Allantoicase

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Giovanni Bernardini - One of the best experts on this subject based on the ideXlab platform.

  • Selective Pressure on the Allantoicase Gene During Vertebrate Evolution
    Journal of Molecular Evolution, 2003
    Co-Authors: Davide Vigetti, Claudio Monetti, Mariangela Prati, Giovanni Bernardini, Giorgio Binelli, Rosalba Gornati
    Abstract:

    During vertebrate evolution, the uric acid degradation pathway has been modified and several enzymes have been lost. Consequently, the end product of purine catabolism varies from species to species. In the past few years, we have focused our attention on vertebrate Allantoicase (an uricolytic pathway enzyme), whose activity is present in certain fish and amphibians only, but whose mRNA we detected also in mammals. As Allantoicase activity disappeared in amniotes, we wonder why these sequences not only remain present in the mammalian genome, but are still transcribed. To elucidate this issue, we have cloned and analyzed comparable cDNA sequences of different organisms from ascidians to mammals. The analysis of the nonsynonymous–synonymous substitution rate that we performed on the coding region comprising exons 3 to 8 by means of maximum likelihood suggested that a certain amount of purifying selection is acting on the Allantoicase sequences. Some implications of the preservation of an apparently unnecessary gene in higher vertebrates are discussed.

  • genomic organization and chromosome localization of the murine and human Allantoicase gene
    Gene, 2002
    Co-Authors: Davide Vigetti, Claudio Monetti, Mariangela Prati, Rosalba Gornati, Giovanni Bernardini
    Abstract:

    Abstract Allantoicase is one of the enzymes involved in uricolysis. The enzymes of this catabolic pathway (i.e. allantoinase, Allantoicase, ureidoglycolate lyase and urease) were lost during vertebrate evolution and the causes for this loss are still unclear. In mammals, as well as in birds and reptiles, the activity of Allantoicase is absent; notwithstanding, we recently cloned human and mouse cDNA sequences with high similarity with previously characterized Allantoicases. In the present paper, we report the genomic organization of the Allantoicase gene in mouse and in man. Both genes are constituted by 11 exons that appear to be very conserved; introns are more variable in length while maintain the same phase but for intron 4. We have also detected a second transcript of the human Allantoicase gene in which exon 1 is absent. Moreover, the mouse gene maps in chromosome 12 at 13.0 cM from the centromere.

  • Property comparison of recombinant amphibian and mammalian Allantoicases.
    FEBS Letters, 2002
    Co-Authors: Davide Vigetti, Claudio Monetti, Mariangela Prati, Giovanni Bernardini, Loredano Pollegioni, Rosalba Gornati
    Abstract:

    Allantoicase is an enzyme involved in uric acid degradation. Although it is commonly accepted that Allantoicase is lost in mammals, birds and reptiles, we have recently identified its transcripts in mice and humans. The mouse mRNA seems capable of encoding a functional Allantoicase, therefore we expressed the Xenopus and mouse Allantoicases (MAlc and XAlc, respectively) in Escherichia coli and characterized the recombinant enzymes. The two recombinant Allantoicases show a similar temperature and pH stability but, although XAlc and MAlc share a 54% amino acid identity, they differ in sensitivity to bivalent cations, in substrate affinity and in the level of expression in tissues (as revealed by means of Western blot analysis). We propose that the loss of Allantoicase activity in mouse is due to a low substrate affinity and to a reduced expression level of the enzyme.

