Sulphate removal induces a major conformational change in Leishmania mexicana pyruvate kinase in the crystalline state

We report X-ray structures of pyruvate kinase from Leishmania mexicana (LmPYK) that are trapped in different conformations. These, together with the previously reported structure of LmPYK in its inactive (T-state) conformation, allow comparisons of three different conformers of the same species of pyruvate kinase (PYK). Four new site point mutants showing the effects of side-chain alteration at subunit interfaces are also enzymatically characterised. The LmPYK tetramer crystals grown with ammonium sulphate as precipitant adopt an active-like conformation, with sulphate ions at the active and effector sites. The sulphates occupy positions similar to those of the phosphates of ligands bound to active (R-state) and constitutively active (nonallosteric) PYKs from several species, and provide insight into the structural roles of the phosphates of the substrates and effectors. Crystal soaking in sulphate-free buffers was found to induce major conformational changes in the tetramer. In particular, the unwinding of the Aα6′ helix and the inward hinge movement of the B domain are coupled with a significant widening (4 Å) of the tetramer caused by lateral movement of the C domains. The two new LmPYK structures and the activity studies of site point mutations described in this article are consistent with a developing picture of allosteric activity in which localised changes in protein flexibility govern the distribution of conformer families adopted by the tetramer in its active and inactive states.     

Reducing plant abiotic and biotic stress: Drought and attacks of greenbugs, corn leaf aphids and virus disease in dryland sorghum

Multi-year spatial overlay patterns of plants, insects and soil water may yield insights for management for reducing biotic and abiotic stresses in dryland crops. A study of non-irrigated grain sorghum (Sorghum bicolor (L.) Moench) was conducted in a Pullman clay loam on the semi-arid High Plain of Texas during 2002–2005. The objectives of the 4-year study were to understand the mechanisms of plant spatial and temporal responses to stress from drought, infestations of greenbug, corn leaf aphid (CLA) and maize dwarf mosaic virus (MDMV) disease and soil water content (SWC) heterogeneity, and to reduce plant biotic and abiotic stress using their underlying relationships in space and time. Infrared IRt/c sensed-canopy temperature was measured at 18 or 54 sites along transects in a 6 m × 6 m grid across the years. Greenbugs, CLA, MDMV, SWC and hyperstectral reflectance were determined at each IRt/c site. Natural infestations of greenbugs and CLA on sorghum occurred in early July and insect populations peaked in late July or early August. Insect attacks resulted in plant water stress and sorghum yield loss except a late replanting in early July in 2004. Sorghum grain yield was negatively correlated with canopy temperature, greenbug and CLA (−0.38 < r < −0.75, P < 0.05), and positively correlated with SWC and plant near infrared reflectance (0.25 < r < 0.67, P < 0.05). The IRt/c temperature decreased with SWC but increased with greenbugs and CLA (0.26 < R² < 0.64). Crosscorrelation analysis showed that these insect, crop, and soil variables were correlated in space within 48–54 m. Late planting in July or spray control in late July or early August would be options to reduce dryland sorghum water stress and yield loss from drought and insect attacks.     

Diatom plastids possess a phosphoribulokinase with an altered regulation and no oxidative pentose phosphate pathway

The chloroplast enzyme phosphoribulokinase (PRK; EC 2.7.1.19) is part of the Calvin cycle (reductive pentose phosphate pathway) responsible for CO(2) fixation in photosynthetic organisms. In green algae and vascular plants, this enzyme is light regulated via reversible reduction by reduced thioredoxin. We have sequenced and characterized the gene of the PRK from the marine diatom Odontella sinensis and found that the enzyme has the conserved cysteine residues necessary for thioredoxin-dependent regulation. Analysis of enzymatic activity of partially purified diatom enzyme and of purified protein obtained by native overexpression in Escherichia coli, however, revealed that under natural redox conditions the diatom enzyme is generally active. Treatment of the enzyme with strong oxidants results in inhibition of the enzyme, which is reversible by subsequent incubation with reducing agents. We determined the redox midpoint potentials of the regulatory cysteine in the PRK from O. sinensis in comparison to the respective spinach (Spinacia oleracea) enzyme and found a more positive redox potential for the diatom PRK, indicating that in vivo this enzyme might not be regulated by thioredoxin. We also demonstrate that in protease-treated diatom plastids, activities of enzymes of the oxidative pentose phosphate pathway are not detectable, thus reducing the need for a tight regulation of the Calvin cycle in diatoms. We discuss our results in the context of rearrangements of the subcellular compartmentation of metabolic pathways due to the peculiar evolution of diatoms by secondary endocytobiosis.     

