Site-directed mutagenesis in rat liver 6-phosphofructo-2-kinase. Mutation at the fructose 6-phosphate binding site affects phosphate activation.

To identify those residues involved in fructose 6-phosphate binding to the kinase domain of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase site-directed mutations were engineered at Lys194, Arg195, Arg230, and Arg238. The mutant enzymes were purified to homogeneity by anion exchange and Blue-Sepharose chromatography and/or substrate elution from phosphocellulose columns. Circular dichroism experiments demonstrated that all of the single amino acid mutations had no effect on the secondary structure of the protein. In addition, when fructose-2,6-bisphosphatase activity was measured, all mutants had Km values for fructose 2,6-bisphosphate, Ki values for fructose 6-phosphate, and maximal velocities similar to that of the wild-type enzyme. Mutation of Arg195----Ala, or His, had little or no effect on the maximal velocity of the kinase but increased the Km for fructose 6-phosphate greater than 3,000-fold. Furthermore, the Ka for phosphate for Arg195Ala was increased 100-fold compared with the wild-type enzyme. Mutation of Lys194----Ala had no effect on maximal velocity or the Km for fructose 6-phosphate. Mutation of either Arg230 or Arg238----Ala increased the maximal velocity and the Km for fructose-6 phosphate of the kinase by 2-3-fold but had no effect on fructose-2,6-bisphosphatase. However, the Km values for ATP of the Arg230Ala and Arg238Ala mutants were 30-40-fold higher than that for the wild-type enzyme. Mutation of Gly48----Ala resulted in a form with no kinase activity, but fructose-2,6-bisphosphatase activity was identical to that of the wild-type enzyme. The results indicate that: 1) Arg195 is a critical residue for the binding of fructose 6-phosphate to the 6-phospho-fructo-2-kinase domain, and that interaction of the sugar phosphate with Arg195 is highly specific since mutation of the adjacent Lys194----Ala had no effect on fructose 6-phosphate binding; 2) Arg195 also play an important role in the binding of inorganic phosphate; and 3) Gly48 is an important residue in the nucleotide binding fold of 6-phosphofructo-2-kinase and that both Arg230 and Arg238 are also involved in ATP binding; and 4) the bifunctional enzyme has two separate and independent fructose 6-phosphate binding sites.     

Folding and Hydrodynamics of a DNA i-Motif from the c-MYC Promoter Determined by Fluorescent Cytidine Analogs

The four-stranded i-motif (iM) conformation of cytosine-rich DNA has importance to a wide variety of biochemical systems that range from their use in nanomaterials to potential roles in oncogene regulation. The iM structure is formed at slightly acidic pH, where hemiprotonation of cytosine results in a stable C-C⁺ basepair. Here, we performed fundamental studies to examine iM formation from a C-rich strand from the promoter of the human c-MYC gene. We used a number of biophysical techniques to characterize both the hydrodynamic properties and folding kinetics of a folded iM. Our hydrodynamic studies using fluorescence anisotropy decay and analytical ultracentrifugation show that the iM structure has a compact size in solution and displays the rigidity of a double strand. By studying the rates of circular dichroism spectral changes and quenching of fluorescent cytidine analogs, we also established a mechanism for the folding of a random coil oligo into the iM. In the course of determining this folding pathway, we established that the fluorescent dC analogs tC° and PdC can be used to monitor individual residues of an iM structure and to determine the pK_a of an iM. We established that the C-C⁺ hydrogen bonding of certain bases initiates the folding of the iM structure. We also showed that substitutions in the loop regions of iMs give a distinctly different kinetic signature during folding compared with bases that are intercalated. Our data reveal that the iM passes through a distinct intermediate form between the unfolded and folded forms. Taken together, our results lay the foundation for using fluorescent dC analogs to follow structural changes during iM formation. Our technique may also be useful for examining folding and structural changes in more complex iMs.     

Targeted integration of foreign genes into repetitive sequences of the Brevibacterium lactofermentum chromosome

Abstract The nucleotide sequence of a gene coding for a 37 kDa subunit of a cytosolic malate dehydrogenase of Trichomonas vaginalis was established. The sequences of a gDNA clone and a cDNA clone, which lacked seven amino-terminal codons, were identical, indicating an absence of introns from the gene. Cell fractionation combined with sequencing of peptide fragments of the purified enzyme showed that the gene codes for an expressed cytosolic enzyme. The derived amino acid sequence was closely related to cytosolic malate dehydrogenases from animals and plants and from the eubacteria Thermus aquaticus and Mycobacterium leprae and was more distant from the enzyme of mitochondria and from Escherichia coli and certain other eubacteria. In phylogenetic reconstructions this enzyme shared a most recent common ancestor with other cytosolic enzymes.     

