Functional characterization and expression of a fructokinase gene from a marine cyanobacterium

PEREZ CENCI, M.; SALERNO, G.L.

Mendoza

Congreso; XLVIII Reunión Anual Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB); 2012

Sociedad Argentina de Investigación Bioquímica y Biología Molecular

Sucrose (Suc) is mainly synthesized by plants, unicellular algae and cyanobacteria.  Two enzymes are responsible for the phosphorylation of fructose and glucose, the Suc cleavage products. Hexokinases preferentially phosphorylate glucose and fructokinase catalyze the transfer of a phosphate group from ATP to fructose to yield fructose 6-phosphate, substrate for Suc synthesis. In the genome of Synechococcus sp. PCC 7002, a unicellular cyanobacterial marine strain, we retrieved an open reading frame (orf) annotated as a member of the PfkB family. After PCR amplification of that sequence from genomic DNA, the amplicon was ligated into an expression vector and heterologously expressed in E. coli cells. The recombinant His-tagged protein purified by affinity chromatography, exhibited frutokinase activity with divalent-cation dependence and specificity for fructose. The Mr of the polypeptide determined by SDS-PAGE was in good agreement with that calculated from the predicted amino-acid sequence. RT-PCR experiments indicated that the orf expression parallels that of the genes for Suc biosynthesis in cells under different conditions. We conclude that the orf corresponds to the encoding sequence of a specific fructokinase, the first characterized from cyanobacteria, which could be providing the phosphorylated substrate for Suc biosynthesis. Supported by PIP134, UNMdP EXA 553/11 and FIBA