesting a role in both arginine and GABA metabolism in vivo The presence of Val79 in the active site of TgOAT in place of Tyr, as in its human counterpart, provides the necessary room to accommodate N-acetylornithine and GABA, resembling the active site arrangement of GABA transaminases. Moreover, mutation of Val79 to Tyr results in a change of substrate preference between GABA, N-acetylornithine and L-ornithine, suggesting a key role of Val79 in defining substrate specificity. The findings that TgOAT possesses parasite-specific structural features as well as differing substrate specificity from its human homologue make it an attractive target for anti-toxoplasmosis inhibitor design that can be exploited for chemotherapeutic intervention., (© 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.)

Micro-nutrient application is essential for normal plant growth while a little is known about manganese (Mn)-induced regulations in morpho-physiological attributes, aroma formation and enzyme involved in 2-acetyl-1-pyrroline (2-AP) biosynthesis in aromatic rice. Present study aimed to examine the influence of four levels of Mn i.e., Mn1 (100 mg MnSO4 pot(-1)), Mn2 (150 mg MnSO4 pot(-1)), Mn3 (200 mg MnSO4 pot(-1)), and Mn4 (250 mg MnSO4 pot(-1)) on the growth, yield formation, quality characters, rice aroma and enzyme involved in 2-acetyl-1-pyrroline biosynthesis in two fragrant rice cultivars i.e., Meixiangzhan and Nongxiang 18. Pots without Mn application were served as control (Ck). Each pot contained 15 kg of soil. Effects on agronomic characters, quality attributes, 2-AP contents and enzymes involved in 2-AP biosynthesis have been studied in early and late season rice. Results depicted that Mn improved rice growth, yield and related characters, and some quality attributes significantly. It further up-regulated proline, pyrroline-5-carboxylic acid (P5C) (precursors of 2-AP), soluble proteins and activities of proline dehydrogenase (ProDH), Δ(1) pyrroline-5-carboxylic acid synthetase (P5CS) ornithine aminotransferase (OAT) that led to enhanced 2-AP production in rice grains. Moreover, higher Mn levels resulted in increased grain Mn contents in both rice cultivars. Along with growth and yield improvement, Mn application significantly improved rice aromatic contents. Overall, Nongxiang 18 accumulated more 2-AP contents than Meixiangzhan in both seasons under Mn application. This study further explored the importance of Mn in rice aroma formation and signifies that micro-nutrients can play significant roles in rice aroma synthesis; however, intensive studies at molecular levels are still needed to understand the exact mechanisms of Mn to improve rice aroma formation., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

Mycobacterium tuberculosis (Mtb) possesses a genetic repertoire for metabolic pathways, which are specific and fit to its intracellular life style. Under in vitro conditions, Mtb is known to use arginine as a nitrogen source, but the metabolic pathways for arginine utilization have not been identified. Here we show that, in the presence of arginine, Mtb upregulates a gene cluster which includes an ornithine aminotransferase (rocD) and Rv2323c, a gene of unknown function. Isotopologue analysis by using 13C- or 15N-arginine revealed that in Mtb arginine is not only used as nitrogen source but also as carbon source for the formation of amino acids, in particular of proline. Surprisingly, rocD, which is widespread in other bacteria and is part of the classical arginase pathway turned out to be naturally deleted in Mtb, but not in non-tuberculous mycobacteria. Mtb lacking Rv2323c showed a growth defect on arginine, did not produce proline from arginine, and incorporated less nitrogen derived from arginine in its core nitrogen metabolism. We conclude that the highly induced pathway for arginine utilization in Mtb differs from that of other bacteria including non-tuberculous mycobacteria, probably reflecting a specific metabolic feature of intracellular Mtb.

