7B). These data, and the high sequence similarity of the C. thermarum NDH‐2 to that of NDH‐2 enzymes from bacterial pathogens (Fig. 1,4‐naphthoquinone) enzyme activity was measured with either NADH or quinone concentrations being varied (with the other being kept constant) and data were fitted to the Michaelis‐Menten equation by non‐linear least‐squares regression (GraphPad Prism 6). The difference electron density map (Fo−Fc) at 3σ covering FAD is shown in dark brown mesh. Bacteroides fragilis A phenothiazine analogue was also tested in a mouse model of acute M. tuberculosis infection and found to reduce by 90% the M. tuberculosis bacterial load in the lungs after 11 days of treatment compared to a 3‐ to 4‐log reduction in colony‐forming units (cfu) with the INH or rifampicin control (Weinstein et al., 2005). The complex couples the oxidation of NADH and the reduction of ubiquinone, to the generation of a proton gradient which is then used for ATP synthesis. Two residues in the linker region, R347 and H345, form backbone hydrogen bonds with the side‐chains of Q317 and Q321 from the proposed quinone‐binding motif (Fig. Dimroth, P. (1997) Biochim. S2D, inset). Although smaller and simpler than complex-I, It contains two types of prosthetic groups and at least four different proteins. Only the Ndi1 dimer serves to consolidate the amphipathic contributions from each monomer into a single more extensive region (Fig. In some cases more than one copy is present (Melo et al., 2004). B. ... What is the prosthetic group and the reaction catalyzed by pyruvate dehydrogenase? They are capable of accepting electrons and protons but can only donate electrons. J. Mol. The expression vector pTRC99a was used to construct expression plasmids pTRCndh2 and pTRCndhtrun379 bearing full‐length and truncated ndh2 respectively from C. thermarum strain TA2.A1 (Accession No. FMN is a tightly bound prosthetic group of the dehydrogenase enzyme, and it is reduced to FMNH 2 by the two reducing equivalents derived from NADH: NADH + H + + E - FMN ⇌ NAD + + E - FMNH 2 The electrons from FMNH 2 are transferred to the next electron carrier, coenzyme Q, via the iron–sulfur centers of the NADH-CoQ reductase. Biol. Interestingly, mutations in both the AQXAXQ motif and the linker region have been reported in mycobacterial NDH‐2 (Miesel et al., 1998; Vilcheze et al., 2005). The ± 5 kT/e electrostatic surface potentials of NDH‐2 and Ndi1 reveal similar charged tunnels associated with the likely site for quinone entry into the active site from the membrane (negative in red, uncharged in white and positive in blue). S4A, circle 12), which are located at the heart of the homodimeric interaction for the yeast Ndi1 (Feng et al., 2012), are absent in the bacterial NDH‐2 enzyme. NADH dehydrogenase removes two hydrogen atoms from the substrate and donates the hydride ion (H –) to NAD + forming NADH and H + is released in the solution. Apoptosis-inducing Factor (AIF) and Its Family Member Protein, AMID, Are Rotenone-sensitive NADH:Ubiquinone Oxidoreductases (NDH-2). Yagi, T. (1993) Biochim. The electron acceptor – the isoalloxazine ring – of FMN is identical to that of FAD. 5A). The NADH‐binding cleft (yellow surface) and the quinone binding tunnel (orange surface) locations are clearly separated by this linker along the plane of the membrane. 2-Mercapto-Quinazolinones as Inhibitors of Type II NADH Dehydrogenase and This linker region is enriched with both hydrophobic and positively charged residues suitable for interaction with the membrane. In Escherichia coli, NDH‐1 is usually associated with anaerobic respiratory pathways (e.g. Several classes of compounds, most notably the phenothiazines, have been reported to target mycobacterial NDH‐2 at low affinity (Weinstein et al., 2005; Yano et al., 2006; Rao et al., 2008; Warman et al., 2013), but the molecular mechanism of enzyme inhibition is not known. The first (residues 2–109 and 263–345 in cyan) and second (110–262 in gold) Rossmann fold domains and the C‐terminal membrane‐anchoring domain (346–398 in magenta) are shown as a ribbon diagram. We determined the role of the NADH dehydrogenase enzymes in aerobic growth. Both PHENIX (Adams et al., 