  • molecular cloning of mouse Allantoicase cdna
    Biochimica et Biophysica Acta, 2001
    Co-Authors: Davide Vigetti, Claudio Monetti, Giovanni Bernardini
    Abstract:

    Abstract The uric acid degradation pathway is progressively lost during vertebrate evolution. In mammals, the end product of this catabolic pathway is allantoin and, therefore, no Allantoicase should be present in mouse tissues. Surprisingly, we have found an expressed sequence tag (EST) from mouse testis with high similarity to Allantoicase. To characterize this transcript, we have completely sequenced the corresponding EST clone insert and found a 1495 bp long cDNA coding for a 414 amino acid long protein. Identities of mouse versus microorganism Allantoicases range from 25 to 30%. Identity reaches 54% when compared to Xenopus Allantoicase. Among the tested tissues, only testis possesses the Allantoicase transcript. Although no deleterious mutations were found in the coding region, no Allantoicase activity could be detected in mouse testis.

  • human Allantoicase gene cdna cloning genomic organization and chromosome localization
    Gene, 2000
    Co-Authors: Davide Vigetti, Claudio Monetti, Roberto Taramelli, Francesco Acquati, Giovanni Bernardini
    Abstract:

    Abstract Uric-acid-degrading enzymes (uricase, allantoinase, Allantoicase, ureidoglycolate lyase and urease) were lost during vertebrate evolution and the causes for this loss are still unclear. We have recently cloned the first vertebrate Allantoicase cDNA from the amphibian Xenopus laevis. Surprisingly, we have found some mammalian expressed sequence tags (ESTs) that show high similarity with Xenopus Allantoicase cDNA. From a human fetal spleen cDNA library and adult kidney EST clone, we have obtained a 1790 nucleotide long cDNA. The 3′ end of this sequence reveals a substantial high identity with the corresponding portion of Xenopus Allantoicase cDNA. In contrast, at the 5′ end the human sequence diverges from that of Xenopus; since no continuous open reading frame can be found in this region, the hypothetical human protein appears truncated at its N-terminus. We proposed that such a transcript could be due to an incorrect splicing mechanism that introduces an intron portion at the 5′ end of human cDNA. Allantoicase cDNA is expressed in adult testis, prostate, kidney and fetal spleen. By comparison with available genomic sequences deposited in database, we have determined that the human Allantoicase gene consists of five exons and spans 8 kb. We have also mapped the gene in chromosome 2.

Davide Vigetti - One of the best experts on this subject based on the ideXlab platform.

  • Selective Pressure on the Allantoicase Gene During Vertebrate Evolution
    Journal of Molecular Evolution, 2003
    Co-Authors: Davide Vigetti, Claudio Monetti, Mariangela Prati, Giovanni Bernardini, Giorgio Binelli, Rosalba Gornati
    Abstract:

    During vertebrate evolution, the uric acid degradation pathway has been modified and several enzymes have been lost. Consequently, the end product of purine catabolism varies from species to species. In the past few years, we have focused our attention on vertebrate Allantoicase (an uricolytic pathway enzyme), whose activity is present in certain fish and amphibians only, but whose mRNA we detected also in mammals. As Allantoicase activity disappeared in amniotes, we wonder why these sequences not only remain present in the mammalian genome, but are still transcribed. To elucidate this issue, we have cloned and analyzed comparable cDNA sequences of different organisms from ascidians to mammals. The analysis of the nonsynonymous–synonymous substitution rate that we performed on the coding region comprising exons 3 to 8 by means of maximum likelihood suggested that a certain amount of purifying selection is acting on the Allantoicase sequences. Some implications of the preservation of an apparently unnecessary gene in higher vertebrates are discussed.

  • genomic organization and chromosome localization of the murine and human Allantoicase gene
    Gene, 2002
    Co-Authors: Davide Vigetti, Claudio Monetti, Mariangela Prati, Rosalba Gornati, Giovanni Bernardini
    Abstract:

    Abstract Allantoicase is one of the enzymes involved in uricolysis. The enzymes of this catabolic pathway (i.e. allantoinase, Allantoicase, ureidoglycolate lyase and urease) were lost during vertebrate evolution and the causes for this loss are still unclear. In mammals, as well as in birds and reptiles, the activity of Allantoicase is absent; notwithstanding, we recently cloned human and mouse cDNA sequences with high similarity with previously characterized Allantoicases. In the present paper, we report the genomic organization of the Allantoicase gene in mouse and in man. Both genes are constituted by 11 exons that appear to be very conserved; introns are more variable in length while maintain the same phase but for intron 4. We have also detected a second transcript of the human Allantoicase gene in which exon 1 is absent. Moreover, the mouse gene maps in chromosome 12 at 13.0 cM from the centromere.

