High-yield expression of pea thioredoxin m and assessment of its efficiency in chloroplast fructose-1,6-bisphosphatase activation.
A cDNA clone encoding pea (Pisum sativum L.) chloroplast thioredoxin (Trx) m and its transit peptide were isolated from a pea cDNA library. Its deduced amino acid sequence showed 70% homology with spinach (Spinacia oleracea L.) Trx m and 25% homology with Trx f from pea and spinach. After subcloning in the Ndel-BamHI sites of pET-12a, the recombinant supplied 20 mg Trx m/L. Escherichia coli culture. This protein had 108 amino acids and was 12,000 D, which is identical to the pea leaf native protein. Unlike pea Trx f, pea Trx m showed a hyperbolic saturation of pea chloroplast fructose-1,6-bisphosphatase (FBPase), with a Trx m/ FBPase molar saturation ratio of about 60, compared with 4 for the Trx f/FBPase quotient. Cross-experiments have shown the ability of pea Trx m to activate the spinach chloroplast FBPase, results that are in contrast with those in spinach found by P. Schürmann, K. Maeda, and A. Tsugita ([1981] Eur J Biochem 116: 37-45), who did not find Trx m efficiency in FBPase activation. This higher efficiency of pea Trx m could be related to the presence of four basic residues (arginine-37, lysine-70, arginine-74, and lysine-97) flanking the regulatory cluster; spinach Trx m lacks the positive charge corresponding to lysine-70 of pea Trx m. This has been confirmed by K70E mutagenesis of pea Trx m, which leads to a 50% decrease in FBPase activation.
López Jaramillo
J
J
Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Granada, Spain.
Chueca
A
A
Jacquot
J P
JP
Hermoso
R
R
Lázaro
J J
JJ
Sahrawy
M
M
López Gorgé
J
J
D000595:Amino Acid Sequence; D054479:Chloroplast Thioredoxins; D002736:Chloroplasts / Q000201:enzymology*; D018076:DNA, Complementary; D004789:Enzyme Activation; D006597:Fructose-Bisphosphatase / Q000378:metabolism*; D008969:Molecular Sequence Data; D018532:Pisum sativum / Q000201:enzymology / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013816:Thermodynamics; D013879:Thioredoxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D054479:Chloroplast Thioredoxins; D018076:DNA, Complementary; D013879:Thioredoxins; D006597:Fructose-Bisphosphatase