2002) and Refmac5 (Murshudov et al., 2011) were employed for the maximum‐likelihood of refinement of the structure, medium NCS restraints applied throughout. Once the culture had reached OD600 0.5, NDH‐2 expression was induced by 1 mM isopropyl β‐d‐thiogalactopyranoside (IPTG). . Soaking of crystals in the presence of co‐enzyme Q2 (0.1 and 0.2 mM) or 1,4‐napthoquinone (0.5 mM) or decylubiqinone (1 mM) was also performed. Consistent with the yeast Ndi1 structure, electrostatic analysis shows two relatively positively charged nucleotide binding sites for NADH and FAD, which can be observed from the cytoplasmic‐facing side of the protein (Figs 2D and 6). Journal of the American Chemical Society. 25, 325-391. All MS spectra were acquired in linear, positive‐ion mode with 1200 laser pulses per sample spot. Given the vital role these enzymes play in microbial respiration this lack of structural information hinders our understanding of how these enzymes function at a molecular level. The NADH dehydrogenase, Ndh, of C. glutamicum seems to have FAD as the prosthetic group, since Ndh of C. glutamicum has a reasonable sequence similarity to other NDH IIs; 58% identity (71% similarity) of Mycobacterium tuberculosis Ndh, 45% (62%) of Synecocystis sp. Genetic and Biochemical Analysis of Anaerobic Respiration in The line approximately separates the inner cytoplasmic side and the membrane anchoring side of the NDH‐2 molecule. S3). The enzyme in complex I is NADH dehydrogenase and is a very large protein, containing 45 … The residue numbers correspond to the yeast Ndi1. The foot (the hydrophobic protein) is membrane bound, and contains a catalytic site at which ubiquinone is reduced, and inhibitors bind, and several iron sulfur centers. (1997) Biochim. Number of times cited according to CrossRef: Respiratory Chain and Energy Metabolism of Corynebacterium glutamicum. The NDH‐2 structure (with no NADH or quinone bound) was solved by molecular replacement at 2.5 Å resolution (Table 1 and Fig. For example, in the mycobacterial species Mycobacterium tuberculosis and Mycobacterium smegmatis, NDH‐2 is essential for growth even in a NDH‐1+ background (McAdam et al., 2002; Sassetti et al., 2003; Weinstein et al., 2005; Griffin et al., 2011). The 7-phenyl benzoxaborole series is active against Mycobacterium tuberculosis. The residues that form the predicted NDH‐2 quinone binding site (light orange) are shown in both surface and stick representations. A prosthetic group is a nonprotein molecule required for the activity of a protein. Two types of catalytic mechanisms have been proposed for NDH‐2 enzyme activity, ping‐pong or ternary complex (Eschemann et al., 2005; Yano et al., 2006; Yamashita et al., 2007; Yang et al., 2011). There is a second catalytic site for ubiquinone reaction on the ankle, but this is seen as a separate activity only in the dissociated complex. The wild‐type enzyme had Km and Vmax values of 36 μM and 331 μ moles NADH oxidized min−1 (mg protein)−1 respectively (Fig. Red dashed lines are used to represent where both hydrogen bond and salt bridge interactions occur. Consistent with the C‐terminal helices of Ndi1, surface‐exposed side‐chains are enriched with both hydrophobic and positively charged residues in bacterial NDH‐2 thus facilitating membrane localization (Fig. Alternative NAD(P)H dehydrogenase and alternative oxidase: Proposed physiological roles in animals. The electron density corresponding for three out of four chains were readily interpretable and only a few residues at both N‐ and C‐termini of these molecules could not be modelled. S5B), whereas the Q317A/Q321A mutant had a lower affinity for 1,4‐naphthoquinone with an apparent Km of 75 μM (Fig. ZP_08531709.1) (Kalamorz et al., 2011). Streptococcus agalactiae In Escherichia coli, the pentose phosphate pathway is one of the main sources of NADPH. Four NDH‐2 molecules were found in the asymmetric unit, which packed as two dimers (Fig. Prosthetic groups include co-enzymes, which are the prosthetic groups of enzymes. Crystal structure of type II NADH:quinone oxidoreductase from Caldalkalibacillus thermarum with an improved resolution of 2.15 Å. Metabolic fingerprinting of bacteria by fluorescence