  • Property comparison of recombinant amphibian and mammalian Allantoicases.
    FEBS Letters, 2002
    Co-Authors: Davide Vigetti, Claudio Monetti, Mariangela Prati, Giovanni Bernardini, Loredano Pollegioni, Rosalba Gornati
    Abstract:

    Allantoicase is an enzyme involved in uric acid degradation. Although it is commonly accepted that Allantoicase is lost in mammals, birds and reptiles, we have recently identified its transcripts in mice and humans. The mouse mRNA seems capable of encoding a functional Allantoicase, therefore we expressed the Xenopus and mouse Allantoicases (MAlc and XAlc, respectively) in Escherichia coli and characterized the recombinant enzymes. The two recombinant Allantoicases show a similar temperature and pH stability but, although XAlc and MAlc share a 54% amino acid identity, they differ in sensitivity to bivalent cations, in substrate affinity and in the level of expression in tissues (as revealed by means of Western blot analysis). We propose that the loss of Allantoicase activity in mouse is due to a low substrate affinity and to a reduced expression level of the enzyme.

  • The purine degradation pathway: possible role in paralytic shellfish toxin metabolism in the cyanobacterium Planktothrix sp. FP1.
    Environment International, 2001
    Co-Authors: F. Pomati, Davide Vigetti, Gianluca Manarolla, Olivia Rossi, Carlo Rossetti
    Abstract:

    Abstract The paralytic shellfish toxins (PSTs) are potent neurotoxic alkaloids and their major biological effect is due to the blockage of voltage-gated sodium channels in excitable cells. They have been recognised as an important health risk for humans, animals, and ecosystems worldwide. The metabolic pathways that lead to the production and the degradation of these toxic metabolites are still unknown. In this study, we investigated the possible link between PST accumulation and the activation of the metabolism that leads to purine degradation in the filamentous freshwater cyanobacterium Planktothrix sp. FP1. The purine catabolic pathway is related to the nitrogen microcycle in water environments, in which cyanobacteria use traces of purines and ureides as a nitrogen source for growth. Thus, the activity of Allantoicase, a key inducible enzyme of this metabolism, was used as tool for assaying the activation of the purine degradation pathway. The enzyme and the pathway were induced by allantoic acid, the direct substrate of Allantoicase, as well as by adenine and, to a lower degree, by urea, one of the main products of purine catabolism. Crude cell extract of Escherichia coli was also employed and showed the best induction of Allantoicase activity. In culture, Planktothrix sp. FP1 showed a differential accumulation of PST in consequence of the induction with different substrates. The cyanobacterial culture induced with allantoic acid accumulated 61.7% more toxins in comparison with the control. On the other hand, the cultures induced with adenine, urea, and the E. coli extract showed low PST accumulation, respectively, 1%, 38%, and 5% of the total toxins content detected in the noninduced culture. A degradation pathway for the PSTs can be hypothesised: as suggested for purine alkaloids in higher plants, saxitoxin (STX) and derivatives may also be converted into xanthine, urea, and further to CO 2 and NH 4 + or recycled in the primary metabolism through the purine degradation pathway.

