Molecular Identification of Carnosine Synthase as ATP-grasp Domain-containing Protein 1 (ATPGD1)

Drożak, Jakub; Veiga‐da‐Cunha, Maria; Vertommen, Didier; Stroobant, Vincent; Schaftingen, Emile Van

doi:10.1074/jbc.m109.095505

Cited by 175 publications

(165 citation statements)

References 37 publications

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“…At the indicated times, an aliquot (200 l) was taken; 100 l of the reaction mixture were loaded onto a 1-ml Dowex AG1-X8 (Cl Ϫ form) to assay radiochemically glutamate consumption and NAAG formation (see "Material and Methods"), and 100 l were used to measure nucleotides by high pressure liquid chromatography as described in Ref. 34. Values are means of three measurements using two and three independent preparations of RIMKLA and RIMKLB, respectively.…”

Section: Resultsmentioning

confidence: 99%

Molecular Identification of N-Acetylaspartylglutamate Synthase and β-Citrylglutamate Synthase

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Stroobant

Lamosa

et al. 2010

Journal of Biological Chemistry

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The purpose of the present work was to determine the identity of the enzymes that synthesize N-acetylaspartylglutamate (NAAG), the most abundant dipeptide present in vertebrate central nervous system (CNS), and ␤-citrylglutamate, a structural analogue of NAAG present in testis and immature brain. Previous evidence suggests that NAAG is not synthesized on ribosomes but presumably is synthesized by a ligase. As attempts to detect this ligase in brain extracts failed, we searched the mammalian genomes for putative enzymes that could catalyze this type of reaction. Mammalian genomes were found to encode two putative ligases homologous to Escherichia coli RIMK, which ligates glutamates to the C terminus of ribosomal protein S6. One of them, named RIMKLA, is almost exclusively expressed in the CNS, whereas RIMKLB, which shares 65% sequence identity with RIMKLA, is expressed in CNS and testis. Both proteins were expressed in bacteria or HEK293T cells and purified. RIMKLA catalyzed the ATP-dependent synthesis of N-acetylaspartylglutamate from N-acetylaspartate and L-glutamate. RIMKLB catalyzed this reaction as well as the synthesis of ␤-citrylglutamate. The nature of the reaction products was confirmed by mass spectrometry and NMR. RIMKLA was shown to produce stoichiometric amounts of NAAG and ADP, in agreement with its belonging to the ATP-grasp family of ligases. The molecular identification of these two enzymes will facilitate progress in the understanding of the function of NAAG and ␤-citrylglutamate. N-Acetylaspartylglutamate (NAAG),3 the most abundant dipeptide in the CNS, is present in brain and in the spinal cord, most particularly in neurons of the anterior horn (1). NAAG can be released from neurons upon calcium depolarization (reviewed in Ref. 1) and is a substrate for two glial peptidases, glutamate carboxypeptidase-II (2) and, to a lower extent, glutamate carboxypeptidase-III (3), which are anchored to the plasma membrane with their catalytic site oriented toward the outside of the cell. NAAG long has been thought to be a neurotransmitter able to bind to the metabotropic glutamate receptors mGluR3 (4). Two recent reports suggest, however, that the effects of NAAG as a neurotransmitter are due to a 0.5% glutamate contamination present in commercial NAAG (5, 6). The function of NAAG is, therefore, presently unknown.␤-Citrylglutamate (BCG), which is structurally close to NAAG, is less well characterized. It was first identified in newborn rat brain, where its concentration reaches 0.5-1 mol/g at birth and then decreases with age (7). BCG also was detected in kidneys and heart and to a much lower extent in intestine, spinal cord, and lung of newborn rats. The content of BCG in all organs decreased with age to the noticeable exception of testes, where its concentration increases during sexual maturation and remains constant in adulthood (8,9). BCG is present not only in the testes of mammals but also in those of amphibians and fishes. There is evidence in germinal cells for a role in spermatogenesis (9). BCG rec...

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Section: Resultsmentioning

confidence: 99%

Molecular Identification of N-Acetylaspartylglutamate Synthase and β-Citrylglutamate Synthase

Collard

Stroobant

Lamosa

et al. 2010

Journal of Biological Chemistry

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“…In addition, other methods, such as stimulation of carnosine synthesis and/or suppression of its degradation by a method of molecular biology, may be effective. In particular, carnosine synthase was recently identified in chickens (Drozak et al, 2010) and this may make molecular studies easier to perform. Further studies are needed to determine how to increase dipeptides in broilers.…”

Section: Discussionmentioning

confidence: 99%

“…Not only that, carnosine seems to contribute to the nutritional quality of meat because dietary carnosine has a hypoglycemic effect (Yamano et al, 2001), hypotensive action (Niijima et al, 2002), antidepressant-like activity (Tomonaga et al, 2008), and anti-aging effects (Gallant et al, 2000). Carnosine is synthesized from β-alanine (β-Ala) and histidine (His) by carnosine synthase (Drozak et al, 2010), and anserine is mainly formed from carnosine by a methylation reaction (Boldyrev and Severin, 1990), while Horinishi et al (1978) reported that a purified enzyme from chickens could methylate His to generate anserine by direct synthesis. On the other hand, carnosinase, which hydrolyzed carnosine or anserine to its constituent amino acids, was confirmed to be present in rats and humans.…”

