Partial purification and properties of <scp>l</scp>-asparagine synthetase from mouse pancreas

Milman, Harry A.; Cooney, David A.

doi:10.1042/bj1810051

Cited by 26 publications

(26 citation statements)

References 18 publications

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“…ganisms that catalyze the transfer of the ␥ amino residue of glutamine to the carboxyl residue of aspartate (19). Glutamine-dependent asparagine synthetase belongs to the purFtype amidotransferase (25,29).…”

Section: Discussionmentioning

confidence: 99%

A Mutation in the Corynebacterium glutamicum ltsA Gene Causes Susceptibility to Lysozyme, Temperature-Sensitive Growth, and l -Glutamate Production

2000

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The Corynebacterium glutamicum mutant KY9714, originally isolated as a lysozyme-sensitive mutant, does not grow at 37°C. Complementation tests and DNA sequencing analysis revealed that a mutation in a single gene of 1,920 bp, ltsA (lysozyme and temperature sensitive), was responsible for its lysozyme sensitivity and temperature sensitivity. The ltsA gene encodes a protein homologous to the glutamine-dependent asparagine synthetases of various organisms, but it could not rescue the asparagine auxotrophy of an Escherichia coli asnA asnB double mutant. Replacement of the N-terminal Cys residue (which is conserved in glutamine-dependent amidotransferases and is essential for enzyme activity) by an Ala residue resulted in the loss of complementation in C. glutamicum. The mutant ltsA gene has an amber mutation, and the disruption of the ltsA gene caused lysozyme and temperature sensitivity similar to that in the KY9714 mutant. L-Glutamate production was induced by elevating growth temperature in the disruptant. These results indicate that the ltsA gene encodes a novel glutamine-dependent amidotransferase that is involved in the mechanisms of formation of rigid cell wall structure and in the L-glutamate production of C. glutamicum.Coryneform bacteria are rod-shaped, nonsporulating, grampositive bacteria that are widely distributed in nature. The nonpathogenic species Corynebacterium glutamicum, Brevibacterium lactofermentum, and Brevibacterium flavum are used for fermentative production of amino acids such as L-glutamic acid (16), L-lysine (21), and L-threonine (26). Recently, B. lactofermentum and B. flavum were reclassified as C. glutamicum (18).C. glutamicum was originally isolated as a glutamate-producing bacterium (16,30). Induction of glutamate excretion by C. glutamicum requires biotin limitation (27) or treatment with penicillin (22) or a fatty acid ester surfactant (28). Since these treatments correlated with alterations in cell surface structure, it had been thought that glutamate leaks passively through a membrane. Recently, several findings that do not agree with the leakage model have been reported (9,12,15). However, the mechanism of the glutamate production and excretion of C. glutamicum is still unknown.Coryneform bacteria have thick cell walls consisting of two layers. The inner layer, which consists mainly of peptidoglycan, invaginates to form the septum. The outer layer, which consists mainly of mycolic acid, breaks after cell division, resulting in postfission movement and the cell arrangements characteristic of these bacteria. C. glutamicum shows high tolerance to lysis by lytic enzymes, such as egg white lysozyme, that catalyze hydrolysis of the ␤-1,4 glycoside bond between the N-acetylglucosamine and N-acetylmuramic acid of peptidoglycan. This is probably because the outer layer, mainly consisting of mycolic acid, gives enough resistance against cell lysis to this bacterium and/or functions as a permeability barrier.As the first step in analyzing the cell wall structure of coryneform bacteria and...

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Section: Discussionmentioning

confidence: 99%

A Mutation in the Corynebacterium glutamicum ltsA Gene Causes Susceptibility to Lysozyme, Temperature-Sensitive Growth, and l -Glutamate Production

2000

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“…It is also an effective inhibitor, along with other glutamine analogues, of glutamine transport (Jayakumar et al 1987;Low et al 1991;Molina et al 1995) and in the regulation of nitrate reductase (McCarty and Bremner 1992). DON substitutes for glutamine in X-ray crystallographic studies of glutaminase (Thangavelu et al 2014) and is an effective inhibitor of g-glutamyl transferase, transamidase, and asparagine synthetase activities (Ghosh et al 1960;Milman and Cooney 1979;Payne and Payne 1984). AZA blocks transfer of nitrogen from glutamine to purine precursors (Aaronson 1959).…”

Section: Identification Of a Gln3 Sequence With The Characteristics Omentioning

confidence: 99%

Nuclear Gln3 Import Is Regulated by Nitrogen Catabolite Repression Whereas Export Is Specifically Regulated by Glutamine

