Characterization of a sodium-regulated glutaminase from cyanobacterium Synechocystis sp. PCC 6803.
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.
Zhou
Jie
J
Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing, 100093, China.
Zhou
JunXia
J
Yang
HaoMeng
H
Yan
ChengShi
C
Huang
Fang
F
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D015964:Gene Expression Regulation, Bacterial; D005972:Glutaminase / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D012964:Sodium / Q000378:metabolism*; D046939:Synechocystis / Q000201:enzymology* / Q000235:genetics
D011994:Recombinant Proteins; D012964:Sodium; D005972:Glutaminase