Section: Introductionmentioning

confidence: 99%

β-Alanine Enhances Brain and Muscle Carnosine Levels in Broiler Chicks

2012

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Carnosine and its methylated derivative anserine are dipeptides present in high levels in chicken muscles. They are antioxidants and putative neurotransmitters. If administration of β-alanine, one of the constituents of carnosine, could increase levels of these dipeptides in the brain and muscles, it may improve brain function and increase commercial values of the chicken meat. In the present study, we investigated whether orally administered β-alanine could increase these dipeptide levels in the brain, Musculus pectoralis superficialis, and plasma in broiler chicks. Broilers (2 days old) were given oral doses of β-alanine (0.176, 0.88, 4.4 and 22 mmol/kg) twice a day for 5 days. Carnosine levels were dose-dependently increased in the brain, Musculus pectoralis superficialis, and plasma while no influence of anserine was detected. These results suggest that supplemental β-alanine could be effective in increasing carnosine levels in the brain and muscle of broiler chicks.

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“…For example, carnosine synthetase uses both, beta-alanine and GABA to synthesize carnosine and homocarnosine, respectively (44). Moreover, using purified carnosine synthetase, Drozak et al (35) could show that the enzyme, which also belongs to the ATP-grasp domain family, accepts different amino acids or amines as the second substrate. The physiological relevance of the additional NAAGS products, if produced at all in vivo, remains to be determined.…”

Section: Discussionmentioning

confidence: 99%

Molecular Characterization of N-Acetylaspartylglutamate Synthetase

Becker

Lodder

Gieselmann

et al. 2010

Journal of Biological Chemistry

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The dipeptide N-acetylaspartyl-glutamate (NAAG) is an abundant neuropeptide in the mammalian brain. Despite this fact, its physiological role is poorly understood. NAAG is synthesized by a NAAG synthetase catalyzing the ATP-dependent condensation of N-acetylaspartate and glutamate. In vitro NAAG synthetase activity has not been described, and the enzyme has not been purified. Using a bioinformatics approach we identified a putative dipeptide synthetase specifically expressed in the nervous system. Expression of the gene, which we named NAAGS (for NAAG synthetase) was sufficient to induce NAAG synthesis in primary astrocytes or CHO-K1 and HEK-293T cells when they coexpressed the NAA transporter NaDC3. Furthermore, coexpression of NAAGS and the recently identified N-acetylaspartate (NAA) synthase, Nat8l, in CHO-K1 or HEK-293T cells was sufficient to enable these cells to synthesize NAAG. Identity of the reaction product of NAAGS was confirmed by HPLC and electrospray ionization tandem mass spectrometry (ESI-MS). High expression levels of NAAGS were restricted to the brain, spinal cord, and testis. Taken together our results strongly suggest that the identified gene encodes a NAAG synthetase. Its identification will enable further studies to examine the role of this abundant neuropeptide in the vertebrate nervous system. N-acetylaspartylglutamate (NAAG)3 is an abundant neuropeptide in the central nervous system of mammals, present in high micromolar to low millimolar concentrations (for review see Refs. 1-3). It was first identified in rabbit and horse brain tissue by Curatolo et al. (4) and in bovine brain by Miyamoto et al. (5). NAAG is present in all regions of the central nervous system of mammals, though the highest concentrations are found in the spinal cord and stem brain (6). Despite its abundance throughout the mammalian nervous system, its physiological role is not fully understood.Because NAAG synthesis in sensory ganglia was not blocked by translation inhibitors, it was assumed that NAAG is not derived from a post-translational process, but is synthesized by a neuron specific NAAG synthetase, catalyzing the condensation of N-acetylaspartate (NAA) and glutamate (Ref. 7; see Fig. 1). After its calcium-dependent release from synaptic terminals, NAAG can be degraded by glutamate carboxypeptidase II (N-acetylated-␣-linked-acidic dipeptidase; GCP-II) or GCP-III, membrane-bound enzymes mainly expressed by astrocytes (for review see Ref. 8). The released NAA is then taken up by glial cells via the high-affinity, sodium-dependent dicarboxylate (NaDC3) transporter (9). In oligodendrocytes, NAA can then be hydrolyzed to aspartate and acetate by aspartoacylase II (8). The released acetate may be used for lipid synthesis by myelinating oligodendrocytes (10,11). To what extent NAA is taken up by astrocytes in vivo and its metabolic fate in these cells is not clear. Deficiency in aspartoacylase II leads to accumulation of NAA, but also NAAG (11), and causes a rare leukodystrophy, Canavan disease (12, 13).Studies on...

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Molecular Identification of Carnosine Synthase as ATP-grasp Domain-containing Protein 1 (ATPGD1)

Cited by 175 publications

References 37 publications

Molecular Identification of N-Acetylaspartylglutamate Synthase and β-Citrylglutamate Synthase

Molecular Identification of N-Acetylaspartylglutamate Synthase and β-Citrylglutamate Synthase

β-Alanine Enhances Brain and Muscle Carnosine Levels in Broiler Chicks

Molecular Characterization of N-Acetylaspartylglutamate Synthetase

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