et al. 2015

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Gln3, a transcription activator mediating nitrogen-responsive gene expression in Saccharomyces cerevisiae, is sequestered in the cytoplasm, thereby minimizing nitrogen catabolite repression (NCR)-sensitive transcription when cells are grown in nitrogen-rich environments. In the face of adverse nitrogen supplies, Gln3 relocates to the nucleus and activates transcription of the NCR-sensitive regulon whose products transport and degrade a variety of poorly used nitrogen sources, thus expanding the cell's nitrogen-acquisition capability. Rapamycin also elicits nuclear Gln3 localization, implicating Target-of-rapamycin Complex 1 (TorC1) in nitrogen-responsive Gln3 regulation. However, we long ago established that TorC1 was not the sole regulatory system through which nitrogen-responsive regulation is achieved. Here we demonstrate two different ways in which intracellular Gln3 localization is regulated. Nuclear Gln3 entry is regulated by the cell's overall nitrogen supply, i.e., by NCR, as long accepted. However, once within the nucleus, Gln3 can follow one of two courses depending on the glutamine levels themselves or a metabolite directly related to glutamine. When glutamine levels are high, e.g., glutamine or ammonia as the sole nitrogen source or addition of glutamine analogues, Gln3 can exit from the nucleus without binding to DNA. In contrast, when glutamine levels are lowered, e.g., adding additional nitrogen sources to glutamine-grown cells or providing repressive nonglutamine nitrogen sources, Gln3 export does not occur in the absence of DNA binding. We also demonstrate that Gln3 residues 64-73 are required for nuclear Gln3 export.

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“…A sparagine, one of the 21 cotranslationally inserted amino acids that make up proteins, is known to be synthesized from aspartate in an ATP-dependent amidation reaction (1). Two mechanistically distinct asparagine synthetases are known (2)(3)(4).…”

mentioning

confidence: 99%

Transfer RNA-dependent amino acid biosynthesis: An essential route to asparagine formation

Min

Pelaschier

Graham

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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Biochemical experiments and genomic sequence analysis showed that Deinococcus radiodurans and Thermus thermophilus do not possess asparagine synthetase (encoded by asnA or asnB), the enzyme forming asparagine from aspartate. Instead these organisms derive asparagine from asparaginyl-tRNA, which is made from aspartate in the tRNA-dependent transamidation pathway [ A sparagine, one of the 21 cotranslationally inserted amino acids that make up proteins, is known to be synthesized from aspartate in an ATP-dependent amidation reaction (1). Two mechanistically distinct asparagine synthetases are known (2-4). The one encoded by asnA utilizes ammonia as amide donor, whereas the asnB-derived protein works with glutamine. These enzymes are present in organisms of all domains, the major one being asparagine synthetase B, which is encoded in different organisms by a small number of related genes. Both enzymes are well studied biochemically (5), and their crystal structures are known (6, 7). Until recently they were assumed to be the sole biosynthetic route to asparagine. However, Thermus thermophilus and Deinococcus radiodurans lack these enzymes; instead they employ a tRNA-dependent transamidation mechanism for conversion of aspartate to asparagine (8,9).Two routes of Asn-tRNA synthesis exist in D. radiodurans (Fig. 1). Similar to many bacteria, Deinococcus contains a tRNA-dependent two-step pathway of Asn-tRNA formation. In the first step a nondiscriminating aspartyl-tRNA synthetase (AspRS)2 generates the misacylated Asp-tRNA Asn species, which then is amidated to the correctly charged Asn-tRNA Asn by the heterotrimeric Asp-tRNA Asn amidotransferase (Asp-AdT; encoded by the gatCAB genes) with glutamine serving as the amide donor (9). In addition, the organism also contains asparaginyl-tRNA synthetase (AsnRS; ref. 10), which is active and produces Asn-tRNA in the canonical aminoacylation reaction (9). The close Deinococcus relative T. thermophilus has similar enzymes and presumably uses the same asparagine biosynthetic routes (8,11,12). It was suggested earlier (8, 9) that the role of Asp-AdT in D. radiodurans and T. thermophilus is to synthesize the cell's entire supply of asparagine, because no asnA or asnB orthologs are present in the genome (10), and because biochemical analysis of crude cell extracts did not reveal the presence of any tRNA-independent asparagine synthetase activity (8, 9). Here we present data from D. radiodurans that prove this role to be correct and propose that tRNA-dependent asparagine synthesis occurs in many bacteria as the sole synthetic route to this essential amino acid. ͞10 g/ml methionine͞25 g/ml histidine͞30 g/ml cysteine͞1 g/ml nicotinic acid͞2 mg/ml fructose. Where necessary, the medium was supplemented with 10 g/ml kanamycin͞2.5 g/ml tetracycline͞3 g/ml chloramphenicol͞20 g/ml asparagine. E. coli strain DH5␣ was grown at 37°C on LB medium (1% tryptone͞0.5% yeast extract͞0.5% NaCl͞1.5% agar) supplemented where necessary with 50 g͞ml ampicillin and 30 g͞ml tetracycline. E. coli strain JF4...

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Partial purification and properties of l-asparagine synthetase from mouse pancreas

Cited by 26 publications

References 18 publications

A Mutation in the Corynebacterium glutamicum ltsA Gene Causes Susceptibility to Lysozyme, Temperature-Sensitive Growth, and l -Glutamate Production

A Mutation in the Corynebacterium glutamicum ltsA Gene Causes Susceptibility to Lysozyme, Temperature-Sensitive Growth, and l -Glutamate Production

Nuclear Gln3 Import Is Regulated by Nitrogen Catabolite Repression Whereas Export Is Specifically Regulated by Glutamine

Transfer RNA-dependent amino acid biosynthesis: An essential route to asparagine formation

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