  • molecular cloning of mouse Allantoicase cdna
    Biochimica et Biophysica Acta, 2001
    Co-Authors: Davide Vigetti, Claudio Monetti, Giovanni Bernardini
    Abstract:

    Abstract The uric acid degradation pathway is progressively lost during vertebrate evolution. In mammals, the end product of this catabolic pathway is allantoin and, therefore, no Allantoicase should be present in mouse tissues. Surprisingly, we have found an expressed sequence tag (EST) from mouse testis with high similarity to Allantoicase. To characterize this transcript, we have completely sequenced the corresponding EST clone insert and found a 1495 bp long cDNA coding for a 414 amino acid long protein. Identities of mouse versus microorganism Allantoicases range from 25 to 30%. Identity reaches 54% when compared to Xenopus Allantoicase. Among the tested tissues, only testis possesses the Allantoicase transcript. Although no deleterious mutations were found in the coding region, no Allantoicase activity could be detected in mouse testis.

Claudio Monetti - One of the best experts on this subject based on the ideXlab platform.

  • Selective Pressure on the Allantoicase Gene During Vertebrate Evolution
    Journal of Molecular Evolution, 2003
    Co-Authors: Davide Vigetti, Claudio Monetti, Mariangela Prati, Giovanni Bernardini, Giorgio Binelli, Rosalba Gornati
    Abstract:

    During vertebrate evolution, the uric acid degradation pathway has been modified and several enzymes have been lost. Consequently, the end product of purine catabolism varies from species to species. In the past few years, we have focused our attention on vertebrate Allantoicase (an uricolytic pathway enzyme), whose activity is present in certain fish and amphibians only, but whose mRNA we detected also in mammals. As Allantoicase activity disappeared in amniotes, we wonder why these sequences not only remain present in the mammalian genome, but are still transcribed. To elucidate this issue, we have cloned and analyzed comparable cDNA sequences of different organisms from ascidians to mammals. The analysis of the nonsynonymous–synonymous substitution rate that we performed on the coding region comprising exons 3 to 8 by means of maximum likelihood suggested that a certain amount of purifying selection is acting on the Allantoicase sequences. Some implications of the preservation of an apparently unnecessary gene in higher vertebrates are discussed.

  • genomic organization and chromosome localization of the murine and human Allantoicase gene
    Gene, 2002
    Co-Authors: Davide Vigetti, Claudio Monetti, Mariangela Prati, Rosalba Gornati, Giovanni Bernardini
    Abstract:

    Abstract Allantoicase is one of the enzymes involved in uricolysis. The enzymes of this catabolic pathway (i.e. allantoinase, Allantoicase, ureidoglycolate lyase and urease) were lost during vertebrate evolution and the causes for this loss are still unclear. In mammals, as well as in birds and reptiles, the activity of Allantoicase is absent; notwithstanding, we recently cloned human and mouse cDNA sequences with high similarity with previously characterized Allantoicases. In the present paper, we report the genomic organization of the Allantoicase gene in mouse and in man. Both genes are constituted by 11 exons that appear to be very conserved; introns are more variable in length while maintain the same phase but for intron 4. We have also detected a second transcript of the human Allantoicase gene in which exon 1 is absent. Moreover, the mouse gene maps in chromosome 12 at 13.0 cM from the centromere.

  • Property comparison of recombinant amphibian and mammalian Allantoicases.
    FEBS Letters, 2002
    Co-Authors: Davide Vigetti, Claudio Monetti, Mariangela Prati, Giovanni Bernardini, Loredano Pollegioni, Rosalba Gornati
    Abstract:

    Allantoicase is an enzyme involved in uric acid degradation. Although it is commonly accepted that Allantoicase is lost in mammals, birds and reptiles, we have recently identified its transcripts in mice and humans. The mouse mRNA seems capable of encoding a functional Allantoicase, therefore we expressed the Xenopus and mouse Allantoicases (MAlc and XAlc, respectively) in Escherichia coli and characterized the recombinant enzymes. The two recombinant Allantoicases show a similar temperature and pH stability but, although XAlc and MAlc share a 54% amino acid identity, they differ in sensitivity to bivalent cations, in substrate affinity and in the level of expression in tissues (as revealed by means of Western blot analysis). We propose that the loss of Allantoicase activity in mouse is due to a low substrate affinity and to a reduced expression level of the enzyme.

