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Mitochondrial NAD dependent malic enzyme from Anopheles
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Pon et al Malaria Journal 2011 10 318,http www malariajournal com content 10 1 318. RESEARCH Open Access,Mitochondrial NAD dependent malic enzyme. from Anopheles stephensi a possible novel target,for malaria mosquito control. Jennifer Pon1 Eleonora Napoli1 Shirley Luckhart3 and Cecilia Giulivi1 2. Background 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 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, 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 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. Conclusions The 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. Keywords malaria mitochondria bioenergetics metabolism inhibitors mosquitoes. Background for flight metabolism in the tsetse fly 3 the mosquito. Recently several pathways for energy production have Aedes aegypti 4 as well as other insects 5 About 20. been identified in mitochondria from Anopheles ste of the glutamate produced by proline oxidation is in. phensi 1 a well studied Anopheles species in the inves turn oxidized by glutamate dehydrogenase 6 whereas. tigation of malaria transmission 2 The mitochondria the remainder undergoes transamination by reaction. dependent energy pathways mainly use proline pyru with pyruvate and the resulting alanine accumulates as. vate a glycerophosphate and acyl carnitine derivatives the proline is utilized The 2 oxoglutarate formed by. as suitable substrates Proline is also the main substrate transamination is further metabolized by the Krebs. cycle Originally pyruvate was thought to be produced. from oxaloacetate by an oxaloacetate decarboxylase 7. Correspondence cgiulivi ucdavis edu, Department of Molecular Biosciences School of Veterinary Medicine but this enzyme was later localized in the cytoplasm.
University of California Davis Davis CA 95616 USA whereas proline oxidation and subsequent reactions all. Full list of author information is available at the end of the article. 2011 Pon et al licensee BioMed Central Ltd This is an Open Access article distributed under the terms of the Creative Commons. Attribution License http creativecommons org licenses by 2 0 which permits unrestricted use distribution and reproduction in. any medium provided the original work is properly cited. Pon et al Malaria Journal 2011 10 318 Page 2 of 12. http www malariajournal com content 10 1 318, take place in the mitochondria 6 consistent with pre Methods. vious studies 1 Mitochondria of cultured cells ASE Chemicals. cell line A stephensi Mos 43 cell line from A ste Organic acids were purchased from Sigma Chemical Co. phensi as well as flight muscle mitochondria of a beetle St Louis USA All reagents were of analytical grade. Popillia japonica which also have the ability to oxidize. proline at a high rate have been shown to contain an Cell maintenance. unusually active malic enzyme 8 The latter species The immortalized A stephensi ASE cell line was grown. utilizes NAD preferentially as a coenzyme and presum in modified Eagle s minimal essential medium E5. ably produces pyruvate by the oxidative decarboxylation supplemented with glucose L glutamine vitamin solu. of malate 8 This mitochondrial enzyme in insects may tion nonessential amino acids penicillin and streptomy. have a critical role in the replenishment of pyruvate for cin and 5 heat inactivated fetal bovine serum at 28 C. either transamination or Krebs cycle with 5 CO2 as described 1 The population doubling. Malic enzyme ME EC 1 1 1 39 catalyses the reversi time of these cells is approximately 18 20 h The cells. ble oxidative decarboxylation of L malate to pyruvate were split 1 10 into E5 medium and grown in 50 ml cul. and CO2 with the concomitant reduction of the cofac ture flasks until confluent These flasks were used to. tor NAD or NADP 9 11 The enzyme requires seed 500 ml culture flasks to prepare 2 billion cells for. divalent cations Mg2 Mn2 or others in the cataly mitochondria preparation Cells harvested for mitochon. sis of this reaction ME activity was first isolated from dria preparation were gently pipetted resuspended in. pigeon liver 12 and has since been found in most liv the medium and transferred to 50 ml tubes Cells were. ing organisms from bacteria to humans Most MEs are pelleted by centrifugation at 800 g for 5 min The super. homotetramers with monomers containing 550 amino natant was removed and the cells were resuspended in. acids and having molecular weights of 60 kDa The a small amount of medium by gentle pipetting and. amino acid sequences of MEs are highly conserved transferred to a sterile holding tube on ice This cycle. across all studied organisms but they lack recognizable was repeated with collection of the concentrated cells. homology to other proteins including other oxidative into one tube until all flasks were processed. decarboxylases The wide distribution of ME activity in. nature and the high degree of sequence conservation Isolation of mitochondria. are consistent with the important biological functions Cells were centrifuged for 1 min at 500 g at 4 C and. of these enzymes such as photosynthesis in C4 plants mitochondria were isolated from pelleted cells as. and even some C3 plants 13 and biosynthesis of fatty described 1 The pellet was weighed and MSHE buffer. acids and steroids in liver and adipose tissues in ani was added at a ratio of 3 ml of MSHE buffer 220 mM. mals In mammals three isoforms of ME have been mannitol 70 mM sucrose 0 5 mM EGTA 0 1 fatty. identified cytosolic NADP dependent ME ME 1 acid free bovine albumin and 2 mM HEPES pH 7 4. 14 mitochondrial NADP dependent ME ME 3 per 1 g of cells The cells were disrupted by gentle. 