Abstract
BackgroundAnopheles stephensi mitochondrial malic enzyme (ME) emerged as having a relevant role in the provision of pyruvate for the Krebs' cycle because inhibition of this enzyme results in the complete abrogation of oxygen uptake by mitochondria. Therefore, the identification of ME in mitochondria from immortalized A. stephensi (ASE) cells and the investigation of the stereoselectivity of malate analogues are relevant in understanding the physiological role of ME in cells of this important malaria parasite vector and its potential as a possible novel target for insecticide development.MethodsTo characterize the mitochondrial ME from immortalized ASE cells (Mos. 43; ASE), mass spectrometry analyses of trypsin fragments of ME, genomic sequence analysis and biochemical assays were performed to identify the enzyme and evaluate its activity in terms of cofactor dependency and inhibitor preference.ResultsThe encoding gene sequence and primary sequences of several peptides from mitochondrial ME were found to be highly homologous to the mitochondrial ME from Anopheles gambiae (98%) and 59% homologous to the mitochondrial NADP+-dependent ME isoform from Homo sapiens. Measurements of ME activity in mosquito mitochondria isolated from ASE cells showed that (i) Vmax with NAD+ was 3-fold higher than that with NADP+, (ii) addition of Mg2+ or Mn2+ increased the Vmax by 9- to 21-fold, with Mn2+ 2.3-fold more effective than Mg2+, (iii) succinate and fumarate increased the activity by 2- and 5-fold, respectively, at sub-saturating concentrations of malate, (iv) among the analogs of L-malate tested as inhibitors of the NAD+-dependent ME catalyzed reaction, small (2- to 3-carbons) organic diacids carrying a 2-hydroxyl/keto group behaved as the most potent inhibitors of ME activity (e.g., oxaloacetate, tartronic acid and oxalate).ConclusionsThe biochemical characterization of Anopheles stephensi ME is of critical relevance given its important role in bioenergetics, suggesting that it is a suitable target for insecticide development.
Highlights
Anopheles stephensi mitochondrial malic enzyme (ME) emerged as having a relevant role in the provision of pyruvate for the Krebs’ cycle because inhibition of this enzyme results in the complete abrogation of oxygen uptake by mitochondria
The critical role of ME in the bioenergetics of mosquitoes along with the deeper understanding of its biochemistry and chemical requirements for inhibition, suggests that this enzyme could became a suitable target for insecticide development
Enzymes significantly associated with insecticide resistance include esterases and cytochrome P450s involved in the oxidative catabolism of insecticides [45,46,47]
Summary
Anopheles stephensi mitochondrial malic enzyme (ME) emerged as having a relevant role in the provision of pyruvate for the Krebs’ cycle because inhibition of this enzyme results in the complete abrogation of oxygen uptake by mitochondria. Mitochondria of cultured cells [ASE cell line (A. stephensi Mos. 43 cell line)] from A. stephensi, as well as flight muscle mitochondria of a beetle (Popillia japonica), which have the ability to oxidize proline at a high rate, have been shown to contain an unusually active malic enzyme [8]. The latter species utilizes NAD+ preferentially as a coenzyme and presumably produces pyruvate by the oxidative decarboxylation of malate [8]. This mitochondrial enzyme in insects may have a critical role in the replenishment of pyruvate for either transamination or Krebs’ cycle
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