  • molecular cloning of mouse Allantoicase cdna
    Biochimica et Biophysica Acta, 2001
    Co-Authors: Davide Vigetti, Claudio Monetti, Giovanni Bernardini
    Abstract:

    Abstract The uric acid degradation pathway is progressively lost during vertebrate evolution. In mammals, the end product of this catabolic pathway is allantoin and, therefore, no Allantoicase should be present in mouse tissues. Surprisingly, we have found an expressed sequence tag (EST) from mouse testis with high similarity to Allantoicase. To characterize this transcript, we have completely sequenced the corresponding EST clone insert and found a 1495 bp long cDNA coding for a 414 amino acid long protein. Identities of mouse versus microorganism Allantoicases range from 25 to 30%. Identity reaches 54% when compared to Xenopus Allantoicase. Among the tested tissues, only testis possesses the Allantoicase transcript. Although no deleterious mutations were found in the coding region, no Allantoicase activity could be detected in mouse testis.

  • human Allantoicase gene cdna cloning genomic organization and chromosome localization
    Gene, 2000
    Co-Authors: Davide Vigetti, Claudio Monetti, Roberto Taramelli, Francesco Acquati, Giovanni Bernardini
    Abstract:

    Abstract Uric-acid-degrading enzymes (uricase, allantoinase, Allantoicase, ureidoglycolate lyase and urease) were lost during vertebrate evolution and the causes for this loss are still unclear. We have recently cloned the first vertebrate Allantoicase cDNA from the amphibian Xenopus laevis. Surprisingly, we have found some mammalian expressed sequence tags (ESTs) that show high similarity with Xenopus Allantoicase cDNA. From a human fetal spleen cDNA library and adult kidney EST clone, we have obtained a 1790 nucleotide long cDNA. The 3′ end of this sequence reveals a substantial high identity with the corresponding portion of Xenopus Allantoicase cDNA. In contrast, at the 5′ end the human sequence diverges from that of Xenopus; since no continuous open reading frame can be found in this region, the hypothetical human protein appears truncated at its N-terminus. We proposed that such a transcript could be due to an incorrect splicing mechanism that introduces an intron portion at the 5′ end of human cDNA. Allantoicase cDNA is expressed in adult testis, prostate, kidney and fetal spleen. By comparison with available genomic sequences deposited in database, we have determined that the human Allantoicase gene consists of five exons and spans 8 kb. We have also mapped the gene in chromosome 2.

Manuel Pineda - One of the best experts on this subject based on the ideXlab platform.

  • Molecular and functional characterization of allantoate amidohydrolase from Phaseolus vulgaris
    Physiologia Plantarum, 2014
    Co-Authors: Juan Luis Díaz-leal, Manuel Pineda, Fernando Torralbo, Francisco Antonio Quiles, Josefa M. Alamillo
    Abstract:

    Allantoate degradation is an essential step for recycling purine-ring nitrogen in all plants, but especially in tropical legumes where the ureides allantoate and allantoin are the main compounds used to store and transport the nitrogen fixed in nodules. Two enzymes, allantoate amidohydrolase (AAH) and allantoate amidinohydrolase (Allantoicase), could catalyze allantoate breakdown, although only AAH-coding sequences have been found in plant genomes, whereas Allantoicase-related sequences are restricted to animals and some microorganisms. A cDNA for AAH was cloned from Phaseolus vulgaris leaves. PvAAH is a single-copy gene encoding a polypeptide of 483 amino acids that conserves all putative AAH active-site domains. Expression and purification of the cDNA in Nicotiana benthamiana showed that the cloned sequence is a true AAH protein that yields ureidoglycine and ammonia, with a Km of 0.46 mM for allantoate. Optimized in vitro assay, quantitative RT-PCR and antibodies raised to the PvAAH protein were used to study AAH under physiological conditions. PvAAH is ubiquitously expressed in common bean tissues, although the highest transcript levels were found in leaves. In accordance with the mRNA expression levels, the highest PvAAH activity and allantoate concentration also occurred in the leaves. Comparison of transcript levels, protein amounts and enzymatic activity in plants grown with different nitrogen sources and upon drought stress conditions showed that PvAAH is regulated at posttranscriptional level. Moreover, RNAi silencing of AAH expression increases allantoate levels in the transgenic hairy roots, indicating that AAH should be the main enzyme involved in allantoate degradation in common bean.

