Binding site on pea chloroplast fructose-1,6-bisphosphatase involved in the interaction with thioredoxin.
When we compare the primary structures of the six chloroplast fructose-1,6-biophosphatases (FBPase) so far sequenced, the existence of a poorly conserved fragment in the region just preceeding the redox regulatory cysteines cluster can be observed. This region is a good candidate for binding of FBPase to its physiological modulator thioredoxin (Td), as this association shows clear differences between species. Using a cDNA clone for pea chloroplast FBPase as template, we have amplified by PCR a DNA insert coding for a 19 amino acid fragment (149Pro-167Gly), which was expressed in pGEMEX-1 as a fusion protein. This protein strongly interacts with pea Td m, as shown by ELISA and Superose 12 gel filtration, depending on pH of the medium. Preliminary assays have shown inhibition of FBPase activity in the presence of specific IgG against the 19 amino acid insert. Surprisingly the fusion protein enhances the FBPase activation in competitive inhibition experiments carried out with FBPase and Td. These results show the fundamental role played by this domain in FBPase-Td binding, not only as docking point for Td, but also by inducing some structural modification in the Td molecule. Taking as model the structural data recently published for spinach photosynthetic FBPase, this sequence from a tertiary and quaternary structural point of view appears available for rearrangement.
Hermoso
R
R
Departamento de Bioquímica, Biologia Celular y Molecular de Plantas, Estación Experimental del Zaidín (CSIC), 18008, Granada, Spain.
Castillo
M
M
Chueca
A
A
Lázaro
J J
JJ
Sahrawy
M
M
Gorgé
J L
JL
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002736:Chloroplasts / Q000201:enzymology*; D004926:Escherichia coli; D006597:Fructose-Bisphosphatase / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D018532:Pisum sativum / Q000201:enzymology*; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013879:Thioredoxins / Q000378:metabolism*
D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D013879:Thioredoxins; D006597:Fructose-Bisphosphatase