Karina E.J. Trípodi

Functional characterization of front‐end desaturases from trypanosomatids depicts the first polyunsaturated fatty acid biosynthetic pathway from a parasitic protozoan

A survey of the three kinetoplastid genome projects revealed the presence of three putative front‐end desaturase genes in Leishmania major, one in Trypanosoma brucei and two highly identical ones (98%) in T. cruzi. The encoded gene products were tentatively annotated as Δ8, Δ5 and Δ6 desaturases for L. major, and Δ6 desaturase for both trypanosomes. After phylogenetic and structural analysis of the deduced proteins, we predicted that the putative Δ6 desaturases could have Δ4 desaturase activity, based mainly on the conserved HX3HH motif for the second histidine box, when compared with Δ4 desaturases from Thraustochytrium, Euglena gracilis and the microalga, Pavlova lutheri, which are more than 30% identical to the trypanosomatid enzymes. After cloning and expression in Saccharomyces cerevisiae, it was possible to functionally characterize each of the front‐end desaturases present in L. major and T. brucei. Our prediction about the presence of Δ4 desaturase activity in the three kinetoplastids was corroborated. In the same way, Δ5 desaturase activity was confirmed to be present in L. major. Interestingly, the putative Δ8 desaturase turned out to be a functional Δ6 desaturase, being 35% and 31% identical to Rhizopus oryzae and Pythium irregulareΔ6 desaturases, respectively. Our results indicate that no conclusive predictions can be made about the function of this class of enzymes merely on the basis of sequence homology. Moreover, they indicate that a complete pathway for very‐long‐chain polyunsaturated fatty acid biosynthesis is functional in L. major using Δ6, Δ5 and Δ4 desaturases. In trypanosomes, only Δ4 desaturases are present. The putative algal origin of the pathway in kinetoplastids is discussed.

Purification and characterization of an NAD-dependent malate dehydrogenase from leaves of the crassulacean acid metabolism plant Aptenia cordifolia

An NAD-malate dehydrogenase (NAD-MDH, EC 1.1.1.37) was purified and characterized from leaves of Aptenia cordifolia L. f. (Schwant). This plant performs crassulacean acid metabolism (CAM), as indicated by: (a) elevated levels of phosphoenolpyruvate carboxylase (PEPC) and NAD(P) malic enzyme; (b) regulation of PEPC compatible with its function during the night; (c) characteristic day/night changes in titratable acidity; and (d) gas exchange profile consistent with that shown by CAM plants. These features remained unchanged by water availability or salt stress, suggesting constitutive CAM. The purified MDH showed a subunit molecular mass of 39.4 kDa, a native mass of 83 kDa (dimer) and a pI of 5.8. It cross-reacted with antibodies against cytosolic malate dehydrogenase (cMDH) from pineapple. Maximum activities for oxaloacetate (OAA) reduction or malate oxidation were observed at pH 7.0 and between pH 7.2 and 8.4, respectively. The enzyme was inhibited by excess OAA, in a pH-dependent manner. A discontinuity was observed in Arrhenius plots at 33 °C, with an activation energy twice as high below this temperature. Although immunologically related, some physical and kinetic dissimilarities between the A. cordifolia and pineapple enzymes suggest that diverse CAM metabolic subtypes may require different MDH isozymes to carry out OAA reduction.

