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Dergiler / Turkish Journal of Biology / 2014 / Cilt: 38 - Sayı: 1

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

Cemal SANDALLI
Ayşegül SARAL
Serdar ÜLKER
Hakan KARAOĞLU
Ali Osman BELDÜZ
Ayşegül ÇOPUR ÇİÇEK
Sayfa
111–117
Yayın Tarihi
2014-05-16
DOI
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The results obtained from this study can be improved upon with the use of different species and substrates.

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Amaç & Hipotez:The cytidine-5'-triphosphate (CTP) synthase (EC 6.4.3.2) gene (pyrG) was cloned and sequenced from the thermophilic bacterium Anoxybacillus gonensis G2 (Ago).
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Temel Bulgular & Veri:The gene is 1590 bp in length and encodes a protein of 530 amino acids, with a molecular mass of 59.5 kDa.
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Sonuç & Katkı:The results obtained from this study can be improved upon with the use of different species and substrates.
🔬Tür:Araştırma Makalesi
📍Kapsam:Genişletilmiş Akademik Örneklem
⚙️Değişkenler:Key words: Anoxybacillus gonensis · cytidine 5'-triphosphate synthase · thermophilic

Özet

The cytidine-5'-triphosphate (CTP) synthase (EC 6.4.3.2) gene (pyrG) was cloned and sequenced from the thermophilic bacterium Anoxybacillus gonensis G2 (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 (Mg+2). AgoCTP synthase (AgoG2CTPs) was overproduced in Escherichia coli BL21 (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.

Abstract

The cytidine-5'-triphosphate (CTP) synthase (EC 6.4.3.2) gene (pyrG) was cloned and sequenced from the thermophilic bacterium Anoxybacillus gonensis G2 (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 (Mg+2). AgoCTP synthase (AgoG2CTPs) was overproduced in Escherichia coli BL21 (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.

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Bu Makaleye Atıf Yap

SANDALLI, C., SARAL, A., ÜLKER, S., KARAOĞLU, H., BELDÜZ, A. O., ÇİÇEK, A. Ç. (2014). Cloning, expression, and characterization of a novel CTP synthase gene from Anoxybacillus gonensis G2. Turkish Journal of Biology, Cilt: 38 - Sayı: 1, 111–117. https://ajindex.com/articles/182543

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