摘要
Glutaminase is widely distributed among microorganisms and mammals with important functions. Lit-tle is known regarding the biochemical properties and functions of the deamidating enzyme glutami-nase 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 Es-cherichia coli. The purified protein possessed glutaminase activity, validating the functional assign-ment of the genomic annotation. The apparent Km 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 Km value was decreased to 12.2 ± 1.9 mmol/L in the presence of Na+. These data demon-strate that the recombinant protein Slr2079 is a glutaminase which is regulated by Na+ through in-creasing its affinity for substrate glutamine. The slr2079 gene was successfully disrupted in Synecho-cystis by targeted mutagenesis and the △slr2079 mutant strain was analyzed. No differences in cell growth and oxygen evolution rate were observed between △slr2079 and the wild type under standard growth conditions, demonstrating slr2079 is not essential in Synechocystis. Under high salt stress condition, however, △slr2079 cells grew 1.25-fold faster than wild-type cells. Moreover, the photosyn-thetic oxygen evolution rate of △slr2079 cells was higher than that of the wild-type. To further charac-terize 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 △slr2079 compared to the wild type. In addition, expression of two key enzymes of ammonium assimi-lation in cyanobacteria, glutamine synthetase (GS) and glutamate synthase (GOGAT) was examined by semi-quantitative RT-PCR. Expression of GOGAT was enhanced in △slr2079 compared to the wild type while GS expression was unchanged. The results indicate that slr2079 functions in the salt stress re-sponse by regulating the expression of salt stress related genes and might not play a major role in glutamine breakdown in Synechocystis.
Glutaminase is widely distributed among microorganisms and mammals with important functions. Lit-tle is known regarding the biochemical properties and functions of the deamidating enzyme glutami-nase 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 Es-cherichia coli. The purified protein possessed glutaminase activity, validating the functional assign-ment of the genomic annotation. The apparent Km 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 Km value was decreased to 12.2 ± 1.9 mmol/L in the presence of Na+. These data demon-strate that the recombinant protein Slr2079 is a glutaminase which is regulated by Na+ through in-creasing its affinity for substrate glutamine. The slr2079 gene was successfully disrupted in Synecho-cystis by targeted mutagenesis and the △slr2079 mutant strain was analyzed. No differences in cell growth and oxygen evolution rate were observed between △slr2079 and the wild type under standard growth conditions, demonstrating slr2079 is not essential in Synechocystis. Under high salt stress condition, however, △slr2079 cells grew 1.25-fold faster than wild-type cells. Moreover, the photosyn-thetic oxygen evolution rate of △slr2079 cells was higher than that of the wild-type. To further charac-terize 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 △slr2079 compared to the wild type. In addition, expression of two key enzymes of ammonium assimi-lation in cyanobacteria, glutamine synthetase (GS) and glutamate synthase (GOGAT) was examined by semi-quantitative RT-PCR. Expression of GOGAT was enhanced in △slr2079 compared to the wild type while GS expression was unchanged. The results indicate that slr2079 functions in the salt stress re-sponse by regulating the expression of salt stress related genes and might not play a major role in glutamine breakdown in Synechocystis.
作者
ZHOU Jie1, ZHOU JunXia1,2, YANG HaoMeng1, YAN ChengShi1 & HUANG Fang1 1 Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China
2 Graduate School of Chinese Academy of Sciences, Beijing 100039, China
基金
Supported by the National Natural Sciences Foundation of China (Grant No. 30500108)
Hundred Talents Program of Chinese Academy of Sciences