Elongation of polyunsaturated fatty acids in trypanosomatids

Leishmania major synthesizes polyunsaturated fatty acids by using Δ6, Δ5 and Δ4 front‐end desaturases, which have recently been characterized [Tripodi KE, Buttigliero LV, Altabe SG & Uttaro AD (2006) FEBS J273, 271–280], and two predicted elongases specific for C18 Δ6 and C20 Δ5 polyunsaturated fatty acids, respectively. Trypanosoma brucei and Trypanosoma cruzi lack Δ6 and Δ5 desaturases but contain Δ4 desaturases, implying that trypanosomes use exogenous polyunsaturated fatty acids to produce C22 Δ4 fatty acids. In order to identify putative precursors of these C22 fatty acids and to completely describe the pathways for polyunsaturated fatty acid biosynthesis in trypanosomatids, we have performed a search in the three genomes and identified four different elongase genes in T. brucei, five in T. cruzi and 14 in L. major. After a phylogenetic analysis of the encoded proteins together with elongases from a variety of other organisms, we selected four candidate polyunsaturated fatty acid elongases. Leishmania major CAJ02037, T. brucei AAX69821 and T. cruzi XP_808770 share 57–52% identity, and group together with C20 Δ5 polyunsaturated fatty acid elongases from algae. The predicted activity was corroborated by functional characterization after expression in yeast. T. brucei elongase was also able to elongate Δ8 and Δ11 C20 polyunsaturated fatty acids. L. major CAJ08636, which shares 33% identity with Mortierella alpinaΔ6 elongase, showed a high specificity for C18 Δ6 polyunsaturated fatty acids. In all cases, a preference for n6 polyunsaturated fatty acids was observed. This indicates that L. major has, as predicted, Δ6 and Δ5 elongases and a complete pathway for polyunsaturated fatty acid biosynthesis. Trypanosomes contain only Δ5 elongases, which, together with Δ4 desaturases, allow them to use eicosapentaenoic acid and arachidonic acid, a precursor that is relatively abundant in the host, for C22 polyunsaturated fatty acid biosynthesis.

Proteomic and metabolomic profiling of Valencia orange fruit after natural frost exposure

The aim of this study was to evaluate the response of orange fruit (Citrus sinensis var. Valencia Late) to freezing stress in planta, both immediately after the natural event and after a week, in order to understand the biochemical and molecular basis of the changes that later derive in internal and external damage symptoms. Using two-dimensional differential gel electrophoresis to analyze exposed and non-exposed fruit, 27 differential protein spots were detected in juice sacs and flavedo, among all comparisons made. Also, primary and secondary metabolites relative contents were analyzed in both tissues by gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry, respectively. Proteins and compounds involved in regulatory functions, iron metabolism, oxidative damage and carbohydrate metabolism were the most affected. Interestingly, three glycolytic enzymes were induced by cold, and there was an increase in fermentation products (volatiles); all of that suggests that more energy generation might be required from glycolysis to counter the cold stress. Moreover, a notable increase in sugar levels was observed after frost, but it was not at the expense of organic acids utilization. Consequently, these results suggest a probable redistribution of photoassimilates in the frost-exposed plants, tending to restore the homeostasis altered by that severe type of stress. Isosinensetin was the most cold-sensitive secondary metabolite because it could not be detected at all after the frost, constituting a possible tool to early diagnose freezing damage.

Characterization of bifunctional sphingolipid Δ4-desaturases/C4-hydroxylases of trypanosomatids by liquid chromatography–electrospray tandem mass spectrometry

Six genes encoding putative sphingolipid desaturases have been identified in trypanosomatid genomes: one in Trypanosoma brucei (TbSLdes protein), one in Trypanosoma cruzi (TcSLdes) and four in Leishmania major (LmSLdes1-4), tandemly arrayed on chromosome 26. The six amino acid sequences showed the three characteristic histidine boxes, with a long spacer between the first and second box, as in fungal desaturases and bifunctional desaturases/hydroxylases, to which they are phylogenetically related. We functionally characterized the trypanosomatid enzymes by their expression in Saccharomyces cerevisiae sur2Δ mutant, which lacks C4-hydroxylase activity. The sphingoid base profile (dinitrophenyl derivatives) of each yeast mutant transformed with each one of the different parasite genes was analyzed by HPLC, using a sur2Δ mutant expressing the Schyzosaccharomyces pombe sphingolipid desaturase (SpSLdes) as positive control. TbSLdes was capable of desaturating endogenous sphingolipids at levels comparable to those found in SpSLdes. By contrast, L. major and T. cruzi enzymes showed either no or negligible activities. Using the HPLC system coupled to electrospray tandem quadrupole/time of flight mass spectrometry we were able to detect significant levels of desaturated and hydroxylated sphingoid bases in extracts of all transformed yeast mutants, except for those transformed with the empty vector. These results indicate that S. pombe, T. brucei, T. cruzi and L. major enzymes are all bifunctional. Using the same methodology, desaturated and hydroxylated sphingoid bases were detected in T. cruzi epimastigotes and L. major promastigote cells, as described previously, and in T. brucei procyclic and bloodstream forms for the first time.