An Insight of RuBisCO Evolution through a Multilevel Approach.

RuBisCO is the most abundant enzyme on earth; it regulates the organic carbon cycle in the biosphere. Studying its structural evolution will help to develop new strategies of genetic improvement in order to increase food production and mitigate CO2 emissions. In the present work, we evaluate how the evolution of sequence and structure among isoforms I, II and III of RuBisCO defines their intrinsic flexibility and residue-residue interactions. To do this, we used a multilevel approach based on phylogenetic inferences, multiple sequence alignment, normal mode analysis, and molecular dynamics. Our results show that the three isoforms exhibit greater fluctuation in the loop between αB and ßC, and also present a positive correlation with loop 6, an important region for enzymatic activity because it regulates RuBisCO conformational states. Likewise, an increase in the flexibility of the loop structure between αB and ßC, as well as Lys330 (form II) and Lys322 (form III) of loop 6, is important to increase photosynthetic efficiency. Thus, the cross-correlation dynamics analysis showed changes in the direction of movement of the secondary structures in the three isoforms. Finally, key amino acid residues related to the flexibility of the RuBisCO structure were indicated, providing important information for its enzymatic engineering.

Electronic structure benchmark calculations of CO2 fixing elementary chemical steps in RuBisCO using the projector-based embedding approach.

Ribulose 1,5-bisphosphate carboxylase-oxygenase (RuBisCO) is the main enzyme involved in atmospheric carbon dioxide (CO2 ) fixation in the biosphere. This enzyme catalyzes a set of five chemical steps that take place in the same active-site within magnesium (II) coordination sphere. Here, a set of electronic structure benchmark calculations have been carried out on a reaction path proposed by Gready et al. by means of the projector-based embedding approach. Activation and reaction energies for all main steps catalyzed by RuBisCO have been calculated at the MP2, SCS-MP2, CCSD, and CCSD(T)/aug-cc-pVDZ and cc-pVDZ levels of theory. The treatment of the magnesium cation with post-HF methods is explored to determine the nature of its involvement in the mechanism. With the high-level ab initio values as a reference, we tested the performance of a set of density functional theory (DFT) exchange-correlation (xc) functionals in reproducing the reaction energetics of RuBisCO carboxylase activity on a set of model fragments. Different DFT xc-functionals show large variation in activation and reaction energies. Activation and reaction energies computed at the B3LYP level are close to the reference SCS-MP2 results for carboxylation, hydration and protonation reactions. However, for the carbon-carbon bond dissociation reaction, B3LYP and other functionals give results that differ significantly from the ab initio reference values. The results show the applicability of the projector-based embedding approach to metalloenzymes. This technique removes the uncertainty associated with the selection of different DFT xc-functionals and so can overcome some of inherent limitations of DFT calculations, complementing, and potentially adding to modeling of enzyme reaction mechanisms with DFT methods.

Comparative modeling and molecular dynamics suggest high carboxylase activity of the Cyanobium sp. CACIAM14 RbcL protein.

Rubisco catalyzes the first step reaction in the carbon fixation pathway, bonding atmospheric CO2/O2 to ribulose 1,5-bisphosphate; it is therefore considered one of the most important enzymes in the biosphere. Genetic modifications to increase the carboxylase activity of rubisco are a subject of great interest to agronomy and biotechnology, since this could increase the productivity of biomass in plants, algae and cyanobacteria and give better yields in crops and biofuel production. Thus, the aim of this study was to characterize in silico the catalytic domain of the rubisco large subunit (rbcL gene) of Cyanobium sp. CACIAM14, and identify target sites to improve enzyme affinity for ribulose 1,5-bisphosphate. A three-dimensional model was built using MODELLER 9.14, molecular dynamics was used to generate a 100 ns trajectory by AMBER12, and the binding free energy was calculated using MM-PBSA, MM-GBSA and SIE methods with alanine scanning. The model obtained showed characteristics of form-I rubisco, with 15 beta sheets and 19 alpha helices, and maintained the highly conserved catalytic site encompassing residues Lys175, Lys177, Lys201, Asp203, and Glu204. The binding free energy of the enzyme-substrate complexation of Cyanobium sp. CACIAM14 showed values around -10 kcal mol(-1) using the SIE method. The most important residues for the interaction with ribulose 1,5-bisphosphate were Arg295 followed by Lys334. The generated model was successfully validated, remaining stable during the whole simulation, and demonstrated characteristics of enzymes with high carboxylase activity. The binding analysis revealed candidates for directed mutagenesis sites to improve rubisco's affinity.