Progeroid disorders overlapping with De Barsy syndrome (DBS) are collectively denoted as autosomal-recessive cutis laxa type 3 (ARCL3). They are caused by biallelic mutations in PYCR1 or ALDH18A1, encoding pyrroline-5-carboxylate reductase 1 and pyrroline-5-carboxylate synthase (P5CS), respectively, which both operate in the mitochondrial proline cycle. We report here on eight unrelated individuals born to non-consanguineous families clinically diagnosed with DBS or wrinkly skin syndrome. We found three heterozygous mutations in ALDH18A1 leading to amino acid substitutions of the same highly conserved residue, Arg138 in P5CS. A de novo origin was confirmed in all six probands for whom parental DNA was available. Using fibroblasts from affected individuals and heterologous overexpression, we found that the P5CS-p.Arg138Trp protein was stable and able to interact with wild-type P5CS but showed an altered sub-mitochondrial distribution. A reduced size upon native gel electrophoresis indicated an alteration of the structure or composition of P5CS mutant complex. Furthermore, we found that the mutant cells had a reduced P5CS enzymatic activity leading to a delayed proline accumulation. In summary, recurrent de novo mutations, affecting the highly conserved residue Arg138 of P5CS, cause an autosomal-dominant form of cutis laxa with progeroid features. Our data provide insights into the etiology of cutis laxa diseases and will have immediate impact on diagnostics and genetic counseling., (Copyright © 2015 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Osmopriming is a pre-sowing treatment that enhances germination performance and stress tolerance of germinating seeds. Brassica napus seeds showed osmopriming-improved germination and seedling growth under salinity stress. To understand the molecular and biochemical mechanisms of osmopriming-induced salinity tolerance, the accumulation of proline, gene expression and activity of enzymes involved in proline metabolism and the level of endogenous hydrogen peroxide were investigated in rape seeds during osmopriming and post-priming germination under control (H2O) and stress conditions (100 mM NaCl). The relationship between gene expression and enzymatic activity of pyrroline-5-carboxylate synthetase (P5CS), ornithine-δ-aminotransferase (OAT) and proline dehydrogenase (PDH) was determined. The improved germination performance of osmoprimed seeds was accompanied by a significant increase in proline content. The accumulation of proline during priming and post-priming germination was associated with strong up-regulation of the P5CSA gene, down-regulation of the PDH gene and accumulation of hydrogen peroxide. The up-regulated transcript level of P5CSA was consistent with the increase in P5CS activity. This study shows, for the first time, the role of priming-induced modulation of activities of particular genes and enzymes of proline turnover, and its relationship with higher content of hydrogen peroxide, in improving seed germination under salinity stress. Following initial stress-exposure, the primed seeds acquired stronger salinity stress tolerance during post-priming germination, a feature likely linked to a 'priming memory'., (Copyright © 2015 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Regulation of proline accumulation in seedlings of rice (Oryza sativa L. cv. Lunishree) was investigated. The increasing concentration of NaCl from 85 mM to 425 mM NaCl progressively increased proline content in rice. The maximum increase in proline content was recorded at 425 mM NaCl concentration as compared to control and other concentrations of NaCl. The highest significant activity of proline synthesizing enzymes, delta1-Pyrrolline-5-carboxylate synthetase, delta1-Pyrrolline-5-carboxylate reductase and Ornithine-δ- aminotransferase with lowest activity of proline hydrolysis enzymes;Proline dehydrogenase was also recorded at 425 mM NaCl salinity over control and other concentrations of NaCI with insignificant increase in the activity of delta1-Pyrrolline-5-carboxylate synthetase and Ornithine-δ-aminotransferase at 85 mM NaCI over control. It was found that the transcript of gene encoded with p5cs is up regulated about 1.35 folds under salinity stress. This gene synthesis an osmo protectant to help the plant resist the change in osmotic imbalances. Externally addition of MnCl2 at 300 mg/220 ml 1/2 strength Hoagland solution, having 1% NaCI, was also seen to increase 893.9% proline content of this variety as compared to control.

Protease inhibitors are involved primarily in defense against pathogens. In recent years, these proteins have also been widely implicated in response of plants to diverse abiotic stresses. Rice chymotrypsin protease inhibitor gene OCPI2 is highly induced under salt and osmotic stresses. The construct containing the complete coding sequence of OCPI2 cloned downstream to CaMV35S promoter was transformed in Arabidopsis and single copy, homozygous transgenic lines were produced. The transgenic plants exhibited significantly enhanced tolerance to NaCl, PEG and mannitol stress as compared to wild type plants. Importantly, the vegetative and reproductive growth of transgenic plants under unstressed, control conditions was also enhanced: transgenic plants were more vigorous than wild type, resulting into higher yield in terms of silique number. The RWC values and membrane stability index of transgenic in comparison to wild type plants was higher. Higher proline content was observed in the AtOCPI2 lines, which was associated with higher transcript expression of pyrroline-5-carboxylate synthase and lowered levels of proline dehydrogenase genes. The chymotrypsin protease activities were lower in the transgenic as against wild type plants, under both unstressed, control as well as stressed conditions. It thus appears that rice chymotrypsin protease inhibitor gene OCPI2 is a useful candidate gene for genetic improvement of plants against salt and osmotic stress., (Copyright © 2015 Elsevier Masson SAS. All rights reserved.)