Age-related decline of sodium-dependent ascorbic acid transport in isolated rat hepatocytes

This study investigated whether the age-related decline in hepatic ascorbic acid (AA) levels in rats was due to altered AA uptake. AA concentrations were 68% lower in freshly isolated hepatocytes from old (24-26 months) versus young (3-5 months; p<0.0005) Fischer 344 rats. When incubated with 100 microM AA, cells from old as compared to young rats showed a 66% decline in both the rate of AA transport and the steady state intracellular levels. Sodium-free media significantly reduced AA uptake, suggesting that the sodium-dependent vitamin C transporter (SVCT) was largely responsible for declines in AA transport. Analysis of SVCT messenger RNA (mRNA) levels shows that one isoform of this transport protein, SVCT1, declines 45% with age, with no significant changes in SVCT2 mRNA levels.These results show for the first time that sodium-dependent AA transport declines during the aging process, which may account for much of the loss in tissue AA content.     

Structural role of the active-site metal in the conformation of Trypanosoma brucei phosphoglycerate mutase

Phosphoglycerate mutases (PGAMs) participate in both the glycolytic and the gluconeogenic pathways in reversible isomerization of 3-phosphoglycerate and 2-phosphoglycerate. PGAMs are members of two distinct protein families: enzymes that are dependent on or independent of the 2,3-bisphosphoglycerate cofactor. We determined the X-ray structure of the monomeric Trypanosoma brucei independent PGAM (TbiPGAM) in its apoenzyme form, and confirmed this observation by small angle X-ray scattering data. Comparing the TbiPGAM structure with the Leishmania mexicana independent PGAM structure, previously reported with a phosphoglycerate molecule bound to the active site, revealed the domain movement resulting from active site occupation. The structure reported here shows the interaction between Asp319 and the metal bound to the active site, and its contribution to the domain movement. Substitution of the metal-binding residue Asp319 by Ala resulted in complete loss of independent PGAM activity, and showed for the first time its involvement in the enzyme's function. As TbiPGAM is an attractive molecular target for drug development, the apoenzyme conformation described here provides opportunities for its use in structure-based drug design approaches. Database Structural data for the Trypanosoma brucei 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (iPGAM) has been deposited with the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank under code 3NVL.     

Channel-forming activities in the glycosomal fraction from the bloodstream form of Trypanosoma brucei

Background

Glycosomes are a specialized form of peroxisomes (microbodies) present in unicellular eukaryotes that belong to the Kinetoplastea order, such as Trypanosoma and Leishmania species, parasitic protists causing severe diseases of livestock and humans in subtropical and tropical countries. The organelles harbour most enzymes of the glycolytic pathway that is responsible for substrate-level ATP production in the cell. Glycolysis is essential for bloodstream-form Trypanosoma brucei and enzymes comprising this pathway have been validated as drug targets. Glycosomes are surrounded by a single membrane. How glycolytic metabolites are transported across the glycosomal membrane is unclear.

Methods/Principal Findings

We hypothesized that glycosomal membrane, similarly to membranes of yeast and mammalian peroxisomes, contains channel-forming proteins involved in the selective transfer of metabolites. To verify this prediction, we isolated a glycosomal fraction from bloodstream-form T.brucei and reconstituted solubilized membrane proteins into planar lipid bilayers. The electrophysiological characteristics of the channels were studied using multiple channel recording and single channel analysis. Three main channel-forming activities were detected with current amplitudes 70–80 pA, 20–25 pA, and 8–11 pA, respectively (holding potential +10 mV and 3.0 M KCl as an electrolyte). All channels were in fully open state in a range of voltages ±150 mV and showed no sub-conductance transitions. The channel with current amplitude 20–25 pA is anion-selective (P _K+/P _Cl−∼0.31), while the other two types of channels are slightly selective for cations (P _K+/P _Cl− ratios ∼1.15 and ∼1.27 for the high- and low-conductance channels, respectively). The anion-selective channel showed an intrinsic current rectification that may suggest a functional asymmetry of the channel''s pore.

Conclusions/Significance

These results indicate that the membrane of glycosomes apparently contains several types of pore-forming channels connecting the glycosomal lumen and the cytosol.     

Frontal GABA levels change during working memory

Functional neuroimaging metrics are thought to reflect changes in neurotransmitter flux, but changes in neurotransmitter levels have not been demonstrated in humans during a cognitive task, and the relationship between neurotransmitter dynamics and hemodynamic activity during cognition has not yet been established. We evaluate the concentration of the major inhibitory (GABA) and excitatory (glutamate + glutamine: Glx) neurotransmitters and the cerebral perfusion at rest and during a prolonged delayed match-to-sample working memory task. Resting GABA levels in the dorsolateral prefrontal cortex correlated positively with the resting perfusion and inversely with the change in perfusion during the task. Further, only GABA increased significantly during the first working memory run and then decreased continuously across subsequent task runs. The decrease of GABA over time was paralleled by a trend towards decreased reaction times and higher task accuracy. These results demonstrate a link between neurotransmitter dynamics and hemodynamic activity during working memory, indicating that functional neuroimaging metrics depend on the balance of excitation and inhibition required for cognitive processing.