Abstract
Chloroplast thioredoxin-f functions efficiently in the light-dependent activation of chloroplast fructose-1, 6-bisphosphatase by reducing a specific disulfide bond located at the negatively charged domain of the enzyme. Around the nucleophile cysteine of the active site (-W-C-G-P-C-), chloroplast thioredoxin-f shows lower density of negative charges than the inefficient modulator Escherichia coli thioredoxin. To examine the contribution of long range electrostatic interactions to the thiol/disulfide exchange between protein-disulfide oxidoreductases and target proteins, we constructed three variants of E. coli thioredoxin in which an acidic (Glu-30) and a neutral residue (Leu-94) were replaced by lysines. After purification to homogeneity, the reduction of the unique disulfide bond by NADPH via NADP-thioredoxin reductase proceeded at similar rates for all variants. However, the conversion of cysteine residues back to cystine depended on the target protein. Insulin and difluoresceinthiocarbamyl-insulin oxidized the sulfhydryl groups of E30K and E30K/L94K mutants more effectively than those of wild type and L94K counterparts. Moreover, the affinity of E30K, L94K, and E30K/L94K E. coli thioredoxin for chloroplast fructose-1,6-bisphosphatase (A0.5 = 9, 7, and 3 microM, respectively) increased with the number of positive charges, and was higher than wild type thioredoxin (A0.5 = 33 microM), though still lower than that of thioredoxin-f (A0.5 = 0.9 microM). We also demonstrated that shielding of electrostatic interactions with high salt concentrations not only brings the A0.5 for all bacterial variants to a limiting value of approximately 9 microM but also increases the A0.5 of chloroplast thioredoxin-f. While negatively charged chloroplast fructose-1,6-bisphosphatase (pI = 4.9) readily interacted with mutant thioredoxins, the reduction rate of rapeseed napin (pI = 11.2) diminished with the number of novel lysine residues. These findings suggest that the electrostatic interactions between thioredoxin and (some of) its target proteins controls the formation of the binary noncovalent complex needed for the subsequent thiol/disulfide exchange.
Highlights
Chloroplast thioredoxin-f functions efficiently in the light-dependent activation of chloroplast fructose-1,6bisphosphatase by reducing a specific disulfide bond located at the negatively charged domain of the enzyme
While negatively charged chloroplast fructose-1,6bisphosphatase readily interacted with mutant thioredoxins, the reduction rate of rapeseed napin diminished with the number of novel lysine residues. These findings suggest that the electrostatic interactions between thioredoxin and its target proteins controls the formation of the binary noncovalent complex needed for the subsequent thiol/disulfide exchange
At variance, when insulin was the final hydrogen acceptor, the rate of NADPH oxidation for E30K and E30K/L94K mutants (5.6 mol1⁄7minϪ1) was persistently higher than for the wild type and L94K Trx (4.6 mol1⁄7minϪ1). That this effect reflected differences in the interaction between Trx and insulin was confirmed in the reaction driven by DTT, as proteins carrying the E30K mutation were again 30% more active than other variants (Fig. 3). All these results demonstrated that novel positive charges near the active site of E. coli Trx did not affect the interaction with NADP-Trx reductase but elicited slight modifications in the affinity of the reduced form for other target proteins [49]
Summary
Chloroplast thioredoxin-f functions efficiently in the light-dependent activation of chloroplast fructose-1,6bisphosphatase by reducing a specific disulfide bond located at the negatively charged domain of the enzyme. While negatively charged chloroplast fructose-1,6bisphosphatase (pI ؍4.9) readily interacted with mutant thioredoxins, the reduction rate of rapeseed napin (pI ؍11.2) diminished with the number of novel lysine residues. These findings suggest that the electrostatic interactions between thioredoxin and (some of) its target proteins controls the formation of the binary noncovalent complex needed for the subsequent thiol/disulfide exchange. The relationship between the phylogenetic origin and the metabolism of chloroplasts remains unknown, it was found that the reduced forms of both Trxs participate in the light-dependent modulation of enzymes These studies led to the view that chloroplast Trx-m preferentially activates and deactivates NADP-malate dehydrogenase and glucose-6-phosphate dehydrogenase, respectively, whereas chloroplast Trx-f is highly efficient in the stimulation of key enzymes of photosynthetic CO2 assimilation (CFBPase, sedoheptulose-1,7-bisphosphatase, phosphoribulokinase, NADP-glyceraldehyde-3-P dehydrogenase).
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