Takahiro Yano - Academia.edu (original) (raw)
Papers by Takahiro Yano
Journal of Biological Chemistry, 1994
The 25-kDa subunit of the proton-translocating NADH-quinone oxidoreductase (NDH-1) of Paracoccus ... more The 25-kDa subunit of the proton-translocating NADH-quinone oxidoreductase (NDH-1) of Paracoccus denitrificans has been expressed in Escherichia coli and purified to homogeneity. EPR studies of the reduced recombinant protein indicated that the expressed subunit contains a single [2Fe-2S] cluster (Yano, T., Sled', V. D., Ohnishi, T., and Yagi, T. (1994) Biochemistry 33,494-499). In this report, the electronic, magnetic, and vibrational properties of the [2Fe-2SI2+,+ center have been investigated by the combination of absorption, circular dichroism, variable-temperature magnetic circular dichroism, electron paramagnetic resonance, and resonance Raman spectroscopies and compared with a range of simple [BFe-2S]-containing proteins. The results are consistent with coordination by two cysteinyl residues at both the reducible and nonreducible iron sites and reveal a striking similarity between the properties of the [2Fe-2S] cluster in the l? denitrificans NDH-1 25-kDa subunit and those of the subclass of ferredoxin-type [2Fe-2S] centers typified by Clostridium pasteurianum 2Fe ferredoxin. The four cyteines residues involved in cluster ligation in these proteins have been tentatively identified based on sequence homology considerations. Paracoccus denitrificans is a Gram-negative soil bacterium (1, 2). Aerobically grown P: denitrificans expresses a mammalian mitochondrial-type respiratory chain (3, 4) which appears to bear only the proton-translocating NADH-quinone (Q)' oxidoreductase (NDH-1) (5-7). I? denitrificans NDH-1 is probably composed of 14 unlike subunits and bears noncovalently bound FMN and at least 5 EPR-detectable iron-sulfur clusters as pros-Grants R01-GM33712 (to T. y.) and R01-GM51962 (to M. K. J.
Angewandte Chemie (International ed. in English), Jan 19, 2018
The generation of ATP through oxidative phosphorylation is an essential metabolic function for My... more The generation of ATP through oxidative phosphorylation is an essential metabolic function for Mycobaterium tuberculosis (Mtb), regardless of the growth environment. The type II NADH dehydrogenase (Ndh-2) is the conduit for electrons into the pathway, and is absent in the mammalian genome, thus making it a potential drug target. Herein, we report the identification of two types of small molecules as selective inhibitors for Ndh-2 through a multicomponent high-throughput screen. Both compounds block ATP synthesis, lead to effects consistent with loss of NADH turnover, and importantly, exert bactericidal activity against Mtb. Extensive medicinal chemistry optimization afforded the best analogue with an MIC of 90 nm against Mtb. Moreover, the two scaffolds have differential inhibitory activities against the two homologous Ndh-2 enzymes in Mtb, which will allow precise control over Ndh-2 function in Mtb to facilitate the assessment of this anti-TB drug target.
![Research paper thumbnail of Discovery of Imidazo[1,2-a]pyridine ethers and Squaramides as Selective and Potent Inhibitors of Mycobacterial Adenosine Triphosphate (ATP) Synthesis](https://attachments.academia-assets.com/110772845/thumbnails/1.jpg)
](Journal of medicinal chemistry, Feb 11, 2017
The approval of bedaquiline to treat tuberculosis has validated adenosine triphosphate (ATP) synt... more The approval of bedaquiline to treat tuberculosis has validated adenosine triphosphate (ATP) synthase as an attractive target to kill Mycobacterium tuberculosis. Herein, we report the discovery of two diverse lead series imidazo[1,2-a]pyridine ethers (IPE) and squaramides (SQA) as inhibitors of mycobacterial ATP-synthesis. Through medicinal chemistry exploration, we established a robust structure activity relationship of these two scaffolds resulting in nanomolar potencies in an ATP-synthesis inhibition assay. A biochemical deconvolution cascade suggested cytochrome c oxidase as the potential target of IPE class of molecules, whereas characterization of spontaneous resistant mutants of SQAs unambiguously identified ATP synthase as its molecular target. Absence of cross resistance against bedaquiline resistant mutants suggested a different binding site for SQAs on ATP synthase. Furthermore, SQAs were found to be non-cytotoxic and demonstrated efficacy in a mouse model of tuberculosis...
MedChemComm, 2016
2,4-Diaminoquinazolines, 2,4-diaminoquinolines and aminopyrazolopyrimidines, inhibitors of mycoba... more 2,4-Diaminoquinazolines, 2,4-diaminoquinolines and aminopyrazolopyrimidines, inhibitors of mycobacterial ATP synthesis, are novel lead molecules towards discovery and development of new anti-tubercular agents.
