Novel Characteristics of Mitochondrial Electron Transport Chain from Eimeria tenella.
ABSTRACT: Eimeria tenella is an intracellular apicomplexan parasite, which infects cecal epithelial cells from chickens and causes hemorrhagic diarrhea and eventual death. We have previously reported the comparative RNA sequence analysis of the E. tenella sporozoite stage between virulent and precocious strains and showed that the expression of several genes involved in mitochondrial electron transport chain (ETC), such as type II NADH dehydrogenase (NDH-2), complex II (succinate:quinone oxidoreductase), malate:quinone oxidoreductase (MQO), and glycerol-3-phosphate dehydrogenase (G3PDH), were upregulated in virulent strain. To study E. tenella mitochondrial ETC in detail, we developed a reproducible method for preparation of mitochondria-rich fraction from sporozoites, which maintained high specific activities of dehydrogenases, such as NDH-2 followed by G3PDH, MQO, complex II, and dihydroorotate dehydrogenase (DHODH). Of particular importance, we showed that E. tenella sporozoite mitochondria possess an intrinsic ability to perform fumarate respiration (via complex II) in addition to the classical oxygen respiration (via complexes III and IV). Further analysis by high-resolution clear native electrophoresis, activity staining, and nano-liquid chromatography tandem-mass spectrometry (nano-LC-MS/MS) provided evidence of a mitochondrial complex II-III-IV supercomplex. Our analysis suggests that complex II from E. tenella has biochemical features distinct to known orthologues and is a potential target for the development of new anticoccidian drugs.
Project description:The only enzyme of the citric acid cycle for which no open reading frame (ORF) was found in the Helicobacter pylori genome is the NAD-dependent malate dehydrogenase. Here, it is shown that in this organism the oxidation of malate to oxaloacetate is catalyzed by a malate:quinone oxidoreductase (MQO). This flavin adenine dinucleotide-dependent membrane-associated enzyme donates electrons to quinones of the electron transfer chain. Similar to succinate dehydrogenase, it is part of both the electron transfer chain and the citric acid cycle. MQO activity was demonstrated in isolated membranes of H. pylori. The enzyme is encoded by the ORF HP0086, which is shown by the fact that expression of the HP0086 sequence from a plasmid induces high MQO activity in mqo deletion mutants of Escherichia coli or Corynebacterium glutamicum. Furthermore, this plasmid was able to complement the phenotype of the C. glutamicum mqo deletion mutant. Interestingly, the protein predicted to be encoded by this ORF is only distantly related to known or postulated MQO sequences from other bacteria. The presence of an MQO shown here and the previously demonstrated presence of a 2-ketoglutarate:ferredoxin oxidoreductase and a succinyl-coenzyme A (CoA):acetoacetyl-CoA transferase indicate that H. pylori possesses a complete citric acid cycle, but one which deviates from the standard textbook example in three steps.
Project description:Like many other bacteria, Corynebacterium glutamicum possesses two types of L-malate dehydrogenase, a membrane-associated malate:quinone oxidoreductase (MQO; EC 22.214.171.124) and a cytoplasmic malate dehydrogenase (MDH; EC 126.96.36.199) The regulation of MDH and of the three membrane-associated dehydrogenases MQO, succinate dehydrogenase (SDH), and NADH dehydrogenase was investigated. MQO, MDH, and SDH activities are regulated coordinately in response to the carbon and energy source for growth. Compared to growth on glucose, these activities are increased during growth on lactate, pyruvate, or acetate, substrates which require high citric acid cycle activity to sustain growth. The simultaneous presence of high activities of both malate dehydrogenases is puzzling. MQO is the most important malate dehydrogenase in the physiology of C. glutamicum. A mutant with a site-directed deletion in the mqo gene does not grow on minimal medium. Growth can be partially restored in this mutant by addition of the vitamin nicotinamide. In contrast, a double mutant lacking MQO and MDH does not grow even in the presence of nicotinamide. Apparently, MDH is able to take over the function of MQO in an mqo mutant, but this requires the presence of nicotinamide in the growth medium. It is shown that addition of nicotinamide leads to a higher intracellular pyridine nucleotide concentration, which probably enables MDH to catalyze malate oxidation. Purified MDH from C. glutamicum catalyzes oxaloacetate reduction much more readily than malate oxidation at physiological pH. In a reconstituted system with isolated membranes and purified MDH, MQO and MDH catalyze the cyclic conversion of malate and oxaloacetate, leading to a net oxidation of NADH. Evidence is presented that this cyclic reaction also takes place in vivo. As yet, no phenotype of an mdh deletion alone was observed, which leaves a physiological function for MDH in C. glutamicum obscure.