15 and mitochondrial NAD P dependent ME homogenization centrifuged at 600 g for 5 min at 4 C. ME 2 10 which can use either NAD and NADP the pellet was discarded and the supernatant was centri. as a cofactor dual specificity but prefers NAD fuged at 10 300 g for 10 min at 4 C The pellet which is. under physiological conditions In invertebrates and in rich in mitochondria was resuspended in a small. particular in insects unusually high activity of NAD volume of MSHE Using this procedure the yield was. linked malic enzyme has been reported in flight mus 7 5 0 5 g mitochondrial protein 10 6 cells Protein. cle mitochondria of the beetle Popillia japonica 8 concentration was determined by using the BCA Protein. and from the tsetse fly and other insects 16 Assay Pierce. Based on previous reports 1 ASE mitochondrial ME, emerged as having a relevant role in the provision of Enzymatic assays. pyruvate for the Krebs cycle because the chemical inhi The ME enzymatic assay was performed using a method. bition of this enzyme resulted in the complete abroga outlined by 17 with the following modifications Mos. tion of oxygen uptake by mitochondria Therefore the quito mitochondria were homogenized in MSHE con. identification of ME in ASE mitochondria and the inves taining 2 mM mercaptoethanol In a 1 ml cuvette 2 g. tigation of the stereoselectivity of malate analogues are ml antimycin 1 mM L malate 0 3 mM NAD 50 mM. relevant in understanding the physiological role of ME HEPES pH 7 8 and 3 mM of MnCl2 unless indicated. in cells of this important malaria parasite vector and its otherwise were added The reaction was initiated with. potential as a possible novel target for insecticide the addition of 40 g of mosquito mitochondrial lysate. development protein The change in absorbance was measured using. Pon et al Malaria Journal 2011 10 318 Page 3 of 12. http www malariajournal com content 10 1 318, the Cary 1E Spectrophotometer at 340 nm for 2 3 min be differentiated based on MS MS analysis alone were. utes control The ME specific activity was calculated grouped to satisfy the principles of parsimony. for each trace utilizing the extinction coefficient for To identify the full length coding sequence for A ste. NADH at 340 nm 6 22 mM 1 cm 1 Each L malate phensi ME the A gambiae ME amino acid sequence. analogue was added to the reaction mixture at the indi Q7QB64 was used as a query by Dr Zhijian Jake Tu. cated concentrations and the change in absorbance was Virginia Tech to identify homologous sequence in the. measured for another 3 5 minutes June 2010 unpublished draft of the A stephensi assem. bly by TBLASTN e value cutoff 1e 7 The region with. Mass spectrometry analysis protein identification and the best match plus 1 kb flanking sequences on either. confirmation of A stephensi encoding sequence side were retrieved. LC MS MS analyses were performed at the Proteomics. Facility of the University of California Davis Genome Statistical analyses. Center Tandem mass spectra were extracted by Bio The experiments were run in duplicate or triplicate and. Works version 3 3 Charge state deconvolution and dei repeated two times in independent experiments Data. sotoping were not performed All MS MS samples were were expressed as mean SEM The data were evalu. analysed using X Tandem 18 19 X Tandem was set ated by using the t test StatSimple v2 0 5 Nidus Tech. up to search the Ensemble A gambiae protein database nologies Toronto Canada with p 0 05 considered as. 13 740 entries assuming the digestion enzyme trypsin statistically significant. X Tandem was searched with a fragment ion mass tol. erance of 0 40 Da and a parent ion tolerance of 1 8 Da Results and discussion. Iodoacetamide derivative of cysteine was specified in X Identification of the mitochondrial ME from A stephensi. Tandem as a fixed modification Deamidation of Asn To characterize the ME in A stephensi cells Multidi. and Gln oxidation of Met and Trp sulphone of Met mensional Protein Identification Technology MudPIT. Trp oxidation to formylkynurenin of Trp and acetylation was used This technique involves digesting mosquito. of the N terminus were specified in X Tandem as vari proteins with trypsin and separating the resulting pep. able modifications Scaffold version Scaffold 3 00 08 tides with two liquid column chromatography steps the. was used to validate MS MS based peptide and protein first being a strong cationic exchange and the second. identifications Peptide identifications were accepted if being reversed phase HPLC As the peptides elute from. they could be established at greater than 90 0 probabil the second column they are sprayed into a linear ion. ity as specified by the Peptide Prophet algorithm 20 trap mass spectrometer The first MS scan assigned. Protein identifications were accepted if they could be each peptide a mass charge ratio The most intense pep. established at greater than 99 0 probability and con tide signals are then fragmented in a second MS MS. tained at least 2 identified peptides Protein probabilities scan which assigns each peptide a unique fingerprint. were assigned by the Protein Prophet algorithm 21 Figure 1 The fingerprints are then analysed against. Proteins that contained similar peptides and could not. Figure 1 Mass spectrometry analyses of ASE ME Mass spectrometry results from MudPIT analyses indicating the peptide sequences. identification probability to A gambiae Q7QB64 peptide modifications identified by spectrum carboxymethylated or CM methionine sulfoxide. or OX and deaminated or DA calculated and actual peptide masses and peptide start and end amino acid from the above indicated A. gambiae sequence, Pon et al Malaria Journal 2011 10 318 Page 4 of 12. http www malariajournal com content 10 1 318, bioinformatics databases NCBI that revealed the pro spp 62 ants Harpegnathos spp 74 Camponotus.