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2014
Methicillin-resistant Staphylococcus aureus (MRSA) is currently one of the principal multiple dru... more Methicillin-resistant Staphylococcus aureus (MRSA) is currently one of the principal multiple drug resistant bacterial pathogens causing serious infections, many of which are life-threatening. Consequently, new therapeutic targets are required to combat such infections. In the current work, we explore the type 2 Nicotinamide adenine dinucleotide reduced form (NADH) dehydrogenases (NDH-2s) as possible drug targets and look at the effects of phenothiazines, known to inhibit NDH-2 from Mycobacterium tuberculosis. NDH-2s are monotopic membrane proteins that catalyze the transfer of electrons from NADH via flavin adenine dinucleotide (FAD) to the quinone pool. They are required for maintaining the NADH/Nicotinamide adenine dinucleotide (NAD +) redox balance and contribute indirectly to the generation of proton motive force. NDH-2s are not present in mammals, but are the only form of respiratory NADH dehydrogenase in several pathogens, including S. aureus. In this work, the two putative ndh genes present in the S. aureus genome were identified, cloned and expressed, and the proteins were purified and characterized. Phenothiazines were shown to inhibit both of the S. aureus NDH-2s with half maximal inhibitory concentration (IC 50) values as low as 8 μM. However, evaluating the effects of phenothiazines on whole cells of S. aureus was complicated by the fact that they are also acting as uncouplers of oxidative phosphorylation. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.
Structure, 2000
The mitochondrial bioenergetics field has experienced an exciting breakthrough with the recent st... more The mitochondrial bioenergetics field has experienced an exciting breakthrough with the recent structure determination of several key membrane complexes. The latest addition to this line of structures, that of quinolfumarate reductase, provides new insights into the mechanism of energy transduction.
Proceedings of the National Academy of Sciences, 1999
The proton-translocating NADH-quinone oxidoreductase (EC 1.6.99.3 ) is the largest and least unde... more The proton-translocating NADH-quinone oxidoreductase (EC 1.6.99.3 ) is the largest and least understood enzyme complex of the respiratory chain. The mammalian mitochondrial enzyme (also called complex I) contains more than 40 subunits, whereas its structurally simpler bacterial counterpart (NDH-1) in Paracoccus denitrificans and Thermus thermophilus HB-8 consists of 14 subunits. A major unsolved question is the location and mechanism of the terminal electron transfer step from iron–sulfur cluster N2 to quinone. Potent inhibitors acting at this key region are candidate photoaffinity probes to dissect NADH-quinone oxidoreductases. Complex I and NDH-1 are very sensitive to inhibition by a variety of structurally diverse toxicants, including rotenone, piericidin A, bullatacin, and pyridaben. We designed (trifluoromethyl)diazirinyl[ 3 H]pyridaben ([ 3 H]TDP) as our photoaffinity ligand because it combines outstanding inhibitor potency, a suitable photoreactive group, and tritium at high ...
Nature Structural & Molecular Biology, 2008
Bacterial polysulfide reductase (PsrABC) is an integral membrane protein complex responsible for ... more Bacterial polysulfide reductase (PsrABC) is an integral membrane protein complex responsible for quinone coupled reduction of polysulfide, a process important in extreme environments such as deep-sea vents and hot springs. We determined the structure of polysulfide reductase from Thermus thermophilus at 2.4 Å resolution, revealing how the PsrA subunit recognizes and reduces its unique poly anionic substrate. The integral membrane subunit PsrC was characterized using the natural substrate menaquinone-7 and inhibitors, providing a comprehensive representation of a quinone binding site and revealing the presence of a water filled cavity connecting the quinone binding site on the periplasmic side to the cytoplasm. These results suggest that polysulfide reductase could be a key energy-conserving enzyme of the T. thermophilus respiratory chain,
Journal of Biological Chemistry, 2004
The proton-pumping NADH-quinone oxidoreductase from Escherichia coli houses nine iron-sulfur clus... more The proton-pumping NADH-quinone oxidoreductase from Escherichia coli houses nine iron-sulfur clusters, eight of which are found in its mitochondrial counterpart, complex I. The extra putative iron-sulfur cluster binding site with a CXXCXXXCX 27 C motif in the NuoG subunit has been assigned to ligate a [2Fe-2S] (N1c). However, we have shown previously that the Thermus thermophilus N1c fragment containing this motif ligates a [4Fe-4S] (Nakamaru-Ogiso, E., Yano, T., Ohnishi, T., and Yagi, T. (2002) J. Biol. Chem. 277, 1680-1688). In the current study, we individually inactivated four sets of the iron-sulfur binding motifs in the E. coli NuoG subunit by replacing all four ligands with Ala. Each mutant subunit, designated ⌬N1b, ⌬N1c, ⌬N4, and ⌬N5, was expressed as maltose-binding protein fusion proteins. After in vitro reconstitution, all mutant subunits were characterized by EPR. Although EPR signals from cluster N1b were not detected in any preparations, we detected two [4Fe-4S] EPR signals with g values of g x,y,z ؍ 1.89, 1.94, and 2.06, and g x,y,z ؍ 1.91, 1.94, and 2.05 at 6-20 K in wild type, ⌬N1b, and ⌬N5. The former signal was assigned to cluster N4, and the latter signal was assigned to cluster N1c because of their disappearance in ⌬N4 and ⌬N1c. Confirming that a [4Fe-4S] cluster ligates to the N1c motif, we propose to replace its misleading [2Fe-2S] name, N1c, with "cluster N7." In addition, because these mutations differently affected the assembly of peripheral subunits by in trans complementation analysis with the nuoG knockout strain, the implicated structural importance of the iron-sulfur binding domains is discussed.
