Identification and characterization of UDP-glucose pyrophosphorylase in cyanobacteria Anabaena sp. PCC 7120

https://doi.org/10.1016/j.jbiosc.2013.10.015Get rights and content

Exopolysaccharides produced by photosynthetic cyanobacteria have received considerable attention in recent years for their potential applications in the production of renewable biofuels. Particularly, cyanobacterial cellulose is one of the most promising products because it is extracellularly secreted as a non-crystalline form, which can be easily harvested from the media and converted into glucose units. In cyanobacteria, the production of UDP-glucose, the cellulose precursor, is a key step in the cellulose synthesis pathway. UDP-glucose is synthesized from UTP and glucose-1-phosphate (Glc-1P) by UDP-glucose pyrophosphorylase (UGPase), but this pathway in cyanobacteria has not been well characterized. Therefore, to elucidate the overall cellulose biosynthesis pathway in cyanobacteria, we studied the putative UGPase All3274 and seven other putative NDP-sugar pyrophosphorylases (NSPases), All4645, Alr2825, Alr4491, Alr0188, Alr3400, Alr2361, and Alr3921 of Anabaena sp. PCC 7120. Assays using the purified recombinant proteins revealed that All3274 exhibited UGPase activity, All4645, Alr2825, Alr4491, Alr0188, and Alr3921 exhibited pyrophosphorylase activities on ADP-glucose, CDP-glucose, dTDP-glucose, GDP-mannose, and UDP-N-acetylglucosamine, respectively. Further characterization of All3274 revealed that the kcat for UDP-glucose formation was one or two orders lower than those of other known UGPases. The activity and dimerization tendency of All3274 increased at higher enzyme concentrations, implying catalytic activation by dimerization. However, most interestingly, All3274 dimerization was inhibited by UTP and Glc-1P, but not by UDP-glucose. This study presents the first in vitro characterization of a cyanobacterial UGPase, and provides insights into biotechnological attempts to utilize the photosynthetic production of cellulose from cyanobacteria.

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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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