  • Manganese is essential for activity of allantoate amidinohydrolase from Chlamydomonas reinhardtii
    Plant Science, 2003
    Co-Authors: Pedro Piedras, Manuel Pineda
    Abstract:

    Abstract Allantoicase (allantoate amidinohydrolase, EC 3.5.3.4) from Chlamydomonas reinhardtii catalyses the degradation of allantoate to (−)ureidoglycolate and of (+)ureidoglycolate to glyoxylate, in both cases with urea as the other product. Allantoicase activity purified in buffers without any cations increased after manganese addition to the assay mixture reaching a saturation maximun with 0.25 mM. The Allantoicase activity was strongly inhibited after EDTA treatment. The activity of the EDTA-treated enzyme was restored after incubation with Mn 2+ , whereas the incubation with Cu 2+ resulted in a fully inactivated preparation. Others cations tested (Ca 2+ , Fe 2+ , Mg 2+ , Zn 2+ , Co 2+ ) had no effect on activity. The same results were obtained independently of the substrate used in all the experiments performed. A manganese-depleted Allantoicase, obtained by purification with manganese free buffers, bound radioactive manganese in vitro . This binding is strong since enzyme retained the radioactive cation after gel chromatography and SDS-PAGE. Results obtained are the first clear demonstration of manganese binding in vitro to any allantoate-degrading enzyme from a photosynthetic organism and that manganese is essential for Allantoicase activity.

  • allantoate amidinohydrolase Allantoicase from chlamydomonas reinhardtii its purification and catalytic and molecular characterization
    Archives of Biochemistry and Biophysics, 2000
    Co-Authors: Pedro Piedras, Miguel Aguilar, Alfonso Munoz, Manuel Pineda
    Abstract:

    Abstract An allantoate-degrading enzyme has been purified to electrophoretic homogeneity for the first time from a photosynthetic organism, the unicellular green algae Chlamydomonas reinhardtii. The purification procedure included a differential protein extraction followed by conventional steps such as ammonium sulfate fractionation, gel filtration, anion exchange chromatography, and preparative electrophoresis. Under the routine assay conditions (7 mM allantoate), specific activity for the purified enzyme was 185 U/mg, which rose to 225 U/mg under kinetic considerations (saturating substrate). Therefore, a turnover number of 4.5 × 104 min−1 can be deduced for the 200-kDa protein. The enzyme is a true Allantoicase (EC 3.5.3.4) that catalyzes the degradation of allantoate to (−)ureidoglycolate and (+)ureidoglycolate to glyoxylate. The enzyme exhibited hyperbolic kinetic for allantoate and ureidoglycolate with Km values of 2 and 0.7 mM, respectively. Vmax of the reaction with allantoate as substrate was nine times higher than that with ureidoglycolate. The native enzyme has a molecular weight of 200 kDa and consists of six identical or similar-sized subunits of 34 kDa each, organized in two trimers of 100 kDa. Each subunit has five cysteine residues, four of which are involved in disulfide bonds, with a total of 12 disulfide bonds in the 200-kDa protein. Allantoate inhibits competitively the reaction with ureidoglycolate as substrate. In addition, buffers and group-specific reagents affect the activity in the same manner irrespective of the substrate used. Those results suggest that both substrates use the same active site. The effect of group-specific reagents suggest that the amino acids histidine, tyrosine, and cysteine are essentials for the Allantoicase activity with both substrates.