tein s identity spp 74 lice Pediculus spp 73 moth Bombyx spp. Six out of seven unique peptides had a 90 match 64 and a nematode Ascaris suum 54 BLAST. with one of two MEs reported for A gambiae Q7QB64 query against the human database revealed a 59 iden. Figure 1 only one of these peptides showed a peptide tity to the human mitochondrial NADP dependent ME. identification probability of approximately 51 These or ME 3 followed by a 57 and a 55 identity to the. peptides provided 26 protein coverage 148 amino human cytosolic ME 1 and the human mitochondrial. acids out of 572 Figure 2 Given the high homology of ME 2 respectively The lower homology of the A stepe. these peptides to A gambiae Q7QB64 Figure 2 this phensi ME with the human mitochondrial counterpart. sequence was used to query the A stephensi genome was not due to the mitochondrial targeting sequence. assembly This analysis retrieved the homologous A ste because its removal from the human isoform and rea. phensi ME sequence Additional file 1 The predicted lignment of the mature protein with the mosquito pro. A stephensi protein sequence Additional file 1 revealed tein did not change the identity 59 similarity 75. a 98 homology 99 positives with A gambiae ME or score 1742 Of note the two human mitochondrial. Q7QB64 Additional file 1 ME isoforms share only 54 of their amino acids which. BLAST query of the complete amino acid sequence of is the same range of homology when comparing human. A stephensi ME to the non redundant NCBI database MEs with A stephensi ME this study or from maize. revealed high identity to MEs from mosquitoes A gam chloroplasts 47 By comparison the highly homolo. biae 96 Anopheles darlingi 94 Aedes aegypti 88 gous A gambiae ME Q7QB64 is 59 identical to the. Culex pungens 87 flies Drosophila spp 75 Glossina human ME 3 57 to human ME 1 and 55 to human. Figure 2 Protein alignments of A gambiae MEs Sequence alignment of the two ME proteins from Anopheles gambiae Q7PRY4 and Q7QB64. Sequence alignments of the two ME proteins sequences as reported in the SwissProt database last visited on April 13 2011 The alignments. were generated using CLUSTAL 55 Boxed amino acids indicate the peptides identified by mass spectrometry from Figure 1. Pon et al Malaria Journal 2011 10 318 Page 5 of 12. http www malariajournal com content 10 1 318, ME 2 whereas the other ME from A gambiae those in human ME 2 The binding site for tartronate. Q7PRY4 has 60 identity to the human ME 1 58 to was also similar to that defined for Ascaris suum ME. ME 3 and 53 to ME 2 As in A gambiae two MEs To gain insight in the activity of ME enzymatic para. have been identified for A darlingi in which one of meters were evaluated in mosquito mitochondria iso. them E3WUS4 is 62 identical to the human ME 1 lated from ASE cells The production of NAD P H was. while the other ME E3WW73 is 60 identical to the followed in the presence of malate and NAD P The. human ME 3 Thus for these mosquito species no ME Km value for L malate was calculated as a function of. 2 ortholog mitochondrial and NAD dependent is total concentrations of the substrate and it was found to. apparent In agreement with these findings only one be 0 12 mM at the pH of 7 8 Table 1 A low activity. mitochondrial ME from A stephensi was detected in the was obtained in the absence of cofactors Mg2 or Mn2. performed MudPIT analyses and genome sequence ana but the addition of either metal increased signifi. lyses However a possible A stephensi ME paralog with cantly the maximum activity 9 to 21 fold however. approximately 60 identity to A gambiae ME Mn2 was a better cofactor than Mg2 2 3 fold of Mg2. Q7QB64 was detected Dr Zhijian Tu personal com alone Table 1 The specific activity of the ASE ME. munication While this encoded protein is potentially enzyme was significantly higher than that of the other. interesting we have focused on the highly homologous two human mitochondrial isoforms the specific activ. A stephensi ME here ities of the human mitochondrial NADP ME and NAD. With the primary sequence of the A stephensi ME a ME have been reported as 12 U mg protein 1 U or. high quality prediction of the 3D structure and biologi unit 1 mol min and 35 U mg protein respectively. cal function of the ME was performed by using a tem whereas the activity of the human cytosolic NADP ME. plate based modelling platform 22 23 The results of is 40 to 55 U mg 15 ASE ME could utilize either. this modelling Additional file 2 revealed that the 3D NAD or NADP however V max with NAD was 3. structure of A stephensi ME with a C score of 1 985 times higher than that with NADP Table 1 consistent. was closely related to that of Ascaris suum 1o0sA TM with the predictions made from the protein similarity. score 0 9645 RMSD 1 43 identity 0 53 coverage 99 search vide supra and structure vide infra. Figure 3 This 3D model of A stephensi ME was based It has been shown that the presence of basic residues. on templates from human ME 1 2aw5B human ME 2 at positions 67 and 91 of human ME 2 are apparently. 1pj3A and 1gz4A and Ascaris suum 1llqA and 1o0sA critical for fumarate dependent activation 24 Figure. Additional file 2 The modelling results also predicted 4A residues marked with asterisks All three human. the binding site for various ligands Additional file 2 In isoforms contain Arg at the equivalent positions 24. particular the predicted binding sites for Mn2 oxalate but only ME 2 is activated by fumarate Thus other fac. and NADP resemble those in pig liver ME 1 whereas tors may contribute to this activation 24 The mutation. those for tartronate malate fumarate and Mg resembled of the amino acid residue Asp 102 had a significant. effect on the fumarate mediated activation of human. ME 2 25 At this position the human ME 1 and ME 3, isoforms have a Ser residue and they do not show any. increase in activity with fumarate The mosquito ME. has a Glu at this position and the Ascaris suum ME has. an Asp suggesting that these isoforms like the human. ME 2 may be activated by fumarate by conserving the. negative charge at this position On the other hand in. the mosquito ME Leu occupies the equivalent position. of Arg 67 all human isoforms and the nematode ME, have an Arg in this position suggesting that mosquito. ME should not be activated by fumarate In addition to. Arg 67 it has been reported that in the nematode mito. chondrial ME the residues that are involved in the. Figure 3 Three dimensional structures of A stephensi and binding of fumarate are Ala 78 Leu 81 and Leu 105. Ascaris suum MEs The 3D structure of A stephensi ME A was. obtained through modelling from the primary sequence from. Figure 4A rows indicated with arrows which in A ste. Additional file 1 and the use of the modeling software I TASSER phensi ME are occupied by Ile Leu and Leu in agree. results shown in Additional file 2 The 3D structure of Ascaris suum ment with the nematode isoform which is activated by. ME was obtained from the protein database and corresponds to the fumarate In agreement with this last prediction succi. accession number 1o0sA Both pdb files were visualized using nate and fumarate increased the activity by 2 to 5 fold. PyMol 1 4 1 56, respectively at sub saturating concentrations of malate. Pon et al Malaria Journal 2011 10 318 Page 6 of 12. http www malariajournal com content 10 1 318, Table 1 Effect of cofactors on Vmax of ME activity.
