CLONING, STRUCTURE AND EXPRESSION OF A PEA CDNA CLONE CODING FOR A PHOTOSYNTHETIC FRUCTOSE-1,6-BISPHOSPHATASE WITH SOME FEATURES DIFFERENT FROM THOSE OF THE LEAF CHLOROPLAST ENZYME

A positive clone against pea (Pisum sativum L.) chloroplast fructose-1 ,6-bisphosphatase (FBPase; EC 3.1.3.11) antibodies was obtained from a copy DNA (cDNA) library in lambda gt11. The insert was 1261 nucleotid es long, and had an open reading frame of 1143 base pairs with coding capability for the whole FBPase subunit and a fragment of a putative p rocessing peptide. An additional 115 base pairs corresponding to a 3'- untranslated region coding for an mRNA poly(A)(+) tail were also found in the clone. The deduced sequence for the FBPase subunit was a 357-a mino-acid protein of molecular mass 39 253 daltons (Da), showing 82-88 % absolute homology with four chloroplastic FBPases sequenced earlier. The 3.1-kilobase (kb) KpnI-SacI fragment of the lambda gt11 derivativ e was subcloned between the KpnI-SacI restriction sites of pTZ18R to y ield plasmid pAMC100. Lysates of Escherichia coli (pAMC100) showed FBP ase activity; this was purified as a 170-kDa protein which, upon sodiu m dodecyl sulfate-polyacrylamide gel electrophoresis, displayed a 44-k Da band. As occurs with native FBPases, this indicates a homotetrameri c structure for the expressed FBPase. When assayed under excess Mg2+ ( 10 mM), the expressed enzyme had a higher affinity for the substrate t han the native pea leaf FBPase; this parameter appears to be substanti ated by a tenfold higher specific activity than that of the native enz yme. However, when activated with dithiothreitol plus saturating conce ntrations of pea thioredoxin (Td) f, both FBPase had similar activitie s, with a 4:1 Td f-FBPase stoichiometry. In contrast to the native pea chloroplast FBPase, the E. coli-expressed enzyme did not react with t he monoclonal antibody GR-PBS. It also had a higher heat sensitivity, with 42% residual activity after heating for 30 min at 60 degrees C, c onditions which preserved the native enzyme in a fully active state. T hese results show the existence of some difference(s) in the conformat ion of the two FBPases; this could be a consequence of a different exp ression of the genomic and cDNA clones, or be due to the need for some factor for the correct assembly of the oligomeric structure of the na tive chloroplast enzyme.