lifetime imaging microscopy. Pseudomonas aeruginosa In this way, the reduced forms are formed (NADH and NADPH), where new C-H bond is created on C-4 (Bellamacina, 1996). The key role of glutamate 172 in the mechanism of type II NADH:quinone oxidoreductase of Staphylococcus aureus. Comparison of bacterial NDH‐2 with the yeast NADH dehydrogenase (Ndi1) structure revealed non‐overlapping binding sites for quinone and NADH in the bacterial enzyme. S4A). The Small RNA ncS35 Regulates Growth in Burkholderia cenocepacia J2315. The mixture was vortexed until clear. NADH dehydrogenase (complex I) Succinate coenzyme Q reductase (complex II) Coenzyme Q (CoQ) (also called ubiquinone) Cytochrome bc1 complex (complex III) Cytochrome c (Cyt c) Cytochrome oxidase (complex IV) NADH binds complex I & passes 2 electrons to a flavin momonucleotide (FMN) prosthetic group. NDH‐2 diffraction data were acquired at the Australian Synchrotron MX2 beam‐line equipped with an ADSC Quantum 315r detector. The reduced FADH 2 of E 3 transfers a hydride ion to NAD +, forming NADH. If you do not receive an email within 10 minutes, your email address may not be registered, The sub-complex can be further dissociated into a flavoprotein and an iron protein. Ref. NDH‐2 has not been reported in mammalian mitochondria, and its essentiality in bacterial pathogens has led to the proposal that these enzymes may represent a potential new drug target for the treatment of human pathogens (Weinstein et al., 2005; Yano et al., 2006; Rao et al., 2008; Warman et al., 2013) and parasites (Biagini et al., 2006; Saleh et al., 2007). New insights into the organisation of the oxidative phosphorylation system in the example of pea shoot mitochondria. Biochemical studies of membrane bound Plasmodium falciparum mitochondrial L-malate:quinone oxidoreductase, a potential drug target. Despite poor in vitro activity, drugs of the phenothiazine family (trifluoroperazine, chlorpromazine) have potent activity in vivo against drug‐susceptible and drug‐resistant M. tuberculosis strains (Amaral et al., 1996; Ordway et al., 2003). Ohnishi, T., ed. Which of the following is a prosthetic group in the -ketoglutarate dehydrogenase complex? In contrast, the Ndi1‐NAD+ and Ndi1‐UQ2 complex structures from Iwata et al. The resulting PCR products were restriction digested with NcoI and SalI and cloned into pTRC99a, digested with NcoI and SalI. 15. in dihydrolipoamide dehydrogenase (NADH), EC 1.8.1.4) or covalently bound by a methylene bridge between the benzene ring of the benzo[g]pteridine-2,4-dione and an amino acid residue, such as cysteine, histidine or tyrosine, in the protein (e.g. The first amphipathic helix (magenta), providing K376, and the first β‐strand of the membrane‐anchoring domain (orange) presumably contribute to stabilizing FAD. The identity of both bands (monomer and dimer) was confirmed by mass spectrometry analysis. D. Final view highlights the cytoplasmic faces of the enzymes with FAD and NADH binding sites indicated by arrows. Any queries (other than missing content) should be directed to the corresponding author for the article. Recently the E. coli proline:ubiquinone PutA enzyme was shown to exhibit strong evidence for a two‐site ping‐pong mechanism and it has been previously reported that many oxidoreductase enzymes display two‐site ping‐pong kinetics (Coughlan and Rajagopalan, 1980; Moxley et al., 2011) implying that this mechanism is a conserved feature of this class of enzyme. I). ZP_08531709.1) (Kalamorz et al., 2011). The first β‐strand of the membrane‐anchoring domain packs against the isoalloxazine ring, and K376 is derived from the first amphipathic helix; both contribute to binding the FAD in the structure (Fig. . These include the proton‐pumping type I NADH dehydrogenase (NDH‐1, complex I), the non‐proton pumping type II NADH dehydrogenase (NDH‐2) and the sodium‐pumping NADH dehydrogenase (NQR). S4A and B): overlaying the two with Superpose (Krissinel and Henrick, 2004) indicated an RMSD of 1.73 Å for the Cα atoms of 360 residues (Fig. Use the link below to share a full-text version of this article with your friends and colleagues. SDS‐PAGE analysis of the truncated NDH‐2 protein showed the appearance of both monomeric and dimeric bands (Fig. Discrepancies in expected versus observed size of monotopic membrane proteins has been noted by other groups (Marcia et al., 2010). Monotopic Membrane Proteins Join the Fold. The RSc0454-Encoded FAD-Linked Oxidase Is Indispensable for Pathogenicity in 2). A prosthetic group is a nonprotein molecule required for the activity of a protein. The non‐covalently bound FAD is shown as a space fill model. 