ABSTRACT
For coping with energetic and synthetic challenges, parasites require high activities of adenylate kinase (AK; ATP + AMP <==> 2 ADP) and GTP:AMP phosphotransferase (GAK; GTP + AMP <==> GDP + ADP). These enzymes were identified in erythrocytic stages of Plasmodium falciparum. The genes encoding PfAK and PfGAK are located on chromosomes 10 and 4, respectively. Molecular cloning and heterologous expression in E. coli yielded enzymatically active proteins of 28.9 (PfAK) and 28.0 kDa (PfGAK). Recombinant PfAK resembles authentic PfAK in its biochemical characteristics including the possible association with a stabilizing protein and the high specificity for AMP as the mononucleotide substrate. Specificity is less stringent for the triphosphate, with ATP as the best substrate (75 U/mg; kcat = 2160 min(-1) at 25 degrees C). PfAK contains the sequence of the amphiphatic helix that is known to mediate translocation of the cytosolic protein into the mitochondrial intermembrane space. PfGAK exhibits substrate preference for GTP and AMP (100 U/mg; kcat = 2800 min(-1) at 25 degrees C); notably, there is no detectable activity with ATP. In contrast to its human orthologue (AK3), PfGAK contains a zinc finger motif and binds ionic iron. The dinucleoside pentaphosphate compounds AP5A and GP5A inhibited PfAK and PfGAK, respectively, with Ki values of approximately 0.2 microM which is more than 250-fold lower than the KM values determined for the nucleotide substrates. The disubstrate inhibitors are useful for studying the enzymatic mechanism of PfAK and PfGAK as well as their function in adenine nucleotide homeostasis; in addition, the chimeric inhibitors represent interesting lead compounds for developing nucleosides to be used as antiparasitic agents.
Subject(s)
Adenylate Kinase/metabolism , Nucleoside-Phosphate Kinase/metabolism , Plasmodium falciparum/enzymology , Adenylate Kinase/antagonists & inhibitors , Adenylate Kinase/genetics , Amino Acid Sequence , Animals , Antimalarials/pharmacology , Base Sequence , DNA, Protozoan/genetics , Energy Metabolism , Enzyme Inhibitors/pharmacology , Genes, Protozoan , Humans , Molecular Sequence Data , Nucleoside-Phosphate Kinase/antagonists & inhibitors , Nucleoside-Phosphate Kinase/genetics , Plasmodium falciparum/genetics , Plasmodium falciparum/growth & development , Plasmodium falciparum/metabolism , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Substrate SpecificityABSTRACT
A growing body of evidence suggests that oxidative stress is a common underlying mechanism in the pathogenesis of neurodegenerative disorders such as Alzheimer's, Huntington's, Creutzfeld-Jakob and Parkinson's diseases. Despite the increasing number of reports finding a causal relation between oxidative stress and neurodegeneration, little is known about the genetic elements that confer protection against the deleterious effects of oxidation in neurons. We have isolated and characterized the Drosophila melanogaster gene sniffer, whose function is essential for preventing age-related neurodegeneration. In addition, we demonstrate that oxidative stress is a direct cause of neurodegeneration in the Drosophila central nervous system and that reduction of sniffer activity leads to neuronal cell death. The overexpression of the gene confers neuronal protection against oxygen-induced apoptosis, increases resistance of flies to experimental normobaric hyperoxia, and improves general locomotor fitness. Sniffer belongs to the family of short-chain dehydrogenase/reductase (SDR) enzymes and exhibits carbonyl reductase activity. This is the first in vivo evidence of the direct and important implication of this enzyme as a neuroprotective agent in the cellular defense mechanisms against oxidative stress.
Subject(s)
Alcohol Oxidoreductases/genetics , Apoptosis/physiology , Drosophila Proteins/genetics , Drosophila melanogaster/genetics , Nerve Degeneration/genetics , Neurons/physiology , Age Factors , Alcohol Oxidoreductases/physiology , Amino Acid Sequence , Animals , Apoptosis/genetics , Blotting, Western , Brain/physiopathology , Drosophila Proteins/physiology , Drosophila melanogaster/physiology , Electrophoresis, Polyacrylamide Gel , Gene Expression , Histocytochemistry , In Situ Nick-End Labeling , Molecular Sequence Data , Neurons/metabolism , Oxidative Stress/genetics , Oxidative Stress/physiology , Oxygen , Reverse Transcriptase Polymerase Chain Reaction , Sequence AlignmentABSTRACT
Defense against oxidative stress in mammals includes the regeneration of the major thiol reductants glutathione and thioredoxin by glutathione reductase and thioredoxin reductase (TrxR), respectively. In contrast, Drosophila, and possibly insects in general, lacks glutathione reductase and must rely solely on the TrxR system. The mammalian TrxRs described so far are selenoproteins that utilize NADPH to reduce protein as well as nonprotein substrates in mitochondria and cytoplasm of cells. We show that a single Drosophila gene, Trxr-1, encodes non-selenocysteine-containing cytoplasmic and mitochondrial TrxR isoforms that differ with respect to their N termini. We generated transcript-specific mutants and used in vivo approaches to explore the biological functions of the two enzyme variants by introducing the corresponding transgenes into different Trxr-1 mutants. The results show that, although the two TrxR isoforms have similar biochemical properties, their biological functions are not interchangeable.
