Cloning, expression, and characterization of a novel CTP synthase gene from Anoxybacillus gonensis G2

Authors : Cemal SANDALLI, Ayşegül SARAL, Serdar ÜLKER, Hakan KARAOĞLU, Ali Osman BELDÜZ, Ayşegül ÇOPUR ÇİÇEK

Pages : 111-117

Doi:10.3906/biy-1304-76

View : 23 | Download : 10

Publication Date : 2014-12-01

Article Type : Research Paper

Abstract :The cytidine-5`-triphosphate insert ignore into journalissuearticles values(CTP); synthase insert ignore into journalissuearticles values(EC 6.4.3.2); gene insert ignore into journalissuearticles values(pyrG); was cloned and sequenced from the thermophilic bacterium Anoxybacillus gonensis G2 insert ignore into journalissuearticles values(Ago);. The gene is 1590 bp in length and encodes a protein of 530 amino acids, with a molecular mass of 59.5 kDa. The amino acid sequence of CTP synthase shares approximately 90%-94% similarity to Bacillus sp., and it belongs to the triad glutamine amidotransferases, which utilize a Cys-His-Glu triad for activity. Multiple sequence alignments revealed that the enzyme includes conserved amino acids responsible for catalytic activity and the binding of a divalent metal ion insert ignore into journalissuearticles values(Mg+2);. AgoCTP synthase insert ignore into journalissuearticles values(AgoG2CTPs); was overproduced in Escherichia coli BL21 insert ignore into journalissuearticles values(DE3); pLysS as recombinant and purified by nickel affinity chromatography. Its biochemical characterization showed that the enzyme had maximal activity at pH 9.0-10.0 and 65 °C. Km, Vmax, and kcat were found to be approximately 12.415 mM, 0.381 U/L, and 0.762 s-1 at 65 °C, respectively. CTP synthase promotes the formation of CTP in dividing cells and is a recognized target for anticancer and antibacterial drugs. The results obtained from this study can be improved upon with the use of different species and substrates.
Keywords : Key words Anoxybacillus gonensis, cytidine 5` triphosphate synthase, thermophilic, NH3 dependent characterization