Addition Km for malate Vmax p value to controls,mM nmol min mg protein 1. MgCl2 3 mM 24 2 0 02,MnCl2 3 mM 0 12 56 3 20 2 0 01. All kinetic parameters were obtained with NAD except the Vmax obtained with NADP value in parentheses The Vmax values were expressed as mean SEM. All other experimental details are described in the Methods. Figure 5B showing no effect at saturating ones not e g glutathione reductase thioredoxin reductase. shown Other amino acids such as Pro had a moderate because it is believed that disrupting the close packing. effect on enzyme activity up to concentrations of 10 of the secondary structure allows the structure to. mM 1 4 fold Surprisingly Glu was found to be a accommodate the phosphate moiety of NADP 29. potent activator increasing ASE ME activity by 4 fold at Furthermore protein engineering of glutathione reduc. saturating concentrations of malate Figure 5B tase in which Ala 179 was replaced by a Gly residue at. One very highly conserved region defined as a part of a an equivalent position in this enzyme both in single as. malate binding domain is shown in Figure 4B This well as in multiple substitutions caused NAD binding. region has been identified on the basis of inhibition preference over that of NADP 32 However this rea. experiments carried out on NADP dependent ME with soning does not seem to apply to all MEs listed in Fig. the competitive inhibitor bromopyruvate 15 Indeed a ure 4D position marked with asterisk In particular all. highly conserved Cys Figure 4B asterisk and the pre human MEs have a third Gly regardless of the cofactor. ceding sequence VYTPTVG Figure 4B bar are present preference indicating that other factors are contributing. in this domain in human nematode plant and A gam to the cofactor preference In support of this statement. biae and A stephensi mosquito isoforms Figure 4B Gly residues in the boxed segments shown in Figures. suggesting that the same type of binding occurs in the 4C 4D when replaced by Val residues rendered abor. mosquito isoforms However recently the role of this tive mutants of maize C4 NADP ME 31 This is con. Cys has been challenged based on crystallography data sistent with the three dimensional model obtained for. 24 This study indicated that this residue is about 13 the maize C4 NADP ME showing that residues at both. away from the substrate analog oxalate suggesting that sites are part of the NADP binding site justifying the. the inhibition of substrate binding upon chemical modi high degree of conservation among all NAD P ME. fication of this Cys is an indirect effect 31, The proposed ADP binding beta alpha beta fold is It has been suggested that two Ala residues spaced by. shown which contains a characteristic arrangement of three amino acids Gly X Gly X X Ala X X X Ala Fig. Gly residues and nonpolar and hydrophilic amino acids ure 4D indicated with a bar are more characteristic for. at specific positions Figure 4C boxed amino acids NADP binding domains relative to NAD binding. 26 A highly conserved Cys residue or the highly domains whereas Gly residues were more consistently. similar Ser in plant NADP ME 2 Figure 4C asterisk identified in NAD dependent enzymes 31 32 How. observed in this region is present in all NADP depen ever all human ME isoforms nematode mitochondrial. dent malic enzymes 27 but is absent from the NAD ME and A stephensi ME have an Ala X X X Ala seg. dependent malic enzymes human ME 2 nematode ment regardless of whether they use NADP or NAD. and mosquito isoforms Therefore the presence or Other residues may also affect binding specificity of. absence of this Cys or Ser residue in this binding fold NAD P in MEs It has been noted that all NADP. apparently distinguishes the preference for NADP over dependent ME isoforms have a conserved Lys at Gln 362. NAD as suggested before by others 28 in human ME 2 Figure 4E asterisk 33 whereas dual. The fingerprint region of NAD P binding sites is specificity MEs have heterogeneous residues at this posi. characterized by a Gly rich sequence Gly X Gly X X tion 34 Figure 4E asterisk This observation combined. Gly Figure 4D boxed amino acids which is the phos with mutational and modelling studies using pigeon ME 1. phate binding consensus 29 30 The first Gly allows led to the supposition that in NADP dependent MEs. the tight turn of the main chain the second allows a NADP specificity is conferred by Lys at this position. close contact to the diphosphate of NAD P and the Mitochondrial NAD MEs of Ascaris and other species. third is important for the close packing of the secondary may have no need to exhibit strict specificity for NAD. structure 29 31 This third Gly is replaced by larger due to the high concentration of NAD relative to NADP. amino acids Ala or Ser in enzymes that utilize NADP within mitochondria and thus the residue at the. Pon et al Malaria Journal 2011 10 318 Page 7 of 12. http www malariajournal com content 10 1 318, Figure 4 Analyses of protein sequence alignments of various MEs Sequence alignments of maize NADP ME 2 chloroplastic NADP ME. and NADP ME 4 human ME 1 ME 2 and ME 3 nematode NAD ME 2 bacterial NAD ME and A stephensi ME mosquito ME The amino. acid sequences of the ME isoforms were analyzed by BLAST against the SwissProt database and the alignments were generated using CLUSTAL. 55 The amino acid residues highlighted in grey share high homology whereas those in bold are identical Key amino acids discussed in the. text are shown with asterisks arrows or bars see text for full description A Sites involved in fumarate activation B Malate binding site C. ADP binding site D NAD P binding domain and sites associated with NAD versus NADP preference E Other sites also associated with. NAD versus NADP preference For complete protein nomenclature see Additional file 3. Pon et al Malaria Journal 2011 10 318 Page 8 of 12. http www malariajournal com content 10 1 318, Figure 5 Effect of L malate analogues on ME activity A Chemical structure of L malate and structural analogues B Effect of L malate.
