Acquisition of a new type of fructose-1,6-bisphosphatase with resistance to hydrogen peroxide in cyanobacteria: molecular characterization of the enzyme from Synechocystis PCC 6803.

We have previously described that Synechococcus PCC 7942 cells contain two fructose-1,6-bisphosphatase isozymes, designated F-I and F-II the former belongs to a new type of fructose-1,6-bisphosphatase, while the latter is a typical enzyme similar to the cytosolic and chloroplastic forms from eukaryotic cells [Tamoi et al., Arch. Biochem. Biophys., 334, 1996, 27-36]. The genes of F-I and F-II were found in three species of cyanobacteria, Synechocystis PCC 6803, Anabaena 7120, and Plectonema boryanum according to the results of Southern hybridization with a probe from the S. 7942 F-I and F-II genes. In Western blotting, antibody raised against the S. 7942 F-I cross-reacted with a protein band corresponding to the F-I in each crude extract from cyanobacterial cells, whereas the antibody against F-II failed to cross-react with any protein band corresponding to the F-II. In cyanobacterial cells, only one form of F-I has been resolved by ion-exchange chromatography at same concentration of NaCl as shown in the F-I of S. 7942. The F-I from Synechocystis 6803 has been purified to electrophoretic homogeneity. The enzyme hydrolyzed both fructose 1,6-bisphosphate and sedoheptulose 1,7-bisphosphate. The apparent K(m) values of the enzyme for fructose 1,6-bisphosphate and sedoheptulose 1,7-bisphosphate were 57 +/- 2.4 and 180 +/- 6.3 microM, respectively. The enzyme activity was inhibited by AMP with a Ki value of 0.57 +/- 0.03 mM for fructose 1,6-bisphosphate and 0.35 +/- 0.02 mM for sedoheptulose 1,7-bisphosphate. The enzyme showed a molecular mass of 168 kDa which was composed of four identical subunits. The activities of FBPase and SBPase from the F-I were resistant to hydrogen peroxide up to 1 mM. The nucleotide sequence of the S. 6803 F-I gene showed an open reading frame of 1164 bp that encoded a protein of 388 amino acid residues (approx. molecular mass of 41.6 kDa). The deduced amino acid sequences had homologous sequences with the S. 7942 F-I.