Construction of chimeric cytosolic fructose-1,6-bisphosphatases by insertion of a chloroplastic redox regulatory cluster.
In order to transform cytosolic fructose-1,6-bisphosphatases (FBPase)(EC 3.1.3.11) into potential reductively-modulated chloroplast-type enzymes, we have constructed four chimeric FBPases, which display structural viability as deduced by previous modelling. In the X1-type BV1 and HL1 chimera the N-half of cytosolic sugar beet (Beta vulgaris L.) and human FBPases was fused with the C-half of the pea (Pisum sativum L.) chloroplast enzyme, which carries the cysteine-rich light regulatory sequence. In the X2-type BV2 and HL2 chimera this regulatory fragment was inserted in the corresponding site of the sugar beet cytosolic and human enzymes. Like the plant cytosolic FBPases, the chimeric enzymes show a low rise of activity by dithiothreitol. Both BV1 and BV2, but not HL1 and HL2, display a negligible activation by Trx f, but neither of them by Trx m. Antibodies raised against the pea chloroplast enzyme showed a positive reaction against the four chimeric FBPases and the human enzyme, but not against the sugar beet one. The four chimera display typical kinetics of cytosolic FBPases, with Km values in the 40-140 microM range. We conclude the existence of a structural capacity of cytosolic FBPases for incorporating the redox regulatory cluster of the chloroplast enzyme. However, the ability of these chimeric FBPases for an in vitro redox regulation seems to be scarce, limiting their use from a biotechnology standpoint in in vivo regulation of sugar metabolism.
Cazalis
R
R
ESAP, 75 voie du Toec, 31076 Toulouse, France.
Chueca
A
A
Sahrawy
M
M
López-Gorgé
J
J
D000595:Amino Acid Sequence; D001483:Base Sequence; D002736:Chloroplasts / Q000201:enzymology*; D003600:Cytosol / Q000201:enzymology*; D017931:DNA Primers; D006597:Fructose-Bisphosphatase / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D006597:Fructose-Bisphosphatase