Historical review of clinical vaccine studies at Oswaldo Cruz Institute and Oswaldo Cruz Foundation - technological development issues

This paper presents, from the perspective of technological development and production, the results of an investigation examining 61 clinical studies with vaccines conducted in Brazil between 1938-2013, with the participation of the Oswaldo Cruz Institute (IOC) and the Oswaldo Cruz Foundation (Fiocruz). These studies have been identified and reviewed according to criteria, such as the kind of vaccine (viral, bacterial, parasitic), their rationale, design and methodological strategies. The results indicate that IOC and Fiocruz have accumulated along this time significant knowledge and experience for the performance of studies in all clinical phases and are prepared for the development of new vaccines products and processes. We recommend national policy strategies to overcome existing regulatory and financing constraints.

Entropy production in oscillatory processes during photosynthesis.

The flow of matter and heat and the rate of enzymatic reactions are examined using two models of photosynthesis that exhibit sustained and damped oscillatory dynamics, with the objective of calculating the rate of entropy generation and studying the effects of temperature and kinetic constants on the thermodynamic efficiency of photosynthesis. The global coefficient of heat transfer and the direct and inverse constants of the formation reaction of the RuBisCO-CO2 complex were used as control parameters. Results show that when the system moves from isothermal to non-isothermal conditions, the transition from a steady state to oscillations facilitates an increase in the energy efficiency of the process. The simulations were carried out for two photosynthetic models in a system on a chloroplast reactor scale.

RBCS1 expression in coffee: Coffea orthologs, Coffea arabica homeologs, and expression variability between genotypes and under drought stress.

BACKGROUND: In higher plants, the inhibition of photosynthetic capacity under drought is attributable to stomatal and non-stomatal (i.e., photochemical and biochemical) effects. In particular, a disruption of photosynthetic metabolism and Rubisco regulation can be observed. Several studies reported reduced expression of the RBCS genes, which encode the Rubisco small subunit, under water stress. RESULTS: Expression of the RBCS1 gene was analysed in the allopolyploid context of C. arabica, which originates from a natural cross between the C. canephora and C. eugenioides species. Our study revealed the existence of two homeologous RBCS1 genes in C. arabica: one carried by the C. canephora sub-genome (called CaCc) and the other carried by the C. eugenioides sub-genome (called CaCe). Using specific primer pairs for each homeolog, expression studies revealed that CaCe was expressed in C. eugenioides and C. arabica but was undetectable in C. canephora. On the other hand, CaCc was expressed in C. canephora but almost completely silenced in non-introgressed ("pure") genotypes of C. arabica. However, enhanced CaCc expression was observed in most C. arabica cultivars with introgressed C. canephora genome. In addition, total RBCS1 expression was higher for C. arabica cultivars that had recently introgressed C. canephora genome than for "pure" cultivars. For both species, water stress led to an important decrease in the abundance of RBCS1 transcripts. This was observed for plants grown in either greenhouse or field conditions under severe or moderate drought. However, this reduction of RBCS1 gene expression was not accompanied by a decrease in the corresponding protein in the leaves of C. canephora subjected to water withdrawal. In that case, the amount of RBCS1 was even higher under drought than under unstressed (irrigated) conditions, which suggests great stability of RBCS1 under adverse water conditions. On the other hand, for C. arabica, high nocturnal expression of RBCS1 could also explain the accumulation of the RBCS1 protein under water stress. Altogether, the results presented here suggest that the content of RBCS was not responsible for the loss of photosynthetic capacity that is commonly observed in water-stressed coffee plants. CONCLUSION: We showed that the CaCe homeolog was expressed in C. eugenioides and non-introgressed ("pure") genotypes of C. arabica but that it was undetectable in C. canephora. On the other hand, the CaCc homeolog was expressed in C. canephora but highly repressed in C. arabica. Expression of the CaCc homeolog was enhanced in C. arabica cultivars that experienced recent introgression with C. canephora. For both C. canephora and C. arabica species, total RBCS1 gene expression was highly reduced with WS. Unexpectedly, the accumulation of RBCS1 protein was observed in the leaves of C. canephora under WS, possibly coming from nocturnal RBCS1 expression. These results suggest that the increase in the amount of RBCS1 protein could contribute to the antioxidative function of photorespiration in water-stressed coffee plants.