  • UPTAKE AND METABOLISM OF ALLANTOIN AND ALLANTOATE BY CELLS OF CHLAMYDOMONAS REINHARDTII (CHLOROPHYCEAE)
    European Journal of Phycology, 1998
    Co-Authors: Pedro Piedras, Miguel Aguilar, Manuel Pineda
    Abstract:

    The green alga Chlamydomonas reinhardtii can use the ureides allantoin and allantoate as sole nitrogen sources. Once the uptake systems for allantoin and allantoate were induced, the uptake and growth rates were identical for the two ureides. However, the enzymatic activities involved in the degradation of the two ureides (allantoinase and Allantoicase) were regulated differently. Allantoinase seems to be constitutive, since it was detected in all the nitrogen sources studied, while Allantoicase behaved as an inducible enzyme, since it was present only in cells cultured in ureides or any metabolic precursor of these compounds. Neither allantoinase nor Allantoicase activities were repressed by ammonium in the presence of ureides. Allantoicase activity was not induced under nitrogen starvation conditions, while it was induced in cells that had been cultured with allantoin or allantoate in the dark. Allantoin uptake showed a pattern similar to that of allantoate under all nutritional and environmental condit...

  • solubilization and extraction of allantoinase and Allantoicase from the green alga chlamydomonas reinhardtii
    Phytochemical Analysis, 1995
    Co-Authors: Pedro Piedras, Jacobo Cardenas, Manuel Pineda
    Abstract:

    Several physical and chemical methods for the solubilization and extraction of allantoinase and Allantoicase from the unicellular green alga Chlamydomonas reinhardtii have been tested and compared. Of all the methods employed (i.e. freezing-thawing, extraction at high ionic strength, disruption with the French press, sonication, and detergent solubilization) sonication gave the best results for extraction of both substances, while sodium deoxycholate extracted allantoinase optimally but not Allantoicase. Allantoinase and Allantoicase were characterized as two separate enzyme proteins differently associated with membranes or located in different cellular compartments. It is also demonstrated that allantoate is enzymatically degraded in vitro by a cell-free algal extract and that Allantoicase is the enzyme responsible for this catabolism.

Pedro Piedras - One of the best experts on this subject based on the ideXlab platform.

  • Manganese is essential for activity of allantoate amidinohydrolase from Chlamydomonas reinhardtii
    Plant Science, 2003
    Co-Authors: Pedro Piedras, Manuel Pineda
    Abstract:

    Abstract Allantoicase (allantoate amidinohydrolase, EC 3.5.3.4) from Chlamydomonas reinhardtii catalyses the degradation of allantoate to (−)ureidoglycolate and of (+)ureidoglycolate to glyoxylate, in both cases with urea as the other product. Allantoicase activity purified in buffers without any cations increased after manganese addition to the assay mixture reaching a saturation maximun with 0.25 mM. The Allantoicase activity was strongly inhibited after EDTA treatment. The activity of the EDTA-treated enzyme was restored after incubation with Mn 2+ , whereas the incubation with Cu 2+ resulted in a fully inactivated preparation. Others cations tested (Ca 2+ , Fe 2+ , Mg 2+ , Zn 2+ , Co 2+ ) had no effect on activity. The same results were obtained independently of the substrate used in all the experiments performed. A manganese-depleted Allantoicase, obtained by purification with manganese free buffers, bound radioactive manganese in vitro . This binding is strong since enzyme retained the radioactive cation after gel chromatography and SDS-PAGE. Results obtained are the first clear demonstration of manganese binding in vitro to any allantoate-degrading enzyme from a photosynthetic organism and that manganese is essential for Allantoicase activity.

