A functional relationship between monothiol glutaredoxins and BolAs has been unraveled by genomic analyses and in several high-throughput studies. Phylogenetic analyses coupled to transient expression of green fluorescent protein (GFP) fusions indicated that, in addition to the sulfurtransferase SufE1, which contains a C-terminal BolA domain, three BolA isoforms exist in Arabidopsis thaliana, BolA1 being plastidial, BolA2 nucleo-cytoplasmic, and BolA4 dual-targeted to mitochondria and plastids. Binary yeast two-hybrid experiments demonstrated that all BolAs and SufE1, via its BolA domain, can interact with all monothiol glutaredoxins. Most interactions between protein couples of the same subcellular compartment have been confirmed by bimolecular fluorescence complementation. In vitro experiments indicated that monothiol glutaredoxins could regulate the redox state of BolA2 and SufE1, both proteins possessing a single conserved reactive cysteine. Indeed, a glutathionylated form of SufE1 lost its capacity to activate the cysteine desulfurase, Nfs2, but it is reactivated by plastidial glutaredoxins. Besides, a monomeric glutathionylated form and a dimeric disulfide-bridged form of BolA2 can be preferentially reduced by the nucleo-cytoplasmic GrxS17. These results indicate that the glutaredoxin-BolA interaction occurs in several subcellular compartments and suggest that a redox regulation mechanism, disconnected from their capacity to form iron-sulfur cluster-bridged heterodimers, may be physiologically relevant for BolA2 and SufE1.

The Calvin-Benson cycle of carbon dioxide fixation in chloroplasts is controlled by light-dependent redox reactions that target specific enzymes. Of the regulatory members of the cycle, our knowledge of sedoheptulose-1,7-bisphosphatase (SBPase) is particularly scanty, despite growing evidence for its importance and link to plant productivity. To help fill this gap, we have purified, crystallized, and characterized the recombinant form of the enzyme together with the better studied fructose-1,6-bisphosphatase (FBPase), in both cases from the moss Physcomitrella patens (Pp). Overall, the moss enzymes resembled their counterparts from seed plants, including oligomeric organization-PpSBPase is a dimer, and PpFBPase is a tetramer. The two phosphatases showed striking structural homology to each other, differing primarily in their solvent-exposed surface areas in a manner accounting for their specificity for seven-carbon (sedoheptulose) and six-carbon (fructose) sugar bisphosphate substrates. The two enzymes had a similar redox potential for their regulatory redoxactive disulfides (−310 mV for PpSBPase vs. −290 mV for PpFBPase), requirement for Mg 2+ and thioredoxin (TRX) specificity (TRX f > TRX m). Previously known to differ in the position and sequence of their regulatory cysteines, the enzymes unexpectedly showed unique evolutionary histories. The FBPase gene originated in bacteria in conjunction with the endosymbiotic event giving rise to mitochondria, whereas SBPase arose from an archaeal gene resident in the eukaryotic host. These findings raise the question of how enzymes with such different evolutionary origins achieved structural similarity and adapted to control by the same light-dependent photosynthetic mechanism-namely ferredoxin, ferredoxin-thioredoxin reductase, and thioredoxin.Calvin-Benson cycle | sedoheptulose-1,7-bisphosphatase | fructose-1,6-bisphosphatase | redox regulation | thiol-disulfide exchange I n oxygenic photosynthesis, CO 2 fixation takes place via the Calvin-Benson cycle consisting of 13 individual reactions that can be separated into carboxylation, reduction, and regeneration phases (1). Considerable effort has focused on a description of the individual enzymes and the overall regulation of the cycle (2, 3). In chloroplasts, the activity of four enzymes of the cycle is linked to light: NADP-glyceraldehyde 3-phosphate dehydrogenase, phosphoribulokinase, fructose-1,6-bisphosphatase (FBPase), and sedoheptulose-1,7-bisphosphatase (SBPase). In some plants, Rubisco is similarly regulated indirectly by Rubisco activase. The activity of each of these enzymes is modulated by the ferredoxin/thioredoxin system -a thiol-based mechanism in which photoreduced ferredoxin provides electrons for the reduction of thioredoxin (TRX) by the enzyme ferredoxin-thioredoxin reductase (FTR) (3-5). TRX, in turn, reduces specific disulfides and thereby activates the regulatory members by thiol-disulfide exchange. Chloroplasts contain several typical thioredoxin subtypes (f, m, x, y, and z) with different target p...

No abstract