Project description:Type II NADH:quinone oxidoreductase (NDH-2) is a proposed drug-target of major pathogenic microorganisms such as Mycobacterium tuberculosis and Plasmodium falciparum. Many NDH-2 inhibitors have been identified, but rational drug development is impeded by the lack of information regarding their mode of action and associated inhibitor-bound NDH-2 structure. We have determined the crystal structure of NDH-2 complexed with a quinolone inhibitor 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO). HQNO is nested into the slot-shaped tunnel of the Q-site, in which the quinone-head group is clamped by Q317 and I379 residues, and hydrogen-bonds to FAD. The interaction of HQNO with bacterial NDH-2 is very similar to the native substrate ubiquinone (UQ1) interactions in the yeast Ndi1-UQ1 complex structure, suggesting a conserved mechanism for quinone binding. Further, the structural analysis provided insight how modifications of quinolone scaffolds improve potency (e.g. quinolinyl pyrimidine derivatives) and suggests unexplored target space for the rational design of new NDH-2 inhibitors.
Project description:Type-II NADH:quinone oxidoreductases (NDH-2s) are membrane proteins involved in respiratory chains and the only enzymes with NADH:quinone oxidoreductase activity expressed in Staphylococcus aureus (S. aureus), one of the most common causes of clinical infections. NDH-2s are members of the two-Dinucleotide Binding Domains Flavoprotein (tDBDF) superfamily, having a flavin adenine dinucleotide, FAD, as prosthetic group and NAD(P)H as substrate. The establishment of a Charge-Transfer Complex (CTC) between the isoalloxazine ring of the reduced flavin and the nicotinamide ring of NAD+ in NDH-2 was described, and in this work we explored its role in the kinetic mechanism using different electron donors and electron acceptors. We observed that CTC slows down the rate of the second half reaction (quinone reduction) and determines the effect of HQNO, an inhibitor. Also, protonation equilibrium simulations clearly indicate that the protonation probability of an important residue for proton transfer to the active site (D302) is influenced by the presence of the CTC. We propose that CTC is critical for the overall mechanism of NDH-2 and possibly relevant to keep a low quinol/quinone ratio and avoid excessive ROS production in vivo.
Project description:Type II NADH-quinone oxidoreductase (NDH-2) catalyzes the transfer electrons from NADH to the quinone pool and plays an essential role in the oxidative phosphorylation system of Mycobacterium tuberculosis (Mtb). The absence of NDH-2 in the mammalian mitochondrial electron transport chain makes this enzyme an attractive target for antibiotic development. To fully establish the kinetic properties of this enzyme, we studied the interaction of Mtb NDH-2 with substrates, NADH, and various quinone analogues and their products in both membrane and soluble environments. These studies, and comparative analyses of the kinetics with thio-NAD(+) and quinone electron acceptors, provided evidence that Mtb NDH-2 catalyzes the transfer electrons from NADH to quinone substrates by a nonclassical, two-site ping-pong kinetic mechanism whereby substrate quinones bind to a site that is distinct from the NADH-binding site. Furthermore, the effects of quinols on Mtb NDH-2 catalytic activity demonstrate the presence of two binding sites for quinone ligands, one favoring the reduced form and the other favoring the oxidized form.
Project description:Oxidation of malate to oxaloacetate in Escherichia coli can be catalyzed by two enzymes: the well-known NAD-dependent malate dehydrogenase (MDH; EC 188.8.131.52) and the membrane-associated malate:quinone-oxidoreductase (MQO; EC 184.108.40.206), encoded by the gene mqo (previously called yojH). Expression of the mqo gene and, consequently, MQO activity are regulated by carbon and energy source for growth. In batch cultures, MQO activity was highest during exponential growth and decreased sharply after onset of the stationary phase. Experiments with the beta-galactosidase reporter fused to the promoter of the mqo gene indicate that its transcription is regulated by the ArcA-ArcB two-component system. In contrast to earlier reports, MDH did not repress mqo expression. On the contrary, MQO and MDH are active at the same time in E. coli. For Corynebacterium glutamicum, it was found that MQO is the principal enzyme catalyzing the oxidation of malate to oxaloacetate. These observations justified a reinvestigation of the roles of MDH and MQO in the citric acid cycle of E. coli. In this organism, a defined deletion of the mdh gene led to severely decreased rates of growth on several substrates. Deletion of the mqo gene did not produce a distinguishable effect on the growth rate, nor did it affect the fitness of the organism in competition with the wild type. To investigate whether in an mqo mutant the conversion of malate to oxaloacetate could have been taken over by a bypass route via malic enzyme, phosphoenolpyruvate synthase, and phosphenolpyruvate carboxylase, deletion mutants of the malic enzyme genes sfcA and b2463 (coding for EC 220.127.116.11 and EC 18.104.22.168, respectively) and of the phosphoenolpyruvate synthase (EC 22.214.171.124) gene pps were created. They were introduced separately or together with the deletion of mqo. These studies did not reveal a significant role for MQO in malate oxidation in wild-type E. coli. However, comparing growth of the mdh single mutant to that of the double mutant containing mdh and mqo deletions did indicate that MQO partly takes over the function of MDH in an mdh mutant.
