Characterization of argininosuccinate lyase (EC 4.3.2.1) from Chlamydomonas reinhardtii.
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ABSTRACT:
SUBMITTER: Farrell K
PROVIDER: S-EPMC1147691 | biostudies-other | 1987 Feb
REPOSITORIES: biostudies-other
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Project description:Post-translational modification of protein cysteine residues is emerging as an important regulatory and signaling mechanism. We have identified numerous putative targets of redox regulation in the unicellular green alga Chlamydomonas reinhardtii. One enzyme, isocitrate lyase (ICL), was identified both as a putative thioredoxin target and as an S-thiolated protein in vivo. ICL is a key enzyme of the glyoxylate cycle that allows growth on acetate as a sole source of carbon. The aim of the present study was to clarify the molecular mechanism of the redox regulation of Chlamydomonas ICL using a combination of biochemical and biophysical methods. The results clearly show that purified C. reinhardtii ICL can be inactivated by glutathionylation and reactivated by glutaredoxin, whereas thioredoxin does not appear to regulate ICL activity, and no inter- or intramolecular disulfide bond could be formed under any of the conditions tested. Glutathionylation of the protein was investigated by mass spectrometry analysis, Western blotting, and site-directed mutagenesis. The enzyme was found to be protected from irreversible oxidative inactivation by glutathionylation of its catalytic Cys(178), whereas a second residue, Cys(247), becomes artifactually glutathionylated after prolonged incubation with GSSG. The possible functional significance of this post-translational modification of ICL in Chlamydomonas and other organisms is discussed.
| S-EPMC2794744 | biostudies-literature
Project description:Argininosuccinate lyase (EC 4.3.2.1) was purified by (NH4)2SO4 fractionation, chromatography on DEAE-cellulose and gel filtration on Sephadex G-200. The final enzyme preparation was purified 46-fold compared with the crude extract. Electrophoresis of this preparation revealed three bands, the major one having the enzyme activity. Analysis of the enzyme by gel filtration and by disc electrophoresis (in two different concentrations of acrylamide) gave mol.wts. of 200000 (+/- 15000) and 190000 (+/- 20000) respectively. Treatment with sodium dodecyl sulphate and mercaptoethanol dissociated the enzyme into subunits of mol.wt. 39000 (+/-2000). The results are indicative of the multimeric structure of the enzyme, which is composed of five (perhaps four or six) identical subunits.
| S-EPMC1183623 | biostudies-other
Project description:The urea cycle consists of six consecutive enzymatic reactions that convert waste nitrogen into urea. Deficiencies of any of these enzymes of the cycle result in urea cycle disorders (UCDs), a group of inborn errors of hepatic metabolism that often result in life-threatening hyperammonemia. Argininosuccinate lyase (ASL) catalyzes the fourth reaction in this cycle, resulting in the breakdown of argininosuccinic acid to arginine and fumarate. ASL deficiency (ASLD) is the second most common UCD, with a prevalence of ~1 in 70,000 live births. ASLD can manifest as either a severe neonatal-onset form with hyperammonemia within the first few days after birth or as a late-onset form with episodic hyperammonemia and/or long-term complications that include liver dysfunction, neurocognitive deficits, and hypertension. These long-term complications can occur in the absence of hyperammonemic episodes, implying that ASL has functions outside of its role in ureagenesis and the tissue-specific lack of ASL may be responsible for these manifestations. The biochemical diagnosis of ASLD is typically established with elevation of plasma citrulline together with elevated argininosuccinic acid in the plasma or urine. Molecular genetic testing of ASL and assay of ASL enzyme activity are helpful when the biochemical findings are equivocal. However, there is no correlation between the genotype or enzyme activity and clinical outcome. Treatment of acute metabolic decompensations with hyperammonemia involves discontinuing oral protein intake, supplementing oral intake with intravenous lipids and/or glucose, and use of intravenous arginine and nitrogen-scavenging therapy. Dietary restriction of protein and dietary supplementation with arginine are the mainstays in long-term management. Orthotopic liver transplantation (OLT) is best considered only in patients with recurrent hyperammonemia or metabolic decompensations resistant to conventional medical therapy.