analogues on ASE mitochondrial ME enzymatic activity The activity of ME was evaluated in the absence and presence of the following. inhibitors compounds at saturating concentrations of all other cofactors and substrates except for succinate and fumarate which are shown at. subsaturating concentrations of malate 0 5 mM 1 mM oxaloacetate 5 mM tartronic acid 1 mM oxalate 2 5 mM alpha ketobutyrate 2 5 mM. beta hydroxybutyrate 2 5 mM alpha hydroxybutyrate 2 5 mM acetoacetate 2 5 mM malonate 10 mM pyruvate 10 mM proline 10 mM. succinate 10 mM glutamate and 5 mM fumarate, corresponding position would not necessarily be con malate with K i values from 80 to 430 M 24 35. strained to Lys Indeed in mosquito ME this study According to Hsieh et al 36 the presence of Lys 346. nematode ME human ME 2 and bacteria ME this in mammalian ME 2 is critical for ATP inhibition Fig. sequence position corresponds to a Lys His Gln and Glu ure 4E arrow because site directed mutagenesis of this. respectively Figure 4E asterisk Recent data clearly indi residue to Ala or Ser diminishes ATP mediated inhibi. cate that the Gln 362 Lys mutant of human ME 2 is a tion This result suggested that the positive charge is. non allosteric non cooperative and NADP specific critical for ATP binding As indicated above in the. enzyme as is ME 1 33 Additionally Gln 362 Lys is human ME 1 and ME 3 as well as the plant MEs in iso. more sensitive to ATP and the inhibition constant is forms that are not inhibited by ATP the equivalent. smaller than that of wild type 33 Sequence alignments positions for Lys 346 are occupied by Ser In the mito. of the nucleotide binding region among MEs have chondrial nematode and mosquito MEs Ile residues are. revealed that in addition to Gln 362 Lys 346 is conserved present in these positions thus no significant effect of. among the NAD dependent malic enzymes human ME ATP would be expected as it has been shown for the. 2 Figure 4E arrow but in NADP dependent MEs Ascaris ME 37 38 In agreement with this prediction. maize human this Lys is replaced by Ser Figure 4E kinetic results showed that ATP was not an inhibitor. arrow In the case of A stephensi ME this residue is with respect to NAD or L malate in A stephensi mito. replaced by Ile as it is in the case of the nematode ME chondrial ME at the concentrations tested 20 to 1 000. Thus in those isoforms with NADP only specificity a M data not shown The Arg 197 Arg 542 and Arg. Lys in position 362 Figure 4E asterisk and a Ser in 346 556 residues in human ME 2 appear to be involved in. Figure 4E arrow are apparently necessary the binding of ATP at the exosite 24 These residues. ATP acts as an active site inhibitor of mitochondrial differ from those in human ME 3 or mosquito ME. ME 2 following a competitive mechanism for NAD and human ME 3 Gln Tyr and Leu and mosquito ME. Pon et al Malaria Journal 2011 10 318 Page 9 of 12. http www malariajournal com content 10 1 318, Gln Thr and His suggesting that this binding is not respectively yielded stronger inhibition of ME Figure. operational in these isoforms 5, Within the diacids the presence of a keto group in. Inhibitors of the A stephensi mitochondrial ME position 2 seemed to be an important factor in control. Several analogues of L malate were tested as inhibitors ling the inhibitory effect of the compounds studied for. of the NAD dependent ME catalyzed reaction Figure lack of this moiety in for example succinate or fuma. 5A Oxalate oxaloacetate tartronate and alpha ketobu rate resulted in a loss of the inhibitory effect The. tyrate were found to be inhibitors of the ME catalyzed change of the 2 keto group of the alpha ketobutyric acid. reaction through a competitive mechanism with respect to 2 hydroxy alpha hydroxybutyrate or the transfer to. to the substrate malate concentration Their Ki values the 3 hydroxy beta hydroxybutyrate resulted in negligi. ranged from 0 1 oxaloacetate and oxalate to 0 6 M ble effect on the ME activity Figure 3B Thus the keto. tartronate Table 2 Alpha hydroxybutyrate and the group in position 2 seems essential for effective inhibi. ketone bodies beta hydroxybutyrate and acetoacetate did tion of ME possibly by favoring interactions with. not exhibit any effect on the ME activity The same out the nicotinamide ring of NAD and or hydrogen bond. come was obtained with either malonate or pyruvate ing with the side chain amide of Asn and the 2 hydro. Figure 5B xyl of the nicotinamide ribose 39 These results. The catalysis by ME generally proceeds in two steps suggest that the extent of inhibition was dependent on. namely the dehydrogenation of malate to produce oxa the size of the analogues 2 to 4 carbons the presence. loacetate and then the decarboxylation to produce pyru of two carboxyl groups along with a 2 hydroxyl or 2. vate In a proposed enzymatic catalysis based on keto moiety important for binding of the substrate ana. structural data the crystallography data the latter step is logue to the enzyme In a study performed with the. believed to occur through the formation of a keto enol enzyme from Flaveria trinervia the presence of a group. intermediate structurally similar to oxalate 39 The with a low pK value 6 to 6 6 probably an H residue. strongest ME inhibitors for which Ki were evaluated i was responsible for the binding of the 2 hydroxyl of. e oxalate oxaloacetate and tartronate are consistent malate and transfer of the hydride to form the inter. with this mechanism for these compounds are either mediate oxaloacetate 41 These results suggest that the. intermediates i e oxaloacetate oxalate or structurally extent of inhibition was dependent on the size of the. similar intermediates tartronate or ketomalonate that analogues 2 to 3 carbons the presence of two carboxyl. fit the tight active site of the enzyme 39 Besides the groups along with a 2 hydroxyl or 2 keto moiety impor. role of size these inhibitors are diacids Thus the pre tant for binding of the substrate analogue to the. sence of an extra carboxyl group appears to be neces enzyme. sary for binding the substrate or inhibitor to the The ME 3 gene is conserved in human chimpanzee. enzyme probably through an induced dipolar type bond dog cow mouse rat a variety of flies and mosquito. with the side chains of Gln and Asn as suggested by species Caenorhabditis elegans Arabidopsis thaliana. others 40 In support of this rationale the presence of and rice The mammalian ME 3 isoform has a strong. the extra carboxylate in malate and oxaloacetate com tissue specific expression mostly in organs with a low. pared to alpha hydroxybutyrate and alpha ketobutyrate division rate e g heart skeletal muscle and brain 15. or with an involvement in steroid hormone biosynthesis. ovary and testes High activity of ME 3 has also been. reported in muscle of crustaceans and fish 42 It has. Table 2 Inhibition constants of various malate analogues been previously reported that the immortalized cells. Compound Ki M from A stephensi from which the ME activity in this. Oxaloacetate 0 12 study was evaluated resembled skeletal muscle cells 1. Oxalate 0 15 Thus the finding of a high activity of this mitochondrial. enzyme is consistent with this previous study Given. Tartronate 0 61, that the reverse reaction i e the carboxylation of pyru. Alpha ketobutyrate n d, vate was 12 times slower than the forward reaction.
