Regulation of Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase Activation by Inorganic Phosphate through Stimulating the Binding of the Activator CO2 to the Activation Sites

The mechanism of the regulation of the activation of ribulose1,5-bisphosphate carboxylase/ oxygenase (RuBisCO) by inorganicphosphate (P_i) in the presence of limiting concentrations ofCO₂ was explored. The activation state of RuBisCO increasedsigmoidally following a biphasic kinetics against the concentrationof P_i in the activation mixture with an intermediary plateauat 2 to 3 mM P_i when the enzyme was activated for 30 min. Theintermediary plateau could not be seen when the preincubationtime was 10 min and the activation was completed at 10 mM P_i.RuBisCO from Euglena also showed a quite similar activationkinetics. The activation was not due to the contaminating CO₂included in the stock P_i solution or in the activation buffercontaining the enzyme. The experiments with 2-carboxyarabinitol1,5-bisphosphate showed that the P_i stimulated activation wasdue to the promotion of binding of the activator CO₂ to theactivation sites. It was also found that P_i increased the affinityof RuBisCO for the activator CO₂ 5.4-fold accompanied by a decreaseof the half-saturating concentration of CO₂ to 1.6 µMat 20 mM MgCl₂. Physiological significance of the effects ofP_i on the activation of RuBisCO is discussed. ²Present address: Laboratory of Plant Molecular Physiology,Research Institute of Innovative Technology for the Earth (RITE),9-2 Kizugawadai, Kizu-cho, Soraku-gun, Kyoto, Japan.     

Irreversible cross-linking of heme to the distal tryptophan of stromal ascorbate peroxidase in response to rapid inactivation by H2O2

Ascorbate peroxidase (APX) isoforms localized in the stroma and thylakoid membrane of chloroplasts play a central role in scavenging reactive oxygen species generated by photosystems. These enzymes are inactivated within minutes by H2O2 when the reducing substrate, ascorbate, is depleted. We found that, when the enzyme is inactivated by H2O2, a heme at the catalytic site of a stromal APX isoform is irreversibly cross-linked to a tryptophan residue facing the distal cavity. Mutation of this tryptophan to phenylalanine abolished the cross-linking and increased the half-time for inactivation from <10 to 62 s. In contrast with H2O2-tolerant peroxidases, rapid formation of the cross-link in APXs suggests that a radical in the reaction intermediate tends to be located in the distal tryptophan so that heme is easily cross-linked to it. This is the first report of a mutation that improves the tolerance of chloroplast APXs to H2O2.     

The rbcX gene product promotes the production and assembly of ribulose-1,5-bisphosphate carboxylase/oxygenase of Synechococcus sp. PCC7002 in Escherichia coli

The operon encoding ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in the cyanobacterium Synechococcus sp. PCC7002 contains three rbc genes, rbcL, rbcX and rbcS, in this order. Introduction of translational frameshift into the rbcX gene resulted in a significant decrease in the production of large (RbcL) and small (RbcS) subunits of the Rubisco protein in Synechococcus sp. PCC7002 and in Escherichia coli. To investigate the function of the rbcX gene product (RbcX), we constructed the expression plasmid for the rbcX gene and examined the effects of RbcX on the recombinant Rubisco production in Escherichia coli. The coexpression experiments revealed that RbcX had marked effects on the production of large and small subunits of Rubisco without any significant influence on the mRNA level of rbc genes and/or the post-translational assembly of the Rubisco protein. The present rbcX coexpression system provides a novel and useful method for investigating the Rubisco maturation pathway.     

Isolation of intact vacuoles and proteomic analysis of tonoplast from suspension-cultured cells of Arabidopsis thaliana

A large number of proteins in the tonoplast, including pumps, carriers, ion channels and receptors support the various functions of the plant vacuole. To date, few proteins involved in these activities have been identified at the molecular level. In this study, proteomic analysis was used to identify new tonoplast proteins. A primary requirement of any organelle analysis by proteomics is that the purity of the isolated organelle needs to be high. Using suspension-cultured Arabidopsis cells (Arabidopsis Col-0 cell suspension), a method was developed for the isolation of intact highly purified vacuoles. No plasma membrane proteins were detected in Western blots of the isolated vacuole fraction, and only a few proteins from the Golgi and endoplasmic reticulum. The proteomic analysis of the purified tonoplast involved fractionation of the proteins by SDS-PAGE and analysis by LC-MS/MS. Using this approach, it was possible to identify 163 proteins. These included well-characterized tonoplast proteins such as V-type H+ -ATPases and V-type H+ -PPases, and others with functions reasonably expected to be related to the tonoplast. There were also a number of proteins for which a function has not yet been deduced.     