Quantitative structure-activity relationship of rubiscolin analogues as delta opioid peptides using comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA).

Three-dimensional quantitative structure-activity relationship (3D-QSAR) studies were carried out on a series of 38 rubiscolins as delta opioid peptides using comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA). Quantitative information on structure-activity relationships is provided for further rational development and direction of selective synthesis. All models were carried out over a training set including 30 peptides. The best CoMFA model included electrostatic and steric fields and had a moderate Q (2) = 0.503. CoMSIA analysis surpassed the CoMFA results: the best CoMSIA model included only the hydrophobic field and had a Q (2) = 0.661. In addition, this model predicted adequately the peptides contained in the test set. Our model identified that the potency of delta opioid activity of rubiscolin analogues essentially exhibited a significant relationship with local hydrophobic and hydrophilic characteristics of amino acids at positions 3, 4, 5, and 6.

Biophysical properties of a synthetic transit peptide from wheat chloroplast ribulose 1,5-bisphosphate carboxylase.

The surface properties of pure RuBisCo transit peptide (RTP) and its interaction with zwitterionic, anionic phospholipids and chloroplast lipids were studied by using the Langmuir monolayer technique. Pure RTP is able to form insoluble films and the observed surface parameters are compatible with an alpha-helix perpendicular to the interface. The alpha-helix structure tendency was also observed by using transmission FT-IR spectroscopy in bulk system of a membrane mimicking environment (SDS). On the other hand, RTP adopts an unordered structure in either aqueous free interface or in the presence of vesicles composed of a zwitterionic phospholipid (POPC). Monolayer studies show that in peptide/lipid mixed monolayers, RTP shows no interaction with zwitterionic phospholipids, regardless of their physical state. Also, with the anionic POPG at high peptide ratios RTP retains its individual surface properties and behaves as an immiscible component of the peptide/lipid mixed interface. This behaviour was also observed when the mixed films were composed by RTP and the typical chloroplast lipids MGDG or DGDG (mono- and di-galactosyldiacylglycerol). Conversely, RTP establishes a particular interaction with phosphatidylglycerol and cardiolipin at low peptide to lipid area covered relation. This interaction takes place with an increase in surface stability and a reduction in peptide molecular area (intermolecular interaction). Data suggest a dynamic membrane modulation by which the peptide fine-tunes its membrane orientation and its lateral stability, depending on the quality (lipid composition) of the interface.

Methyl jasmonate promotes the transient reduction of the levels of 2-Cys peroxiredoxin in Ricinus communis plants.

Jasmonates are signaling molecules that play a key role in the regulation of metabolic processes, reproduction and defense against insects and pathogens. This study investigated the effects of methyl jasmonate on the protein pattern of Ricinus communis plants and the activity of guaiacol peroxidase, an antioxidant enzyme. Methyl jasmonate treatment caused a transient reduction in guaiacol peroxidase activity. A similar response was observed for the levels of 2-Cys peroxiredoxin protein. Moreover, the levels of the small and large chains of Rubisco were also reduced. The transient reduction of the levels and activity of antioxidant enzymes could account for the increase in the levels of H2O2, an important signaling molecule in plant defense.

The 70-kDa heat-shock protein/DnaK chaperone system is required for the productive folding of ribulose-biphosphate carboxylase subunits in Escherichia coli.

We have studied the in vivo requirements of the DnaK chaperone system for the folding of recombinant ribulose-bisphosphate carboxylase/oxygenase in Escherichia coli. Expression of functional dimeric or hexadecameric ribulose-bisphosphate carboxylase from different bacterial sources (including purple bacteria and cyanobacteria) was severely impaired in E. coli dnaK, dnaJ, or grpE mutants. These enzymes were synthesized mostly in soluble, fully enzymatically active forms in wild-type E. coli cells cultured in the temperature range 20-42 degrees C, but aggregated extensively in dnaK null mutants. Co-expression of dnaK, but not groESL, markedly reduced the aggregation of ribulose-bisphosphate carboxylase subunits in dnaK null mutants and restored the enzyme activity to levels found in isogenic wild-type strains. Ribulose-bisphosphate carboxylase expression in wild-type E. coli cells growing at 30 degrees C promoted an enhanced synthesis of stress proteins, apparently by sequestering DnaK from its negative regulatory role in this response. The overall results indicate that the DnaK chaperone system assists in vivo the folding pathway of ribulose-bisphosphate carboxylase large subunits, most probably at its very early stages.