Inhibition studies were performed by varying the concentrations of L malate. from 0 1 mM subsaturating to up to 1 mM saturating while the analogue. not shown the role for ME 3 in the anaplerotic reac. chosen based on its inhibitory activity from Figure 3B was held at several tion pyruvate to malate of the Krebs cycle is expected. fixed concentrations around its inhibition constant Concentrations of the to be negligible. other components in the assay mixture were held at saturation Reciprocal. velocities were plotted as a function of reciprocal substrate concentrations A previous report indicated that addition of tartronic. and all plots were linear in the range of substrate concentrations assayed 0 1 acid completely inhibited the oxygen consumption of. 0 5 mM The lines were calculated from the fits of the experimentally. determined values to the appropriate kinetic parameters Abbreviations n d. Pro supplemented phosphorylating mitochondria 1, not determined This suggests a critical role for ME in a pathway to. Pon et al Malaria Journal 2011 10 318 Page 10 of 12. http www malariajournal com content 10 1 318, provide NADH to the electron transport chain The ME direct studies of insecticide resistance are beyond the. will then be activated with glutamate succinate or scope of the present study it is important to bear in. fumarate as in the case when Pro or other suitable sub mind that inhibition of multiple mitochondrial targets. strate becomes available The physiological function of e g Complex I and ME might result in synergistic. the inhibition by oxalate is not clear given the limiting effects or in attenuation of the development of cross. amount of this substrate in mitochondria If NADH resistance as already observed when multiple unrelated. levels are high and citrate concentration rises when targets for insecticides are tested 53 54. switching from high to low work this would result in. an inhibition of citrate synthase and increases in oxalo Additional material. cetate which in turn would inhibit ME and reduce the. supply of pyruvate and acetylCoA An interesting feature Additional file 1 DNA and protein sequences for A stephensi ME A. of this isoform is that the optimum pH was 7 8 which is Nucleotide sequence for A stephensi ME B Amino acid sequence. deduced from DNA equence C Protein sequence producing significant. probably close to the physiological intramitochondrial alignment to A stephensi ME performed with BLAST. pH value under phosphorylating conditions 43 Additional file 2 Modeling results obtained with A stephensi ME. It is likely that a secondary role of this enzyme in This file contains the results obtained with the simulation suite named I. mosquito mitochondria based on the preferential use of TASSER performed with the primary amino acid sequence of A stephensi. NAD over NADP is to provide NADPH for the, Additional file 3 Additional information on the nomenclature of. detoxification of reactive oxygen radicals generated in proteins listed under Figure 4 This file contains all additional. mitochondria during respiration Glutathione reductase information full name short name E C number and species in regards. activity requires NADPH to recycle oxidized glutathione to the nomenclature of ME shown under Figure 4. for the glutathione peroxidase system 44 and mito, chondrial NAD ME might contribute to the generation. of reducing equivalents List of abbreviations, ASE Anopheles stephensi Mos 43 cell line ME malic enzyme ME 1 cytosolic.
