Chloroplast FBPase and SBPase are thioredoxin-linked enzymes with similar architecture but different evolutionary histories

Abstract

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...

Arabidopsis and Chlamydomonas phosphoribulokinase crystal structures complete the redox structural proteome of the Calvin–Benson cycle

“…In fact, each of these five proteins is regulated by TRX in a different way. In FBPase and SBPase, the TRX-binding site is far from the conserved sugar-binding domain (5). In FBPase, upon reduction, the substrate-binding site adopts a catalytically competent conformation due to the loosening of secondary structures (4,5).…”

“…In FBPase and SBPase, the TRX-binding site is far from the conserved sugar-binding domain (5). In FBPase, upon reduction, the substrate-binding site adopts a catalytically competent conformation due to the loosening of secondary structures (4,5). In AB-GAPDH, the redox-active cysteines are found in the highly flexible CTE, which is characteristic of B subunits and, when oxidized, the last residue in the CTE blocks the NADPH-binding site (7).…”

“…Structural comparisons with ancient PRKs indicate that the presence of flexible regions close to regulatory cysteines is a unique feature that is shared by TRXdependent CB cycle enzymes, suggesting that the evolution of the PRK structure has resulted in a global increase in protein flexibility for photosynthetic eukaryotes. structure to achieve the same TRX-dependent redox control (5). In the three available crystal structures of enzymes regulated by TRX, the pair of redox-sensitive cysteines was found to be located in different regions.…”

“…In the three available crystal structures of enzymes regulated by TRX, the pair of redox-sensitive cysteines was found to be located in different regions. In AB-GAPDH, these cysteines are located in a C-terminal extension (CTE) (7); in SBPase, they are present at the dimer interface (5); and in FBPase, they are solvent-exposed and distant from the sugar-binding site (4,5).…”

Computational simulation of the reactive oxygen species and redox network in the regulation of chloroplast metabolism

“…The TRX complement of Arabidopsis plastids comprises 20 TRX and TRX-like proteins with representatives of the f-, m-, x-, y-, z-group of TRX, TRX-like proteins which include chloroplast drought-induced stress protein of 32 kDa (CDSP32), Lilium1-4 (ACHT1-4) and TRX-like [3]. TRX-f1 and TRX-f2 function in activation of Calvin-Benson-Cycle (CBC) enzymes and γ-subunit of F-ATP synthase [4]. TRX-m1, -m2, -m3 and -m4 are suggested to regulate targets which control the NADPH/NADP ratio [5] which is linked to their ability to efficiently activate the NADPH-dependent malate dehydrogenase [6].…”