HIGH-YIELD EXPRESSION OF PEA THIOREDOXIN-M AND ASSESSMENT OF ITS EFFICIENCY IN CHLOROPLAST FRUCTOSE-1,6-BISPHOSPHATASE ACTIVATION

A cDNA clone encoding pea (Pisom 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 (Spin acia oleracea L.) Trx m and 25% homology with Trx f from pea and spina ch. After subcloning in the Ndel-BamHI sites of pET-12a, the recombina nt supplied 20 mg Trx m/L Escherichia coli culture. This protein had 1 08 amino acids and was 12,000 D, which is identical to the pea leaf na tive protein. Unlike pea Trx f, pea Trx m showed a hyperbolic saturati on 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. Schurmann, K. Maeda, and A . Tsugita ([1981] Eur J Biochem 116: 37-45), who did not find Trx m ef ficiency in FBPase activation. This higher efficiency of pea Trx m cou ld be related to the presence of four basic residues (arginine-37, lys ine-70, arginine-74, and lysine-97) flanking the regulatory cluster; s pinach Trx m lacks the positive charge corresponding to lysine-70 of p ea Trx m. This has been confirmed by K70E mutagenesis of pea Trx m, wh ich leads to a 50% decrease in FBPase activation.