NADP dependent ME ME 2 mitochondrial NAD P dependent ME ME 3. Conclusions mitochondrial NADP dependent ME MS mass spectrometry MS MS. The critical role of ME in the bioenergetics of mosqui tandem MS mtDNA mitochondrial DNA MudPIT Multidimensional Protein. toes along with the deeper understanding of its bio Identification Technology. chemistry and chemical requirements for inhibition Acknowledgements. suggests that this enzyme could became a suitable target This study was supported by the University of California Mosquito Research. for insecticide development In this regard structural Program UC MRP grant 07 019 3 1 and by a National Institutes of Health. NIH National Institute of Allergy and Infectious Diseases NIAID grant. analogues of L malate especially C2 or C3 organic dia AI073745 We thank the technical expertise of Ashley Horton for preparation. cids with a 2 hydroxyl or 2 keto group behaved as the of ASE cells and Ms Catherine Ross Inta for isolating mitochondria from ASE. most potent inhibitors of ME activity e g oxaloacetate cells We thank Dr Zhijian Jake Tu for analysis of the A stephensi ME. homolog The draft A stephensi genome assembly was obtained through a. tartronic acid and oxalate collaborative effort led by Drs Zhijian Jake Tu and Igor Sharakhov at. Enzymes significantly associated with insecticide resis Virginia Tech. tance include esterases and cytochrome P450s involved. Author details, in the oxidative catabolism of insecticides 45 47 Mito 1. Department of Molecular Biosciences School of Veterinary Medicine. chondrial Complex I inhibitors seem to circumvent the University of California Davis Davis CA 95616 USA 2M I N D Institute School. aforementioned resistance 5 48 49 However recent of Medicine University of California Davis Sacramento CA 95817 USA. Department of Medical Microbiology and Immunology School of Medicine. studies of pyrethroid resistant A gambiae demonstrated University of California Davis Davis CA 95616 USA. that exposure to permethrin induced strong upregula. tion of genes that encode subunits of Complex I 50 Authors contributions. JP carried out all enzymatic analyses and performed statistical analyses EN. whereas the presence of MAP kinase Pmk1 and PKA supervised directly the technical work performed statistical analyses and. conferred resistance to Schizosaccharomyces pombe to helped to draft the manuscript SL provided all biological materials. rotenone a potent Complex I inhibitor 51 In addition contributed with substantial intellectual input in regards to the malaria field. and revised the manuscript critically CG conceived of the study participated. whereas most cases of insecticide resistance were usually in its design and coordination performed all sequence alignment and. associated with mutations in the nuclear genome a role analyses protein modelling and interpretation and wrote the manuscript All. for maternally inherited mtDNA has emerged as a pos authors read and approved the final manuscript. sible mediator of resistance to Complex I inhibitors and Competing interests. insecticides 47 52 These observations suggest that Drs Giulivi and Luckhart filed a disclosure and record of invention with the. Complex I associated resistance may already occur in University of California Davis on 04 11 2008 entitled Inhibitors of mosquito. malic enzyme as insecticides UC Case 2008 673 1, natural populations Thus this possibility should be. considered when resistance to current insecticides and Received 13 October 2011 Accepted 26 October 2011. development of novel insecticides are evaluated While Published 26 October 2011. Pon et al Malaria Journal 2011 10 318 Page 11 of 12. http www malariajournal com content 10 1 318, References metabolism plant Mesembryanthemum crystallinum Eur J Biochem 1992. 1 Giulivi C Ross Inta C Horton AA Luckhart S Metabolic pathways in 208 259 266. Anopheles stephensi mitochondria Biochem J 2008 415 309 316 28 Winning BM Bourguignon J Leaver CJ Plant mitochondrial NAD. 2 Luckhart S Vodovotz Y Cui L Rosenberg R The mosquito Anopheles dependent malic enzyme cDNA cloning deduced primary structure of. stephensi limits malaria parasite development with inducible synthesis of the 59 and 62 kDa subunits import gene complexity and expression. nitric oxide Proc Natl Acad Sci USA 1998 95 5700 5705 analysis J Biol Chem 1994 269 4780 4786. 3 Bursell E Aspects of the flight metabolism of tsetse flies Glossina Comp 29 Bellamacina CR The nicotinamide dinucleotide binding motif a. Biochem Physiol 1966 19 809 818 comparison of nucleotide binding proteins FASEB J 1996 10 1257 1269. 4 Scaraffia PY Wells MA Proline can be utilized as an energy substrate 30 Rossman MG Liljas A Br nd n CI Banaszak LJ The Enzymes New York. during flight of Aedes aegypti females J Insect Physiol 2003 49 591 601 Academic Press 1975 61 112. 5 Schuler F Casida JE The insecticide target in the PSST subunit of 31 Detarsio E Wheeler MC Campos Bermudez VA Andreo CS Drincovich MF. Complex I Pest Manag Sci 2001 57 932 940 Maize C4 NADP malic enzyme Expression in Escherichia coli and. 6 Bursell E Slack E Oxidation of proline by sarcosomes of the tsetse fly characterization of site directed mutants at the putative nucleoside. Glossina morsitans Insect Biochem 1976 6 159 167 binding sites J Biol Chem 2003 278 13757 13764. 7 Bursell E Oxaloacetic carboxylase in flight musculature of the tsetse fly 32 Scrutton NS Berry A Perham RN Redesign of the coenzyme specificity of. Comp Biochem Physiol 1965 16 259 266 a dehydrogenase by protein engineering Nature 1990 343 38 43. 8 Hansford RG Johnson RN The nature and control of the tricarboxylate 33 Hsieh JY Liu GY Chang GG Hung HC Determinants of the dual cofactor. cycle in beetle flight muscle Biochem J 1975 148 389 402 specificity and substrate cooperativity of the human mitochondrial NAD. 9 Chou WY Huang SM Liu YH Chang GG Cloning and expression of P dependent malic enzyme functional roles of glutamine 362 J Biol. pigeon liver cytosolic NADP dependent malic enzyme cDNA and some Chem 2006 281 23237 23245. of its abortive mutants Arch Biochem Biophys 1994 310 158 166 34 Kuo CC Tsai LC Chin TY Chang GG Chou WY Lysine residues 162 and. 10 Loeber G Infante AA Maurer Fogy I Krystek E Dworkin MB Human NAD 340 are involved in the catalysis and coenzyme binding of NADP. dependent mitochondrial malic enzyme cDNA cloning primary dependent malic enzyme from pigeon Biochem Biophys Res Comm. structure and expression in Escherichia coli J Biol Chem 1991 2000 270 821 825. 