The results of this modelling (Additional file 2) revealed that this 3D structure of em A

The results of this modelling (Additional file 2) revealed that this 3D structure of em A. Fenretinide 4. 1475-2875-10-318-S3.PDF (73K) GUID:?0B800D8D-C58F-4BC5-829B-2FBF4BDD581F Abstract Background em Anopheles stephensi /em 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 em A. stephensi /em (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. Methods To 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. Results The encoding gene sequence and main sequences of several peptides from mitochondrial ME were found to be highly homologous to the mitochondrial ME from em Anopheles gambiae /em (98%) and 59% homologous to the mitochondrial NADP+-dependent ME isoform from em Homo sapiens /em . Measurements of ME activity in mosquito mitochondria isolated from ASE cells showed that ( em i /em ) em Vmax /em with NAD+ was 3-fold higher than that with NADP+, ( em ii /em ) addition of Mg2+ or Mn2+ increased the em Vmax /em by 9- to 21-fold, with Mn2+ 2.3-fold more effective than Mg2+, ( em iii /em ) succinate and fumarate increased the activity by 2- and 5-fold, respectively, at sub-saturating concentrations of malate, ( em iv /em ) 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). Conclusions The biochemical characterization of em Anopheles stephensi /em ME is usually of crucial relevance given its important role in bioenergetics, suggesting that it is a suitable target for insecticide development. strong class=”kwd-title” Keywords: malaria, mitochondria, bioenergetics, metabolism, inhibitors, mosquitoes Background Recently, several pathways for energy production have been recognized in mitochondria from em Anopheles stephensi /em [1], a well-studied em Anopheles /em species in the investigation of malaria transmission [2]. The mitochondria-dependent energy pathways mainly use proline, pyruvate, -glycerophosphate, and acyl-carnitine derivatives as suitable substrates. Proline is also the main substrate for airline flight metabolism in the tsetse travel [3], the mosquito em Aedes aegypti /em [4] as well as other insects [5]. About 20% of the glutamate produced by proline oxidation is usually in turn oxidized by glutamate dehydrogenase [6], whereas the remainder undergoes transamination by reaction with pyruvate and the producing alanine accumulates as the proline is usually utilized. The 2-oxoglutarate created by transamination is usually further metabolized by the Krebs’ cycle. Originally pyruvate was thought to be produced from oxaloacetate by an oxaloacetate decarboxylase [7], but this enzyme was later localized in the cytoplasm whereas proline oxidation and subsequent reactions all take place in the mitochondria [6], consistent with previous studies [1]. Mitochondria of cultured cells [ASE cell collection ( em A. Colec11 stephensi /em Mos. 43 cell collection)] from em A /em . em stephensi /em , as well as flight muscle mass mitochondria of a beetle ( em Popillia japonica /em ), which also 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. Malic enzyme (ME; EC 1.1.1.39) catalyses the reversible oxidative Fenretinide decarboxylation of em L /em -malate to pyruvate and CO2 with the concomitant reduction of the cofactor NAD+ or NADP+ [9-11]. The enzyme requires divalent cations (Mg2+, Mn2+, or others) in the catalysis of this reaction. ME activity was first isolated from pigeon liver [12] and has since been found in most living organisms, from bacteria to humans. Most MEs are homotetramers, with monomers made up of 550 amino acids and having molecular weights of 60 kDa. The amino acid sequences of MEs are highly conserved across all analyzed organisms, but they lack recognizable homology to other proteins, including other oxidative decarboxylases. The wide distribution of ME activity in nature and the high degree of sequence conservation are consistent with the important biological functions of these enzymes, such as photosynthesis in C4 plants and even some C3 plants [13] and biosynthesis of fatty acids and steroids in liver and adipose tissues in animals. In mammals, three isoforms of ME have been identified–cytosolic NADP+-dependent ME (ME-1; [14]), mitochondrial NADP+-dependent ME (ME-3; [15]), and mitochondrial NAD(P)+-dependent ME (ME-2; [10]), which can use either NAD+ and.gambiae /em ME (“type”:”entrez-protein”,”attrs”:”text”:”Q7QB64″,”term_id”:”74921284″,”term_text”:”Q7QB64″Q7QB64) was detected (Dr. Background em Anopheles stephensi /em 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 em A. stephensi /em (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. Methods To 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. Results The encoding gene sequence and primary sequences of several peptides from mitochondrial ME were found to be highly homologous to the mitochondrial ME from em Anopheles gambiae /em (98%) and 59% homologous to the mitochondrial NADP+-dependent ME isoform from em Homo sapiens /em . Measurements of ME activity in mosquito mitochondria isolated from ASE cells showed that ( em i /em ) em Vmax /em with NAD+ was 3-fold higher than that with NADP+, ( em ii /em ) addition of Mg2+ or Mn2+ increased the em Vmax /em by 9- to 21-fold, with Mn2+ 2.3-fold more effective than Mg2+, ( em iii /em ) succinate and fumarate increased the activity by 2- and 5-fold, respectively, at sub-saturating concentrations of malate, ( em iv /em ) 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). Conclusions The biochemical characterization of em Anopheles stephensi /em ME is of critical relevance given its important role in bioenergetics, suggesting that it is a suitable target for insecticide development. strong class=”kwd-title” Keywords: malaria, mitochondria, bioenergetics, metabolism, inhibitors, mosquitoes Background Recently, several pathways for energy production have been identified in mitochondria from em Anopheles stephensi /em [1], a well-studied em Anopheles /em species in the investigation of malaria transmission [2]. The mitochondria-dependent energy pathways mainly use proline, pyruvate, -glycerophosphate, and acyl-carnitine derivatives as suitable substrates. Proline is also the main substrate for flight metabolism in the tsetse fly [3], the mosquito em Aedes aegypti /em [4] as well as other insects [5]. About 20% of the glutamate produced by proline oxidation is in turn oxidized by glutamate dehydrogenase [6], whereas the remainder undergoes transamination by reaction with pyruvate and the resulting alanine accumulates as the proline is utilized. The 2-oxoglutarate formed by transamination is further metabolized by the Krebs’ cycle. Originally pyruvate was thought to be produced from oxaloacetate by an oxaloacetate decarboxylase [7], but this enzyme was later localized in the cytoplasm whereas proline oxidation and subsequent reactions all take place in the mitochondria [6], consistent Fenretinide with previous studies [1]. Mitochondria of cultured cells [ASE cell line ( Fenretinide em A. stephensi /em Mos. 43 cell line)] from em A /em . em stephensi /em , as well as flight muscle mitochondria of a beetle ( em Popillia japonica /em ), which also 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. Malic enzyme (ME; EC 1.1.1.39) catalyses the reversible oxidative decarboxylation of em L /em -malate to pyruvate and CO2 with the concomitant reduction of the cofactor NAD+ or NADP+ [9-11]. The enzyme requires divalent cations (Mg2+, Mn2+, or others) in the catalysis of this reaction. ME activity was first isolated from pigeon liver [12] and has since been found in most living organisms, from bacteria to humans. Most MEs are homotetramers, with monomers containing 550 amino acids Fenretinide and having molecular weights of 60 kDa. The amino acid sequences of MEs are highly.