Cyclic electron flow within PSII functions in intact chloroplasts from spinach leaves

Using thylakoid membranes, we previously demonstrated that accumulated electrons in the photosynthetic electron transport system induces the electron flow from the acceptor side of PSII to its donor side only in the presence of a pH gradient ((Delta)pH) across the thylakoid membranes. This electron flow has been referred to as cyclic electron flow within PSII (CEF-PSII) [Miyake and Yokota (2001) Plant Cell Physiol. 42: 508]. In the present study, we examined whether CEF-PSII operates in isolated intact chloroplasts from spinach leaves, by correlating the quantum yield of PSII [Phi(PSII)] with the activity of the linear electron flow [V(O(2))]. The addition of the protonophore nigericin to the intact chloroplasts decreased Phi(PSII), but increased V(O(2)), and relative electron flux in PSII [Phi(PSII) x PFD] and V(O(2)) were proportional to one another. Phi(PSII) x PFD at a given V(O(2)) was much higher in the presence of (Delta)pH than that in its absence. These effects of nigericin on the relationship between Phi(PSII) x PFD and V(O(2)) are consistent with those previously observed in thylakoid membranes, indicating the occurrence of CEF-PSII also in intact chloroplasts. In the presence of (Delta)pH, CEF-PSII accounted for the excess electron flux in PSII that could not be attributed to photosynthetic linear electron flow. The activity of CEF-PSII increased with increased light intensity and almost corresponded to that of the water-water cycle (WWC), implying that CEF-PSII can dissipate excess photon energy in cooperation with WWC to protect PSII from photoinhibition under limited photosynthesis conditions.     

An evolutionally conserved Lys122 is essential for function in Rhodospirillum rubrum bona fide RuBisCO and Bacillus subtilis RuBisCO-like protein

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and RuBisCO-like protein (RLP) catalyze similar enolase-type reactions. Both enzymes have a conserved non-catalytic Lys122 or Arg122 on the β-strand E lying in the interface between the N- and C-terminal domains. We used site-directed mutagenesis to analyze the function of Lys122 in the form II Rhodospirillum rubrum RuBisCO (RrRuBisCO) and Bacillus subtilis RLP (BsRLP). The K122R mutant of RrRuBisCO had a 40% decrease in k_cat for carboxylase activity, a 2-fold increase in K_m for CO₂, and a 1.9-fold increase in K_m for ribulose-1,5-bisphosphate. K122M and K122E mutants of RrRuBisCO were almost inactive. None of the substitutions affected the thermal stability of RrRuBisCO. The K122R mutant of BsRLP had a 32% decrease in k_cat and lower thermal stability than the wild-type enzyme. The K122M and K122E mutants of BsRLP failed to form a catalytic dimer. Our results suggest that the lysine residue is essential for function in both enzymes, although in each case, its role is likely distinct.     

Metabolic engineering to produce phytochromes with phytochromobilin, phycocyanobilin, or phycoerythrobilin chromophore in Escherichia coli

By co-expression of heme oxygenase and various bilin reductase(s) in a single operon in conjunction with apophytochrome using two compatible plasmids, we developed a system to produce phytochromes with various chromophores in Escherichia coli. Through the selection of different bilin reductases, apophytochromes were assembled with phytochromobilin, phycocyanobilin, and phycoerythrobilin. The blue-shifted difference spectra of truncated phytochromes were observed with a phycocyanobilin chromophore compared to a phytochromobilin chromophore. When the phycoerythrobilin biosynthetic enzymes were co-expressed, E. coli cells accumulated orange-fluorescent phytochrome. The metabolic engineering of bacteria for the production of various bilins for assembly into phytochromes will facilitate the molecular analysis of photoreceptors.