Identification and characterization of UDP-glucose pyrophosphorylase in cyanobacteria Anabaena sp. PCC 7120
Section snippets
Strains and cultures
Escherichia BL21(DE3) (Life Technologies, Carlsbad, CA, USA) was used as the parental strain for recombinant protein expression. Transformed cells were cultured with shaking (120 rpm) in Luria–Bertani (LB) medium, supplemented with 100 μg mL−1 ampicillin (pET22b) or 20 μg mL−1 kanamycin (pET28a).
Cloning and construction of plasmids
Coding regions (without the stop codon) of the genes were amplified from the genomic DNA of Anabaena sp. PCC 7120 by PCR, using primers containing the corresponding restriction sites (Table S1). The
Cloning, recombinant expression, and purification
Five cyanobacterial genes were identified as UDP-glucose pyrophosphorylase or UTP–glucose-1-phosphate uridylyltransferase by annotation in the genome database (CyanoBase): all3274, Ava_4927, Npun_F1146, Tery_2112, and NIES39_K04100. We investigated whether All3274 (Gene ID; 1106872) in Anabaena sp. PCC 7120 is indeed a UGPase, and whether other candidate UGPases exist. The gene all3274 is 882 bp long and is predicted to encode a protein of 293 amino acids with a molecular mass of 32.6 kDa.
Discussion
All3274 is the first cyanobacterial UGPase characterized in detail in vitro. This study highlights several interesting features in comparison to other known bacterial UGPases. First, All3274 is a UGPase with high specificity for UTP and does not utilize dTTP to yield dTDP-glucose (Fig. 2), whereas many bacterial UGPases can react with dTTP, as well as UTP 4, 8, 10, 22, 23. Second, the UDP-Glc formation activity of All3274-His is lower than those of the known enzymes, and Km for Glc-1P is much
Acknowledgments
This research was supported by Precursory Research for Embryonic Science and Technology (PRESTO) in Japan Science and Technology Agency (JST) program. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. This work was also supported by the Programme for Promotion of Basic and Applied Researches for Innovations in Bio-oriented Industry.
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