Purpose: In humans, deficiency of ornithine-δ-aminotransferase (OAT) results in progressive degeneration of the neural retina (gyrate atrophy) with blindness in the fourth decade. In this study, we used the Xenopus embryonic developmental model to study functions of the OAT gene on embryonic development., Methods: We cloned and sequenced full-length OAT cDNA from Xenopus oocytes (X-OAT) and determined X-OAT expression in various developmental stages of Xenopus embryos and in a variety of adult tissues. The phenotype, gene expression of neural developmental markers, and enzymatic activity were detected by gain-of-function and loss-of-function manipulations., Results: We showed that X-OAT is essential for Xenopus embryonic development, and overexpression of X-OAT produces a ventralized phenotype characterized by a small head, lack of axial structure, and defective expression of neural developmental markers. Using X-OAT mutants based on mutations identified in humans, we found that substitution of both Arg 180 and Leu 402 abrogated both X-OAT enzymatic activity and ability to modulate the developmental phenotype. Neurogenesis is inhibited by X-OAT during Xenopus embryonic development., Conclusions: Neurogenesis is inhibited by X-OAT during Xenopus embryonic development, but it is essential for Xenopus embryonic development. The Arg 180 and Leu 402 are crucial for these effects of the OAT molecule in development.

Background: Arginine may play important roles in tumor progression by providing ornithine for polyamine biosynthesis, required for cell growth. The aim of this work was to determine the expression of arginine metabolic pathway enzymes in head and neck squamous cell carcinoma (HNSCC) in northeast India., Materials and Methods: The expressions of arginase isoforms (ARG1 and ARG2), ornithine aminotransferase (OAT) and ornithine decarboxylase (ODC) were examined in fifty paired HNSCC and adjacent non-tumor tissues by immunohistochemistry. Immunocytochemistry, semiquantitative reverse transcription sq-PCR and quantitative real-time qPCR were used to assess protein and mRNA expressions in peripheral blood of fifty HNSCC patients and hundred controls., Results: ARG1 and ODC protein and mRNA were strongly expressed in peripheral blood from HNSCC patients. No ARG2 expression was observed. In vivo, expression of ARG1, ARG2 and ODC was significantly higher in tumor than in non-tumor tissues. Most tumors expressed low levels of OAT, with no difference in tissues or blood, compared to controls. The absolute extent of maximal ARG1 upregulation with qPCR showed 6.23 fold increase in HNSCC., Conclusions: These findings strongly suggest that in HNSCCs, the ARG1 pathway is stimulated leading to the formation of polyamines as indicated by higher ODC expression, which promote tumor growth.

Delta 1-pyrroline-5-carboxylate synthetase (P5CS) catalyzes the conversion of glutamate (Glu) to Glu semialdehyde (GSA). GSA spontaneously cyclizes to form P5C. P5C is then reduced to proline (Pro) or is converted to ornithine, the intermediate for arginine (Arg) biosynthesis. In the present study, a full-length Ldp5cs complementary DNA was cloned from the Colorado potato beetle Leptinotarsa decemlineata, a notorious insect defoliator of potato in most potato-growing regions of the world. Ldp5cs encodes a 792-amino-acid protein which shares high identity to homologues from other insect species. Quantitative reverse transcription polymerase chain reaction revealed that Ldp5cs was ubiquitously expressed in the eggs, first to fourth-instar larvae, wandering larvae, pupae and sexually mature adults. In the adults, Ldp5cs mRNA levels were higher in the fat body, foregut, midgut and hindgut, moderate in the ventral ganglion, lower in the thorax muscles, epidermis and Malpighian tubules. Two double-stranded RNAs (dsRNAs) (dsLdp5cs1 and dsLdp5cs2) targeting Ldp5cs were constructed and bacterially expressed. Ingestion during 3 consecutive days of dsLdp5cs1 or dsLdp5cs2 successfully silenced Ldp5cs, significantly reduced the contents of Pro and Arg in the hemolymph, decreased flight speed and shortened flight distance of the resulting adults. Furthermore, knocking down Ldp5cs significantly increased adult mortality. Thus, our results suggest that identified Ldp5cs encodes a functional P5CS enzyme that is involved in the biosynthesis of Pro and Arg in L. decemlineata., (Copyright © 2014 Elsevier Ltd. All rights reserved.)