Seed priming with iron and zinc in bread wheat: effects in germination,mitosis and grain yield

Currently, the biofortification of crops like wheat with micronutrients such as iron (Fe) and zinc (Zn) is extremely important due to the deficiencies of these micronutrients in the human diet and in soils. Agronomic biofortification with Fe and Zn can be done through different exogenous strategies such as soil application, foliar spraying, and seed priming. However, the excess of these micronutrients can be detrimental to the plants. Therefore, in the last decade, a high number of studies focused on the evaluation of their phytotoxic effects to define the best strategies for biofortification of bread wheat. In this study, we investigated the effects of seed priming with different dosages (1 mg L^?1 to 8 mg L^?1) of Fe and/or Zn in germination, mitosis and yield of bread wheat cv. ‘Jordão’ when compared with control. Overall, our results showed that: micronutrient dosages higher than 4 mg L^?1 negatively affect the germination; Fe and/or Zn concentrations higher than 2 mg L^?1 significantly decrease the mitotic index and increase the percentage of dividing cells with anomalies; treatments performed with 8 mg L^?1 of Fe and/or 8 mg L^?1 Zn caused negative effects in germination, mitosis and grain yield. Moreover, seed priming with 2 mg L^?1 Fe?+?2 mg L^?1 Zn has been shown to be non-cytotoxic, ensuring a high rate of germination (80%) and normal dividing cells (90%) as well as improving tillering and grain yield. This work revealed that seed priming with Fe and Zn micronutrients constitutes a useful and alternative approach for the agronomic biofortification of bread wheat.     

Metabolic fingerprinting analysis of oil palm reveals a set of differentially expressed metabolites in fatal yellowing symptomatic and non-symptomatic plants

Introduction

Oil palm (E. guineensis), the most consumed vegetable oil in the world, is affected by fatal yellowing (FY), a condition that can lead to the plant’s death. Although studies have been performed since the 1980s, including investigations of biotic and abiotic factors, FY’s cause remains unknown and efforts in researches are still necessary.

Objectives

This work aims to investigate the metabolic expression in plants affected by FY using an untargeted metabolomics approach.

Method

Metabolic fingerprinting analysis of oil palm leaves was performed using ultra high liquid chromatography–electrospray ionization–mass spectrometry (UHPLC–ESI–MS). Chemometric analysis, using principal component analysis (PCA) and partial least square discriminant analysis (PLS-DA), was applied to data analysis. Metabolites identification was performed by high resolution mass spectrometry (HRMS), MS/MS experiments and comparison with databases and literature.

Results

Metabolomics analysis based on MS detected more than 50 metabolites in oil palm leaf samples. PCA and PLS-DS analysis provided group segregation and classification of symptomatic and non-symptomatic FY samples, with a great external validation of the results. Nine differentially expressed metabolites were identified as glycerophosphorylcholine, arginine, asparagine, apigenin 6,8-di-C-hexose, tyramine, chlorophyllide, 1,2-dihexanoyl-sn-glycero-3-phosphoethanolamine, proline and malvidin 3-glucoside-5-(6″-malonylglucoside). Metabolic pathways and biological importance of those metabolites were assigned.

Conclusion

Nine metabolites were detected in a higher concentration in non-symptomatic FY plants. Seven are related to stress factors i.e. plant defense and nutrient absorption, which can be affected by the metabolic depression of these compounds. Two of those metabolites (glycerophosphorylcholine and 1,2-dihexanoyl-sn-glycero-3-phosphoethanolamine) are presented as potential biomarkers, since they have no known direct relation to plant stress.

     

Territory size as a main driver of male-mating success in an Amazonian nurse frog (<Emphasis Type="Italic">Allobates paleovarzensis</Emphasis>, Dendrobatoidea)

In polygamous mating systems, it is most often males that compete for the opposite sex, using strategies to provide access to as many females as possible. Females, on the other hand, constitute the sex that exerts the choice and so require a means of accessing the quality of a potential partner in comparison to its competitors. A common challenge in sexual selection studies is to identify the most relevant trait for mating success, since many are correlated with each other. In addition, little is known about how the female accesses the aspects related to male quality. In this context, we tested the role of different male characteristics on mating success in a natural environment using the Amazonian frog Allobates paleovarzensis as a model. A multiple linear regression model showed a positive relationship between territory size and number of male matings, while calling persistence was slightly related to the mating success. We did not detect a relation of the number of matings with the distance to the nearest body of water nor with male body size. Additionally, we observed that territory size was not related to calling persistence, but had a positive relation with the duration of the couple’s courting process. Thus, we conclude that: (1) territory size is the main determinant of male-mating success, and this is not correlated with the other attributes tested; and (2) females access the size of the males’ territory through the courting process that precedes oviposition.     

Structural basis of Smad1 activation by receptor kinase phosphorylation.

Phosphorylation of Smad1 at the conserved carboxyl terminal SVS sequence activates BMP signaling. Here we report the crystal structure of the Smad1 MH2 domain in a conformation that reveals the structural effects of phosphorylation and a molecular mechanism for activation. Within a trimeric subunit assembly, the SVS sequence docks near two putative phosphoserine binding pockets of the neighboring molecule, in a position ready to interact upon phosphorylation. The MH2 domain undergoes concerted conformational changes upon activation, which signal Smad1 dissociation from the receptor kinase for subsequent heteromeric assembly with Smad4. Biochemical and modeling studies reveal unique favorable interactions within the Smad1/Smad4 heteromeric interface, providing a structural basis for their association in signaling.