Project description:Type II NADH:quinone oxidoreductase (NDH-2) is a respiratory enzyme found in the electron-transport chain of many species, with the exception of mammals. It is a 40-70?kDa single-subunit monotopic membrane protein that catalyses the oxidation of NADH and the reduction of quinone molecules via the cofactor FAD. NDH-2 is a promising new target for drug development given its essential role in many bacterial species and intracellular parasites. Only two bacterial NDH-2 structures have been reported and these structures are at moderate resolution (2.3-2.5?Å). In this communication, a new crystallization platform is reported that produced high-quality NDH-2 crystals that diffracted to high resolution (2.15?Å). The high-resolution NDH-2 structure was used for in silico quinone substrate-docking studies to investigate the binding poses of menadione and ubiquinone molecules. These studies revealed that a very limited number of molecular interactions occur at the quinone-binding site of NDH-2. Given that the conformation of the active site is well defined, this high-resolution structure is potentially suitable for in silico inhibitor-compound screening and ligand-docking applications.
Project description:The proton-translocating NADH-quinone oxidoreductase (complex I/NDH-1) is a multisubunit enzymatic complex. It has a characteristic L-shaped form with two domains, a hydrophilic peripheral domain and a hydrophobic membrane domain. The membrane domain contains three antiporter-like subunits (NuoL, NuoM, and NuoN, Escherichia coli naming) that are considered to be involved in the proton translocation. Deletion of either NuoL or NuoM resulted in an incomplete assembly of NDH-1 and a total loss of the NADH-quinone oxidoreductase activity. We have truncated the C terminus segments of NuoM and NuoL by introducing STOP codons at different locations using site-directed mutagenesis of chromosomal DNA. Our results suggest an important structural role for the C-terminal segments of both subunits. The data further advocate that the elimination of the last transmembrane helix (TM14) of NuoM and the TM16 (at least C-terminal seven residues) or together with the HL helix and the TM15 of the NuoL subunit lead to reduced stability of the membrane arm and therefore of the whole NDH-1 complex. A region of NuoL critical for stability of NDH-1 architecture has been discussed.
Project description:The respiratory NADH:quinone oxidoreductase (complex I) (NDH-1) is a multisubunit enzyme that translocates protons (or in some cases Na+) across energy-conserving membranes from bacteria or mitochondria. We studied the reaction of the Na+-translocating complex I from the enterobacterium Klebsiella pneumoniae with N,N'-dicyclohexylcarbodiimide (DCCD), with the aim of identifying a subunit critical for Na+ binding. At low Na+ concentrations (0.6 mM), DCCD inhibited both quinone reduction and Na+ transport by NDH-1 concurrent with the covalent modification of a 30-kDa polypeptide. In the presence of 50 mM Na+, NDH-1 was protected from inhibition by DCCD, and the modification of the 30-kDa polypeptide with [14C]DCCD was prevented, indicating that Na+ and DCCD competed for the binding to a critical carboxyl group in NDH-1. The 30-kDa polypeptide was assigned to NuoH, the homologue of the ND1 subunit from mitochondrial complex I. It is proposed that Na+ binds to the NuoH subunit during NADH-driven Na+ transport by NDH-1.
Project description:Aspartate, which is converted from oxaloacetate (OAA) by aspartate aminotransferase, is considered an important precursor for purine salvage and pyrimidine de novo biosynthesis, and is thus indispensable for the growth of Plasmodium parasites at the asexual blood stages. OAA can be produced in malaria parasites via two routes: (i) from phosphoenolpyruvate (PEP) by phosphoenolpyruvate carboxylase (PEPC) in the cytosol, or (ii) from fumarate by consecutive reactions catalyzed by fumarate hydratase (FH) and malate:quinone oxidoreductase (MQO) in the mitochondria of malaria parasites. Although PEPC-deficient Plasmodium falciparum and Plasmodium berghei (rodent malaria) parasites show a growth defect, the mutant P. berghei can still cause experimental cerebral malaria (ECM) with similar dynamics to wild-type parasites. In contrast, the importance of FH and MQO for parasite viability, growth and virulence is not fully understood because no FH- and MQO-deficient P. falciparum has been established. In this study, the role of FH and MQO in the pathogenicity of asexual-blood-stage Plasmodium parasites causing cerebral malaria was examined.First, FH- and MQO-deficient parasites were generated by inserting a luciferase-expressing cassette into the fh and mqo loci in the genome of P. berghei ANKA strain. Second, the viability of FH-deficient and MQO-deficient parasites that express luciferase was determined by measuring luciferase activity, and the effect of FH or MQO deficiency on the development of ECM was examined. While the viability of FH-deficient P. berghei was comparable to that of control parasites, MQO-deficient parasites exhibited considerably reduced viability. FH activity derived from erythrocytes was also detected. This result and the absence of phenotype in FH-deficient P. berghei parasites suggest that fumarate can be metabolized to malate by host or parasite FH in P. berghei-infected erythrocytes. Furthermore, although the growth of FH- and MQO-deficient parasites was impaired, the development of ECM was suppressed only in mice infected with MQO-deficient parasites.These findings suggest that MQO-mediated mitochondrial functions are required for development of ECM of asexual-blood-stage Plasmodium parasites.