Mutagenesis at Two Distinct Phosphate-Binding Sites Unravels Their Differential Roles in Regulation of Rubisco Activation and Catalysis

Abstract

Orthophosphate (P i ) has two antagonistic effects on ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), stimulation of activation and inhibition of catalysis by competition with the substrate RuBP. The enzyme binds P i at three distinct sites, two within the catalytic site (where 1P and 5P of ribulose 1,5-bisphosphate [RuBP] bind), and the third at the latch site (a positively charged pocket involved in active-site closure during catalysis). We examined the role of the latch and 5P sites in regulation of Rubisco activation and catalysis by introducing specific mutations in the enzyme of the cyanobacterium Synechocystis sp. strain PCC 6803. Whereas mutations at both sites abolished the P i -stimulated Rubisco activation, substitution of residues at the 5P site, but not at the latch site, affected the P i inhibition of Rubisco catalysis. Although some of these mutations substantially reduced the catalytic turnover of Rubisco and increased the K m (RuBP), they had little to moderate effect on the rate of photosynthesis and no effect on photoautotrophic growth. These findings suggest that in cyanobacteria, Rubisco does not limit photosynthesis to the extent previously estimated. These results indicate that both the latch and 5P sites participate in regulation of Rubisco activation, whereas P i binding only at the 5P site inhibits catalysis in a competitive manner.Activation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC4.1.1.39) is essential for catalysis and occurs by carbamylation of Lys-201 and binding of Mg 2ϩ by the carbanion (14). Several effectors, mostly anions such as orthophosphate (P i ), sugar-phosphates, sulfate, and NADPH, modulate the activation process via a mechanism that is still controversial (2,3,5,11,12,16,18,20,21,26). It is accepted that these compounds evoke activation by elevating the carbamylation level, presumably by slowing the rate of enzyme deactivation (3, 12, 18). Nonetheless, evidence was brought that in addition to stimulation of Rubisco carbamylation, P i also enhances the activation of the enzyme without a parallel increase in the carbamylation level (16,21). Whereas McCurry et al. (18) reported monophasic, hyperbolic P i concentration-dependent activation of Rubisco, nonmonotonously biphasic kinetics was obtained in other, more recent studies (2, 16), indicating that P i stimulates enzyme activation via a mechanism that involves multiple interacting sites (28).Paradoxically, in addition to the stimulatory effect on activation, the effectors also inhibit the catalytic activity of the enzyme by competing with the substrate ribulose 1,5-bisphosphate (RuBP) (3, 16). Based on the observation of McCurry et al. (18) and the ability of the transition state analog carboxyarabinitol-bisphosphate to prevent 6-phosphogluconate from binding, Badger and Lorimer (3) suggested that inhibition of activity and stimulation of activation are both induced by effector binding to the catalytic site. Although this would imply that RuBP competitively inhibits the stimulation...

Rubisco mutagenesis provides new insight into limitations on photosynthesis and growth in Synechocystis PCC6803

“…aApparent K m (CO 2 ), determined at 21% O 2 .bNM, not measured.cResults taken from Marcus et al (2005).…”

Transcriptional Modulation of Ethylene Response Factor Protein JERF3 in the Oxidative Stress Response Enhances Tolerance of Tobacco Seedlings to Salt, Drought, and Freezing

“…The enhanced expression of glutathione peroxidase NtGPX and cytosolic and chloroplastic ascorbate peroxidases (NtAPX1 and NtAPX2) in unstressed OE seedlings further supports the idea that JERF3 might transcriptionally regulate the expression of genes related to oxidative reactions. Furthermore, Rubisco, a key enzyme in photosynthesis, catalyzes ribulose-1,5-bisphosphate and assimilates CO 2 and also catalyzes the oxygenase reaction of ribulose-1,5-bisphosphate into glycolate, which is metabolized in peroxisome (Ruuska et al, 2000;Marcus et al, 2005). This process helps scavenge ROS (Apel and Hirt, 2004).…”

Bacterial Carbonic Anhydrases as Drug Targets: Toward Novel Antibiotics?

“…But the best studied bacterial α-CA is the one from the gastric pathogen provoking ulcer and gastric cancer, H. pylori , hpαCA (Chirică et al, 2002; Marcus et al, 2005; Shahidzadeh et al, 2005; Nishimori et al, 2006, 2007) – see Table 1. The genome project of H. pylori identified in fact two different classes of CAs, with different subcellular localization: a periplasmic α-class CA (hpαCA) and a cytoplasmic β-class CA (hpβCA; Nishimori et al, 2008).…”