| S-EPMC3709024 | biostudies-literature
Project description:Deficiency of argininosuccinate lyase (ASL) causes argininosuccinic aciduria, an urea cycle defect that may present with a severe neonatal onset form or with a late onset phenotype. To date phenotype-genotype correlations are still not clear because biochemical assays of ASL activity correlate poorly with clinical severity in patients. We employed a yeast-based functional complementation assay to assess the pathogenicity of 12 missense ASL mutations, to establish genotype-phenotype correlations, and to screen for intragenic complementation. Rather than determining ASL enzyme activity directly, we have measured the growth rate in arginine-free medium of a yeast ASL(null) strain transformed with individual mutant ASL alleles. Individual haploid strains were also mated to obtain diploid, "compound heterozygous" yeast. We show that the late onset phenotypes arise in patients because they harbor individual alleles retaining high residual enzymatic activity or because of intragenic complementation among different mutated alleles. In these cases complementation occurs because in the hybrid tetrameric enzyme at least one active site without mutations can be formed or because the differently mutated alleles can stabilize each other, resulting in partial recovery of enzymatic activity. Functional complementation in yeast is simple and reproducible and allows the analysis of large numbers of mutant alleles. Moreover, it can be easily adapted for the analysis of mutations in other genes involved in urea cycle disorders.
| S-EPMC2781438 | biostudies-literature
Project description:ObjectiveArgininosuccinate lyase (ASL) gene mutations account for argininosuccinic aciduria (ASA). This study aimed to design a minigene construct of ASL gene in order to investigate the impact of variants on splicing.MethodsThe peripheral blood samples were collected from the family members, and genomic DNA was extracted for gene diagnosis using the total exon sequencing method. The novel mutation gene was cloned into pEGFP-C1 vector, and the pathogenicity of the mutation was examined in cultured cells in vitro.ResultsThe clinical diagnosis of the proband as ASA was clear. Two pathogenic mutations, c.281G>T (p.Arg94Leu) and c.208-15 T>A were detected in the ASL gene, and the two mutations had not been reported. The minigene expression in vitro confirmed that c.208-15 T>A could cause aberrant splicing, resulting in the retention of 13 bp in intron 2 to exon 3.ConclusionTwo new pathogenic mutations of ASL gene, c.208-15 T>A and c.281G>T, were found in an ASA family, which enriches the mutational profile of the ASL gene and provides a basis for genetic diagnosis of ASA. Minigenes are optimal approaches to determine whether the intron mutation can cause aberrant splicing.
| S-EPMC6533879 | biostudies-literature
Project description:The unicellular green alga Chlamydomonas reinhardtii has a special type of anaerobic metabolism that is quite unusual for eukaryotes. It has two oxygen-sensitive [Fe-Fe] hydrogenases (EC 1.12.7.2) that are coupled to photosynthesis and, in addition, a formate- and ethanol-producing fermentative metabolism, which was proposed to be initiated by pyruvate formate-lyase (Pfl; EC 2.3.1.54). Pfl enzymes are commonly found in prokaryotes but only rarely in eukaryotes. Both the hydrogen- and the formate/ethanol-producing pathways are involved in a sustained anaerobic metabolism of the alga, which can be induced by sulfur depletion in illuminated cultures. Before now, the presence of a Pfl protein in C. reinhardtii was predicted from formate secretion and the homology of the deduced protein of the PFL1 gene model to known Pfl enzymes. In this study, we proved the formate-producing activity of the putative Pfl1 enzyme by heterologous expression of the C. reinhardtii PFL1 cDNA in Escherichia coli and subsequent in vitro activity tests of the purified protein. Furthermore, a Pfl-deficient E. coli strain secretes formate when expressing the PFL1 cDNA of C. reinhardtii. We also examined the Pfl1 fermentation pathway of C. reinhardtii under the physiological condition of sulfur depletion. Genetic and biochemical analyses show that sulfur-depleted algae express genes encoding enzymes acting downstream of Pfl1 and also potentially ethanol-producing enzymes, such as pyruvate decarboxylase (EC 4.1.1.1) or pyruvate ferredoxin oxidoreductase (EC 1.2.7.1). The latter enzymes might substitute for Pfl1 activity when Pfl1 is specifically inhibited by hypophosphite.
| S-EPMC2268514 | biostudies-literature
Project description:Telomeres are repeated sequences found at the end of the linear chromosomes of most eukaryotes and are required for chromosome integrity. Expression of the reverse-transcriptase telomerase allows for extension of telomeric repeats to counteract natural telomere shortening. Although Chlamydomonas reinhardtii, a photosynthetic unicellular green alga, is widely used as a model organism in photosynthesis and flagella research, and for biotechnological applications, the biology of its telomeres has not been investigated in depth. Here, we show that the C. reinhardtii (TTTTAGGG)n telomeric repeats are mostly nondegenerate and that the telomeres form a protective structure, with a subset ending with a 3' overhang and another subset presenting a blunt end. Although telomere size and length distributions are stable under various standard growth conditions, they vary substantially between 12 genetically close reference strains. Finally, we identify CrTERT, the gene encoding the catalytic subunit of telomerase and show that telomeres shorten progressively in mutants of this gene. Telomerase mutants eventually enter replicative senescence, demonstrating that telomerase is required for long-term maintenance of telomeres in C. reinhardtii.