266 3016 3021 35 Hsu WC Hung HC Tong L Chang GG Dual functional roles of ATP in the. 11 Rao GSJ Coleman DE Kulkarni G Goldsmith EJ Cook PF Harris BG NAD human mitochondrial malic enzyme Biochemistry 2004 43 7382 7390. malic enzyme from Ascaris suum Sequence and structural studies 36 Hsieh JY Liu GY Hung HC Influential factor contributing to the isoform. Protein Pept Lett 2000 7 297 304 specific inhibition by ATP of human mitochondrial NAD P dependent. 12 Ochoa S Mehler A Kornberg A Reversible oxidative decarboxylation of malic enzyme functional roles of the nucleotide binding site Lys346. malic acid J Biol Chem 1947 167 871 872 FEBS J 2008 275 5383 5392. 13 Hibberd JM Quick WP Characteristics of C4 photosynthesis in stems and 37 Coleman DE Rao GSJ Goldsmith EJ Cook PF Harris BG Crystal structure. petioles of C3 flowering plants Nature 2002 415 451 454 of the malic enzyme from Ascaris suum complexed with nicotinamide. 14 Chang GG Wang JK Huang TM Lee HJ Chou WY Meng CL Purification adenine dinucleotide at 2 3 resolution Biochemistry 2002 41 6928 6938. and characterization of the cytosolic NADP dependent malic enzyme 38 Landsperger WJ Harris BG NAD malic enzyme Regulatory properties of. from human breast cancer cell line Eur J Biochem 1991 202 681 688 the enzyme from Ascaris suum J Biol Chem 1976 251 3599 3602. 15 Loeber G Maurer Fogy I Schwendenwein R Purification cDNA cloning 39 Yang Z Floyd DL Loeber G Tong L Structure of a closed form of human. and heterologous expression of the human mitochondrial NADP malic enzyme and implications for catalytic mechanism Nat Struct Biol. dependent malic enzyme Biochem J 1994 304 687 692 2000 7 251 257. 16 Hoek JB Pearson DJ Olembo NK NAD linked malic enzyme EC 1 1 1 39 in 40 Schimerlik MI Cleland WW Inhibition and alternate substrate studies on. flight muscle of the tsetse fly Glossina and other insects Biochem J 1976 the mechanism of malic enzyme Biochemistry 1977 16 565 570. 160 253 262 41 Holaday AS Lowder GW Effect of pH on the kinetic parameters of NADP. 17 Spampinato CP Colombo SL Andreo CS Interaction of analogues of malic enzyme from a C 4 Flaveria Asteraceae species Plant Physiol 1989. substrate with NADP malic enzyme from maize leaves Photosynth Res 90 401 405. 1994 39 67 73 42 Skorkowski EF Mitochondrial malic enzyme from crustacean and fish. 18 Bjornson RD Carriero NJ Colangelo C Shifman M Cheung K H Miller PL muscle Comp Biochem Physiol B 1988 90 19 24. 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CASP7 Proteins 2007 69 Suppl 8 108 117 48 Song C Scharf ME Mitochondrial impacts of insecticidal formate esters. 24 Yang Z Lanks CW Tong L Molecular mechanism for the regulation of in insecticide resistant and insecticide susceptible Drosophila. human mitochondrial NAD P dependent malic enzyme by ATP and melanogaster Pest Manag Sci 2009 65 697 703. fumarate Structure 2002 10 951 960 49 Nauen R Bretschneider T New modes of action of insecticides Pestic. 25 Hsieh JY Su KL Ho PT Hung HC Long range interaction between the Outlook 2002 13 241 245. enzyme active site and a distant allosteric site in the human 50 Vontas J Blass C Koutsos AC David JP Kafatos FC Louis C Hemingway J. mitochondrial NAD P dependent malic enzyme Arch Biochem Biophys Christophides GK Ranson H Gene expression in insecticide resistant and. 2009 487 19 27 susceptible Anopheles gambiae strains constitutively or after insecticide. 26 Wierenga RK Terpstra P Hol WGJ Prediction of the occurrence of the exposure Insect Mol Biol 2005 14 509 521. ADP binding bab fold in proteins using an amino acid sequence 51 Wang Y Gulis G Buckner S Johnson PC Sullivan D Busenlehner L. fingerprint J Mol Biol 1986 187 101 107 Marcus S The MAP kinase Pmk1 and protein kinase A are required for. 27 Cushman JC Characterization and expression of a NADP malic enzyme rotenone resistance in the fission yeast Schizosaccharomyces pombe. cDNA induced by salt stress from the facultative crassulacean acid Biochem Biophys Res Comm 2010 399 123 128. Pon et al Malaria Journal 2011 10 318 Page 12 of 12. http www malariajournal com content 10 1 318, 52 Van Leeuwen T Vanholme B Van Pottelberge S Van Nieuwenhuyse P. Nauen R Tirry L Denholm I Mitochondrial heteroplasmy and the. evolution of insecticide resistance non Mendelian inheritance in action. Proc Natl Acad Sci USA 2008 105 5980 5985, 53 Oxborough RM Kitau J Matowo J Mndeme R Feston E Boko P Odjo A.
Metonnou CG Irish S N Guessan R et al Evaluation of indoor residual. spraying with the pyrrole insecticide chlorfenapyr against pyrethroid. susceptible Anopheles arabiensis and pyrethroid resistant Culex. quinquefasciatus mosquitoes Trans R Soc Trop Med Hyg 2010 104 639 645. 54 Farenhorst M Mouatcho JC Kikankie CK Brooke BD Hunt RH Thomas MB. Koekemoer LL Knols BG Coetzee M Fungal infection counters insecticide. resistance in African malaria mosquitoes Proc Natl Acad Sci USA 2009. 106 17443 17447, 55 Thompson JD Higgins DG Gibson TJ CLUSTAL W improving the. sensitivity of progressive multiple sequence alignment through. sequence weighting position specific gap penalties and weight matrix. choice Nucleic Acids Res 1994 22 4673 4680, 56 Schrodinger LLC The PyMOL Molecular Graphics System Version 1 3r1. doi 10 1186 1475 2875 10 318, Cite this article as Pon et al Mitochondrial NAD dependent malic. enzyme from Anopheles stephensi a possible novel target for malaria. mosquito control Malaria Journal 2011 10 318,Submit your next manuscript to BioMed Central. and take full advantage of,Convenient online submission.
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