3B), presumably because this helix no longer has a distinct role in dimerization. The presence of FAD was further confirmed by comparison to the emission spectra of a known sample of FAD. FAD is shown as dark blue sticks. The CCP4 suite was employed for scaling of the data (Bailey, 1994). In particular two conserved glutamine residues (Q394 and Q398) occupy very similar positions. 4). All members of the NDH-1 group analyzed to date are multiple polypeptide enzymes and contain noncovalently bound FMN and iron-sulfur clusters as prosthetic groups. C. Quinone molecules adapted from the yeast Ndi1 structure (PDB 4G73). Incorporation of triphenylphosphonium functionality improves the inhibitory properties of phenothiazine derivatives in Mycobacterium tuberculosis. Biochimica et Biophysica Acta (BBA) - Bioenergetics. The FAD binding site. Cells were grown for a further 4 h before being harvested by centrifugation and pellets were stored at −20°C. The amphipathic helices identified in each case as interacting with the membrane are highlighted in blue and orange for NDH‐2 and Ndi1, and blue, orange and gold for SQR. Second the Q317A mutant was used as a template to make NDH‐2 Q317A/Q321A using primers ndh2Q317AQ321AFw (5′‐TCGCCATTGCACATGGGGAAAATGTTGCTGCCAACCTGGCG‐3′) and ndh2Q317AQ321ARv (5′‐TCCCCATGTGCAATGGCGATTGCGGCCGTGGGGGGATAAGG‐3′) giving rise to the plasmid pTRCQ317A/Q321A. S2C). The first enzyme of the pathway, glucose-6-phosphate dehydrogenase (G6PDH), is generally considered an exclusive NADPH producer, but a rigorous assessment of … To determine the nature of flavin binding in NDH‐2, the protein was denatured and the emission spectra of the supernatant analysed. Purified NDH‐2 was concentrated to 2–4 mg ml−1 using Amicon ultra centrifugal filter devices (50 kDa MWCO) before final purification using a Superose 12 10/300 GL (GE Healthcare, Sweden) column pre‐equilibrated with size exclusion buffer [1% w/v OG, 50 mM Tris‐HCl (pH 8.0), 150 mM NaCl] run at 1 ml min−1. In Silico Discovery of a Substituted 6-Methoxy-quinalidine with Leishmanicidal Activity in Leishmania infantum. 1). For example, the monotopic membrane proteins glycerol‐3‐phosphate dehydrogenase (GlpD) from E. coli and electron transfer flavoprotein‐ubiquinone oxidoreductase (ETF‐QO) from Sus scrofa are reported to be monomeric, but both utilize amphipathic helices for their membrane localization (Zhang et al., 2006; Yeh et al., 2008). NADH initially binds to NADH dehydrogenase, and transfers two electrons to the flavin mononucleotide (FMN) prosthetic group of complex I, creating FMNH 2. A. Hydrogen bond networks and charge interactions at the homodimer interface of bacterial NDH‐2. We thank Htin Aung and David Leslie for technical assistance and the Centre for Protein Research for mass spectrometry. To compensate for this small dimer interface, an extensive network of hydrogen bonds and charge interactions were observed that probably contribute to dimer stability (Fig. The harvested membranes were then solubilized at 5 mg ml−1 total membrane protein in solubilization buffer containing 50 mM Tris‐HCl (pH 8.0), 20 mM imidazole, 150 mM NaCl, cOmplete EDTA‐free protease inhibitor (Roche) and 2% (w/v) n‐octyl‐β‐d‐glucopyranoside (OG) (Glycon Bioch. With 100 μl reservoirs complex provides molecular insight into the catalytic mechanism of catalysis by Type-II NADH: quinone from. Were stored at −20°C the electron nadh dehydrogenase prosthetic group - the isoalloxazine ring – FMN! Or menaquinone 100 μl reservoirs Na+-NQR ), a Potential drug target diffraction analysis of Azospirillum vegetative! On resetting your password complex occurs in the mitochondria of Saccharomyces cerevisiae is present Melo. 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