| S-EPMC6549138 | biostudies-literature
Project description:The single-cell green alga Chlamydomonas reinhardtii harbors twelve truncated hemoglobins (Cr-TrHbs). Cr-TrHb1-1 and Cr-TrHb1-8 have been postulated to be parts of the nitrogen assimilation pathway, and of a NO-dependent signaling pathway, respectively. Here, spectroscopic and reactivity properties of Cr-TrHb1-1, Cr-TrHb1-2, and Cr-TrHb1-4, all belonging to clsss 1 (previously known as group N or group I), are reported. The ferric form of Cr-TrHb1-1, Cr-TrHb1-2, and Cr-TrHb1-4 displays a stable 6cLS heme-Fe atom, whereas the hexa-coordination of the ferrous derivative appears less strongly stabilized. Accordingly, kinetics of azide binding to ferric Cr-TrHb1-1, Cr-TrHb1-2, and Cr-TrHb1-4 are independent of the ligand concentration. Conversely, kinetics of CO or NO2- binding to ferrous Cr-TrHb1-1, Cr-TrHb1-2, and Cr-TrHb1-4 are ligand-dependent at low CO or NO2- concentrations, tending to level off at high ligand concentrations, suggesting the presence of a rate-limiting step. In agreement with the different heme-Fe environments, the pH-dependent kinetics for CO and NO2-binding to ferrous Cr-TrHb1-1, Cr-TrHb1-2, and Cr-TrHb1-4 are characterized by different ligand-linked protonation events. This raises the question of whether the simultaneous presence in C. reinhardtii of multiple TrHb1s may be related to different regulatory roles.
| S-EPMC4439042 | biostudies-literature
Project description:The urea cycle consists of six consecutive enzymatic reactions that convert waste nitrogen into urea. Deficiencies of any of these enzymes of the cycle result in urea cycle disorders (UCD), a group of inborn errors of hepatic metabolism that often result in life threatening hyperammonemia. Argininosuccinate lyase (ASL) is a cytosolic enzyme which catalyzes the fourth reaction in the cycle and the first degradative step, that is, the breakdown of argininosuccinic acid to arginine and fumarate. Deficiency of ASL results in an accumulation of argininosuccinic acid in tissues, and excretion of argininosuccinic acid in urine leading to the condition argininosuccinic aciduria (ASA). ASA is an autosomal recessive disorder and is the second most common UCD. In addition to the accumulation of argininosuccinic acid, ASL deficiency results in decreased synthesis of arginine, a feature common to all UCDs except argininemia. Arginine is not only the precursor for the synthesis of urea and ornithine as part of the urea cycle but it is also the substrate for the synthesis of nitric oxide, polyamines, proline, glutamate, creatine, and agmatine. Hence, while ASL is the only enzyme in the body able to generate arginine, at least four enzymes use arginine as substrate: arginine decarboxylase, arginase, nitric oxide synthetase (NOS) and arginine/glycine aminotransferase. In the liver, the main function of ASL is ureagenesis, and hence, there is no net synthesis of arginine. In contrast, in most other tissues, its role is to generate arginine that is designated for the specific cell's needs. While patients with ASA share the acute clinical phenotype of hyperammonemia, encephalopathy, and respiratory alkalosis common to other UCD, they also present with unique chronic complications most probably caused by a combination of tissue specific deficiency of arginine and/or elevation of argininosuccinic acid. This review article summarizes the clinical characterization, biochemical, enzymatic, and molecular features of this disorder. Current treatment, prenatal diagnosis, diagnosis through the newborn screening as well as hypothesis driven future treatment modalities are discussed.
| S-EPMC3073162 | biostudies-literature
Project description:The most motile phototrophic organisms exhibit photo-induced behavioral responses (photobehavior) to inhabit better light conditions for photosynthesis. The unicellular green alga Chlamydomonas reinhardtii is an excellent model organism to study photobehavior. Several years ago, we found that C. reinhardtii cells reverse their phototactic signs (i.e., positive and negative phototaxis) depending on the amount of reactive oxygen species (ROS) accumulated in the cell. However, its molecular mechanism is unclear. In this study, we isolated seven mutants showing positive phototaxis, even after the induction of negative phototaxis (ap1~7: always positive) to understand the ROS-dependent regulatory mechanism for the phototactic sign. We found no common feature in the mutants regarding their growth, high-light tolerance, and photosynthetic phenotypes. Interestingly, five of them grew faster than the wild type. These data suggest that the ROS-dependent regulation of the phototactic sign is not a single pathway and is affected by various cellular factors. Additionally, the isolation and analyses of mutants with defects in phototactic-sign regulation may provide clues for their application to the efficient cultivation of algae.
| S-EPMC8309459 | biostudies-literature