RESUMO
Dihydroxyacetone (DHA) can be used as an energy source by many cell types; however, it is toxic at high concentrations. The enzyme dihydroxyacetone kinase (DAK) has shown to be involved in DHA detoxification and osmoregulation. Among protozoa of the genus Trypanosoma, T. brucei, which causes sleeping sickness, is highly sensitive to DHA and does not have orthologous genes to DAK. Conversely, T. cruzi, the etiological agent of Chagas Disease, has two putative ATP-dependent DAK (TcDAKs) sequences in its genome. Here we show that T. cruzi epimastigote lysates present a DAK specific activity of 27.1 nmol/min/mg of protein and that this form of the parasite is able to grow in the presence of 2 mM DHA. TcDAK gene was cloned and the recombinant enzyme (recTcDAK) was expressed in Escherichia coli. An anti-recTcDAK serum reacted with a protein of the expected molecular mass of 61 kDa in epimastigotes. recTcDAK presented maximal activity using Mg+2, showing a Km of 6.5 µM for DHA and a K0.5 of 124.7 µM for ATP. As it was reported for other DAKs, recTcDAK activity was inhibited by FAD with an IC50 value of 0.33 mM. In conclusion, TcDAK is the first DAK described in trypanosomatids confirming another divergent metabolism between T. brucei and T. cruzi.
Assuntos
Fosfotransferases (Aceptor do Grupo Álcool)/isolamento & purificação , Trypanosoma cruzi/enzimologia , Sequência de Aminoácidos , Animais , Western Blotting , Chlorocebus aethiops , Di-Hidroxiacetona/metabolismo , Di-Hidroxiacetona/toxicidade , Eletroforese em Gel de Poliacrilamida , Imunofluorescência , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Osmorregulação , Fosfotransferases (Aceptor do Grupo Álcool)/química , Fosfotransferases (Aceptor do Grupo Álcool)/classificação , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma cruzi/efeitos dos fármacos , Células VeroRESUMO
Reed canary grass (RCG) is a perennial grass traditionally cultivated for forage. It is also used as fuel to produce energy in Finland and Sweden, and other countries have expressed interest in the cultivation of RCG. In China, arable land is limited. Salinity is considered to be a major factor limiting plant crop development and productivity. To boost biofuel production of RCG and extend its range in saline soil, we seek to improve its salt tolerance. Proline acts as an osmolyte that accumulates when plants are subjected to abiotic stress. P5CS plays a crucial role in proline biosynthesis. We isolated a P5CS gene from RCG, designated B231P5CS (GenBank accession No. JQ622685). B231P5CS is a fragment (971 bp) that encodes a 323-amino acid polypeptide. We also cloned an actin gene fragment from RCG as a reference gene in expression analysis of B231P5CS gene. Expression analysis revealed that B231P5CS transcripts were upregulated in leaves after treatment with salt (200 mM NaCl) and that transcript levels of B231P5CS reached a maximum 12 h after exposure, which was 14.69 times the level in control plants. The trends of expression were exactly opposite in roots; transcripts were downregulated after salt treatment. Proline concentration increased in leaves after stress. In contrast, proline content of roots decreased up to 3.6-fold relative to controls. Changes in proline concentration after stress were correlated with B231P5CS expression. Our results suggest that B231P5CS is a stress-inducible gene and plays a non-redundant role in plant development. This gene may be used to improve stress tolerance of RGC and other bioenergy feedstock.
Assuntos
Glutamato-5-Semialdeído Desidrogenase/genética , Complexos Multienzimáticos/genética , Phalaris/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Proteínas de Plantas/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA Complementar/química , DNA Complementar/genética , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Glutamato-5-Semialdeído Desidrogenase/classificação , Glutamato-5-Semialdeído Desidrogenase/metabolismo , Dados de Sequência Molecular , Complexos Multienzimáticos/classificação , Complexos Multienzimáticos/metabolismo , Phalaris/enzimologia , Phalaris/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/classificação , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Filogenia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/classificação , Proteínas de Plantas/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Prolina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tolerância ao Sal/genética , Análise de Sequência de DNA , Cloreto de Sódio/farmacologia , Estresse Fisiológico/genética , Fatores de TempoRESUMO
In several archaea of the Euryarchaeota, the glycolytic flux proceeds through a modified version of the Embden-Meyerhof pathway, where the phosphofructokinase and glucokinase enzymes use ADP as the phosphoryl donor. These enzymes are homologous to each other. In the hyperthermophilic methanogenic archaeon Methanocaldococcus jannaschii, it has been possible to identify only one homolog for these enzymes, which shows both ADP-dependent glucokinase and phosphofructokinase activity. This enzyme has been proposed as an ancestral form in this family. In this work we studied the evolution of this protein family using the Bayesian method of phylogenetic inference and real value evolutionary trace in order to test the ancestral character of the bifunctional enzyme. Additionally, to search for specificity determinants of these two functions, we have modeled the bifunctional protein and its interactions with both sugar substrates using protein-ligand docking and restricted molecular dynamics. The results show that the evolutionary story of this family is complex. The root of the family is located inside the glucokinase group, showing that the bifunctional enzyme is not an ancestral form, but could be a transitional form from glucokinase to phosphofructokinase, due to its basal location within the phosphofructokinase group. The evolutionary trace and the molecular modeling experiments showed that the specificity for fructose 6-phosphate is mainly related to the stabilization of a negative charge in the phosphate group, whereas the specificity for glucose is related to the presence of some histidines instead of glutamines/asparagines and to the interaction of this ligand with a glutamic acid residue corresponding to Glu82 in the bifunctional enzyme.