Molecular characterization and resistance to hydrogen peroxide of two fructose-1,6-bisphosphatases from Synechococcus PCC 7942.
In Synechococcus PCC 7942 cells, two catalytic fructose-1,6-bisphosphatase isoenzymes, designated F-I and F-II, have been resolved by chromatography on a HiLoad 26/10 Q Sepharose column at 0.24 and 0.34 M of NaCl, respectively; the former represented the major part of the total extractable enzyme activity. F-I has been purified to electrophoretic homogeneity from the cells. F-I and F-II had respective molecular masses of 160 and 150 kDa and each enzyme was composed of four identical subunits. F-I hydrolyzed both fructose 1,6-bisphosphate and sedoheptulose 1,7-bisphosphate, whereas F-II hydrolyzed only fructose 1,6-bisphosphate. The apparent Km values of F-I and F-II for fructose 1,6-bisphosphate were 52 +/- 4.5 and 25 +/- 1.5 microM, respectively. F-I was inhibited by AMP with a Ki value of 0.26 mM, but F-II was not affected by AMP. The F-I failed to cross-react by Western blotting with the antibody raised against F-II; similarly, the F-II did not react with the F-I antibody. The genes encoding F-I and F-II were cloned from the chromosomal DNA of Synechococcus PCC 7942. A 1068-bp open reading frame, encoding F-I of 356 amino acid residues (approx molecular mass of 38.3 kDa) was observed. The nucleotide sequence of the F-II gene showed an open reading frame of 1017 bp that encodes a protein of 339 amino acid residues (approx molecular mass of 37.2 kDa). The recombinant enzymes expressed in Escherichia coli as well as the native enzymes of F-I and F-II from Synechococcus PCC 7942 cells were resistant to 1 mM hydrogen peroxide unlike the light-activated higher plant chloroplast enzymes.
Tamoi
M
M
Department of Food and Nutrition, Faculty of Agriculture, Kinki University, Nakamachi, Nara, Japan.
Ishikawa
T
T
Takeda
T
T
Shigeoka
S
S
D000595:Amino Acid Sequence; D000818:Animals; D000907:Antibodies, Bacterial; D001483:Base Sequence; D000458:Cyanobacteria / Q000201:enzymology* / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D006597:Fructose-Bisphosphatase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D017386:Sequence Homology, Amino Acid
D000907:Antibodies, Bacterial; D017931:DNA Primers; D004269:DNA, Bacterial; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D011994:Recombinant Proteins; D006861:Hydrogen Peroxide; D006597:Fructose-Bisphosphatase