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 nucleotides 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 processing 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-amino-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 derivative was subcloned between the KpnI-SacI restriction sites of pTZ18R to yield plasmid pAMC100. Lysates of Escherichia coli (pAMC100) showed FBPase activity; this was purified as a 170-kDa protein which, upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, displayed a 44-kDa band. As occurs with native FBPases, this indicates a homotetrameric structure for the expressed FBPase. When assayed under excess Mg2+ (10 mM), the expressed enzyme had a higher affinity for the substrate than the native pea leaf FBPase; this parameter appears to be substantiated by a tenfold higher specific activity than that of the native enzyme. However, when activated with dithiothreitol plus saturating concentrations of pea thioredoxin (Td) f, both FBPase had similar activities, 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 the monoclonal antibody GR-PB5.(ABSTRACT TRUNCATED AT 250 WORDS)
Carrasco
J L
JL
Department of Plant Biochemistry, Estación Experimental del Zaidín (CSIC), Granada, Spain.
Chueca
A
A
Prado
F E
FE
Hermoso
R
R
Lázaro
J J
JJ
Ramos
J L
JL
Sahrawy
M
M
López Gorgé
J
J
D000595:Amino Acid Sequence; D001483:Base Sequence; D002736:Chloroplasts / Q000201:enzymology*; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004926:Escherichia coli; D007887:Fabaceae / Q000235:genetics*; D006597:Fructose-Bisphosphatase / Q000235:genetics*; D008969:Molecular Sequence Data; D010788:Photosynthesis; D010946:Plants, Medicinal; D011994:Recombinant Proteins
D018076:DNA, Complementary; D011994:Recombinant Proteins; D006597:Fructose-Bisphosphatase