Isolation of a novel, phosphate-activated glutaminase from Bacillus pasteurii

Klein, M

doi:10.1016/s0378-1097(01)00477-3

Cited by 11 publications

(17 citation statements)

References 9 publications

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“…Amino functional groups on aliphatic and heterocyclic compounds can be removed by hydrolases (23,37); however, to our knowledge, 5NAA deaminase is the first hydrolase that can catalyze the hydrolytic removal of an amino group attached to the benzene ring. It seems likely that the -NO 2 and -COOH groups synergistically polarize 5NAA due to the electronic effect, which makes the amino group of 5NAA equivalent to an "imino group" and, thus, facilitates the hydrolysis.…”

Section: Discussionmentioning

confidence: 99%

Molecular and Biochemical Characterization of the 5-Nitroanthranilic Acid Degradation Pathway in Bradyrhizobium sp. Strain JS329

Spain

2011

J Bacteriol

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Biodegradation pathways of synthetic nitroaromatic compounds and anilines are well documented, but little is known about those of nitroanilines. We previously reported that the initial step in 5-nitroanthranilic acid (5NAA) degradation by Bradyrhizobium sp. strain JS329 is a hydrolytic deamination to form 5-nitrosalicylic acid (5NSA), followed by ring fission catalyzed by 5NSA dioxygenase. The mechanism of release of the nitro group was unknown. In this study, we subcloned, sequenced, and expressed the genes encoding 5NAA deaminase (5NAA aminohydrolase, NaaA), 5NSA dioxygenase (NaaB) and lactonase (NaaC), the key genes responsible for 5NAA degradation. Sequence analysis and enzyme characterization revealed that NaaA is a hydrolytic metalloenzyme with a narrow substrate range. The nitro group is spontaneously eliminated as nitrite concomitant with the formation of a lactone from the ring fission product of 5NSA dioxygenation. The elimination of the nitro group during lactone formation is a previously unreported mechanism for denitration of nitro aliphatic compounds.

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Section: Discussionmentioning

confidence: 99%

Molecular and Biochemical Characterization of the 5-Nitroanthranilic Acid Degradation Pathway in Bradyrhizobium sp. Strain JS329

Spain

2011

J Bacteriol

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“…The difference in deamination mechanisms among 5NAA-, anthranilate-, and aniline-degrading enzymes might be caused by steric or electronic effects of the nitro group in the 5NAA molecule. Aminohydrolases are commonly involved in elimination of amino groups from aliphatic and heterocyclic compounds (16,28) but have not been reported to act on aromatic compounds. Purification and characterization of the novel enzyme will be required to determine its substrate range and the mechanism of the unusual reaction.…”

Section: Discussionmentioning

confidence: 99%

Biodegradation of 5-Nitroanthranilic Acid by Bradyrhizobium sp. Strain JS329

Spain

2010

Appl Environ Microbiol

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Biodegradation of synthetic compounds has been studied extensively, but the metabolic diversity required for catabolism of many natural compounds has not been addressed. 5-Nitroanthranilic acid (5NAA), produced in soil by Streptomyces scabies, is also the starting material for synthetic dyes and other nitroaromatic compounds. Bradyrhizobium JS329 was isolated from soil by selective enrichment with 5NAA. When grown on 5NAA, the isolate released stoichiometric amounts of nitrite and half of the stoichiometric amounts of ammonia. Enzyme assays indicate that the initial step in 5NAA degradation is an unusual hydrolytic deamination for formation of 5-nitrosalicylic acid (5NSA). Cloning and heterologous expression revealed the genes that encode 5NAA deaminase (naaA) and the 5NSA dioxygenase (naaB) that cleaves the aromatic ring of 5NSA without prior removal of the nitro group. The results provide the first clear evidence for the initial steps in biodegradation of amino-nitroaromatic compounds and reveal a novel deamination reaction for aromatic amines.

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“…The effect of phosphate on the glutaminase acitivity was investigated. Phosphate is a specific activator of all animal glutaminases and one of microbial glutaminases from Bacillus pasteurii [27] . The effect of phosphate on the glutaminase activity was investigated.…”

Section: Biochemical Characterization Of the Recombinant Slr2079mentioning

confidence: 99%

Characterization of a sodium-regulated glutaminase from cyanobacterium Synechocystis sp. PCC 6803

et al. 2008

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Glutaminase is widely distributed among microorganisms and mammals with important functions. Little is known regarding the biochemical properties and functions of the deamidating enzyme glutaminase in cyanobacteria. In this study a putative glutaminase encoded by gene slr2079 in Synechocystis sp. PCC 6803 was investigated. The slr2079 was expressed as histidine-tagged fusion protein in Escherichia coli. The purified protein possessed glutaminase activity, validating the functional assignment of the genomic annotation. The apparent K (m) value of the recombinant protein for glutamine was 26.6 +/- 0.9 mmol/L, which was comparable to that for some of other microbial glutaminases. Analysis of the purified protein revealed a two-fold increase in catalytic activity in the presence of 1 mol/L Na(+). Moreover, the K (m) value was decreased to 12.2 +/- 1.9 mmol/L in the presence of Na(+). These data demonstrate that the recombinant protein Slr2079 is a glutaminase which is regulated by Na(+) through increasing its affinity for substrate glutamine. The slr2079 gene was successfully disrupted in Synechocystis by targeted mutagenesis and the Deltaslr2079 mutant strain was analyzed. No differences in cell growth and oxygen evolution rate were observed between Deltaslr2079 and the wild type under standard growth conditions, demonstrating slr2079 is not essential in Synechocystis. Under high salt stress condition, however, Deltaslr2079 cells grew 1.25-fold faster than wild-type cells. Moreover, the photosynthetic oxygen evolution rate of Deltaslr2079 cells was higher than that of the wild-type. To further characterize this phenotype, a number of salt stress-related genes were analyzed by semi-quantitative RT-PCR. Expression of gdhB and prc was enhanced and expression of desD and guaA was repressed in Deltaslr2079 compared to the wild type. In addition, expression of two key enzymes of ammonium assimilation in cyanobacteria, glutamine synthetase (GS) and glutamate synthase (GOGAT) was examined by semi-quantitative RT-PCR. Expression of GOGAT was enhanced in Deltaslr2079 compared to the wild type while GS expression was unchanged. The results indicate that slr2079 functions in the salt stress response by regulating the expression of salt stress related genes and might not play a major role in glutamine breakdown in Synechocystis.

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Isolation of a novel, phosphate-activated glutaminase from Bacillus pasteurii

Cited by 11 publications

References 9 publications

Molecular and Biochemical Characterization of the 5-Nitroanthranilic Acid Degradation Pathway in Bradyrhizobium sp. Strain JS329

Molecular and Biochemical Characterization of the 5-Nitroanthranilic Acid Degradation Pathway in Bradyrhizobium sp. Strain JS329

Biodegradation of 5-Nitroanthranilic Acid by Bradyrhizobium sp. Strain JS329

Characterization of a sodium-regulated glutaminase from cyanobacterium Synechocystis sp. PCC 6803

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