Chloroplast fructose 1,6-bisphosphatase with changed redox modulation: comparison of the Galdieria enzyme with cysteine mutants from spinach.
Spinach fructose 1,6-bisphosphatase (FBPase, EC 3.1.3.11), a redox-modulated chloroplast enzyme and part of the Calvin cycle, and three different Cys mutants were expressed in E. coli. The properties of the purified proteins were compared to those of native and recombinant chloroplast FBPase from the red alga Galdieria sulphuraria. In spinach chloroplast FBPase, Cys(155) and Cys(174) are engaged in the formation of the disulfide bridge. The corresponding mutants are active when expressed in E. coli, while C179S is inactive and can be reductively activated as can the wild-type enzyme. The active C174S mutant, however, could be inactivated by oxidation, and reactivated, but only by reduction, not alternatively with high pH and high Mg(2+) as is the case for the wild-type enzyme. In the sequence of Galdieria FBPase, the Cys that corresponds to Cys(179) in the spinach enzyme is lacking. However, the Galdieria FBPase, in contrast to the spinach Cys(179) mutant, does not show any indication for a comparable redox modulation of its activity. Instead, oxidation only leads to partial inactivation without any qualitative changes in enzyme properties. Upon reduction, the lost activity can be recovered.
Reichert
Angelika
A
Paradigm Genetics, Inc, 108 Alexander Drive, Research Triangle Park, NC 27709, USA.
Dennes
André
A
Vetter
Susanne
S
Scheibe
Renate
R
D000595:Amino Acid Sequence; D002736:Chloroplasts / Q000201:enzymology*; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004926:Escherichia coli / Q000378:metabolism; D006597:Fructose-Bisphosphatase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D008274:Magnesium; D008969:Molecular Sequence Data; D009154:Mutation; D010084:Oxidation-Reduction; D000461:Rhodophyta / Q000201:enzymology*; D016415:Sequence Alignment; D018724:Spinacia oleracea / Q000201:enzymology / Q000235:genetics*
D004220:Disulfides; D006597:Fructose-Bisphosphatase; D008274:Magnesium; D003545:Cysteine