  • allantoate amidinohydrolase Allantoicase from chlamydomonas reinhardtii its purification and catalytic and molecular characterization
    Archives of Biochemistry and Biophysics, 2000
    Co-Authors: Pedro Piedras, Miguel Aguilar, Alfonso Munoz, Manuel Pineda
    Abstract:

    Abstract An allantoate-degrading enzyme has been purified to electrophoretic homogeneity for the first time from a photosynthetic organism, the unicellular green algae Chlamydomonas reinhardtii. The purification procedure included a differential protein extraction followed by conventional steps such as ammonium sulfate fractionation, gel filtration, anion exchange chromatography, and preparative electrophoresis. Under the routine assay conditions (7 mM allantoate), specific activity for the purified enzyme was 185 U/mg, which rose to 225 U/mg under kinetic considerations (saturating substrate). Therefore, a turnover number of 4.5 × 104 min−1 can be deduced for the 200-kDa protein. The enzyme is a true Allantoicase (EC 3.5.3.4) that catalyzes the degradation of allantoate to (−)ureidoglycolate and (+)ureidoglycolate to glyoxylate. The enzyme exhibited hyperbolic kinetic for allantoate and ureidoglycolate with Km values of 2 and 0.7 mM, respectively. Vmax of the reaction with allantoate as substrate was nine times higher than that with ureidoglycolate. The native enzyme has a molecular weight of 200 kDa and consists of six identical or similar-sized subunits of 34 kDa each, organized in two trimers of 100 kDa. Each subunit has five cysteine residues, four of which are involved in disulfide bonds, with a total of 12 disulfide bonds in the 200-kDa protein. Allantoate inhibits competitively the reaction with ureidoglycolate as substrate. In addition, buffers and group-specific reagents affect the activity in the same manner irrespective of the substrate used. Those results suggest that both substrates use the same active site. The effect of group-specific reagents suggest that the amino acids histidine, tyrosine, and cysteine are essentials for the Allantoicase activity with both substrates.

  • UPTAKE AND METABOLISM OF ALLANTOIN AND ALLANTOATE BY CELLS OF CHLAMYDOMONAS REINHARDTII (CHLOROPHYCEAE)
    European Journal of Phycology, 1998
    Co-Authors: Pedro Piedras, Miguel Aguilar, Manuel Pineda
    Abstract:

    The green alga Chlamydomonas reinhardtii can use the ureides allantoin and allantoate as sole nitrogen sources. Once the uptake systems for allantoin and allantoate were induced, the uptake and growth rates were identical for the two ureides. However, the enzymatic activities involved in the degradation of the two ureides (allantoinase and Allantoicase) were regulated differently. Allantoinase seems to be constitutive, since it was detected in all the nitrogen sources studied, while Allantoicase behaved as an inducible enzyme, since it was present only in cells cultured in ureides or any metabolic precursor of these compounds. Neither allantoinase nor Allantoicase activities were repressed by ammonium in the presence of ureides. Allantoicase activity was not induced under nitrogen starvation conditions, while it was induced in cells that had been cultured with allantoin or allantoate in the dark. Allantoin uptake showed a pattern similar to that of allantoate under all nutritional and environmental condit...

  • solubilization and extraction of allantoinase and Allantoicase from the green alga chlamydomonas reinhardtii
    Phytochemical Analysis, 1995
    Co-Authors: Pedro Piedras, Jacobo Cardenas, Manuel Pineda
    Abstract:

    Several physical and chemical methods for the solubilization and extraction of allantoinase and Allantoicase from the unicellular green alga Chlamydomonas reinhardtii have been tested and compared. Of all the methods employed (i.e. freezing-thawing, extraction at high ionic strength, disruption with the French press, sonication, and detergent solubilization) sonication gave the best results for extraction of both substances, while sodium deoxycholate extracted allantoinase optimally but not Allantoicase. Allantoinase and Allantoicase were characterized as two separate enzyme proteins differently associated with membranes or located in different cellular compartments. It is also demonstrated that allantoate is enzymatically degraded in vitro by a cell-free algal extract and that Allantoicase is the enzyme responsible for this catabolism.