Journal of Biological Chemistry, 1999
The proton-translocating NADH-quinone oxidoreductase (NDH-1) of Paracoccus denitrificans is compo... more The proton-translocating NADH-quinone oxidoreductase (NDH-1) of Paracoccus denitrificans is composed of at least 14 subunits (NQO1-14) and is located in the cytoplasmic membrane. In the present study, topological properties and stoichiometry of the 7 subunits (NQO1-6 and NQO9) of the P. denitrificans NDH-1 in the membranes were investigated using immunological techniques. Treatments with chaotropic reagents (urea, NaI, or NaBr) or with alkaline buffer (pH 10-12) resulted in partial or complete extraction of all the subunits from the membranes. Of interest is that when NaBr or urea were used, the NQO6 and NQO9 subunits remained in the membranes, whereas the other subunits were completely extracted, suggesting their direct association with the membrane part of the enzyme complex. Both deletion study and homologous expression study of the NQO9 subunit provided a clue that its hydrophobic Nterminal stretch plays an important role in such an association. In light of this observation and others, topological properties of the subunits in the NDH-1 enzyme complex are discussed. In addition, determination of stoichiometry of the peripheral subunits of the P. denitrificans NDH-1 was completed by radioimmunological methods. All the peripheral subunits are present as one molecule each in the enzyme complex. These results estimated the total number of cofactors in the P. denitrificans NDH-1; the enzyme complex contains one molecule of FMN and up to eight iron-sulfur clusters, 2؋[2Fe-2S] and 6؋[4Fe-4S], provided that the NQO6 subunit bears one [4Fe-4S] cluster.
Journal of Biological Chemistry, 2001
The proton-translocating NADH-quinone oxidoreductase (NDH-1) of Thermus thermophilus HB-8 is comp... more The proton-translocating NADH-quinone oxidoreductase (NDH-1) of Thermus thermophilus HB-8 is composed of 14 subunits (designated Nqo1-14). This NDH-1 houses nine putative iron-sulfur binding sites, eight of which are generally found in bacterial NDH-1 and its mitochondrial counterpart (complex I). The extra site contains a CXXCXXXCX 27 C motif and is located in the Nqo3 subunit. This motif was originally found in Escherichia coli NDH-1 and was assigned to a binuclear cluster (g z, y, x ؍ 2.00, 1.95, 1.92) and named N1c. In this report, the Thermus Nqo3 fragment containing this motif was heterologously overexpressed, using a glutathione S-transferase fusion system. This fragment contained a small amount of iron-sulfur cluster, whose content was significantly increased by in vitro reconstitution. The UV-visible and EPR spectroscopic properties of this fragment indicate that the ligated iron-sulfur cluster is tetranuclear with nearly axial symmetry (g ʈ,Ќ ؍ 2.045, ϳ1.94). Site-directed mutants show that all four cysteines participate in the ligation of a [4Fe-4S] cluster. Considering the fact that the same motif coordinates only tetranuclear clusters in other enzymes so far known, we propose that the CXXCXXXCX 27 C motif in the Nqo3 subunit most likely ligates the [4Fe-4S] cluster. Bacterial proton-translocating NADH-quinone (Q) oxidoreductase (NDH-1) 1 and its mitochondrial counterpart (com
![Research paper thumbnail of Characterization of the Putative 2×[4Fe-4S]-binding NQO9 Subunit of the Proton-translocating NADH-Quinone Oxidoreductase (NDH-1) of Paracoccus denitrificans](https://attachments.academia-assets.com/110772834/thumbnails/1.jpg)
](Journal of Biological Chemistry, 1999
Molecular properties of the NQO9 subunit of Paracoccus denitrificans NDH-1, which is predicted to... more Molecular properties of the NQO9 subunit of Paracoccus denitrificans NDH-1, which is predicted to contain 2؋[4Fe-4S] clusters, were investigated using recombinant expression techniques and EPR spectroscopy. The full-length form of NQO9 subunit co-expressed with thioredoxin in Escherichia coli at ambient temperature was found dominantly in the cytoplasmic membrane with low amplification. Genetic deletion of relatively hydrophobic and less conserved N-terminal stretches (30 or 40 amino acid residues long) of the NQO9 subunit resulted in the overexpression of the truncated soluble form of the subunit in a high yield in the cytoplasm. The purified soluble form of the NQO9 subunit contained only a small quantity of Fe and S 2؊ (2.0-2.2 mol each per mol of subunit). However, the iron-sulfur content was considerably increased by in vitro reconstitution. The reconstituted NQO9 subunit contained 7.6-7.7 mol each of Fe and S 2؊ per molecule and exhibited optical absorption spectra similar to those of 2؋[4Fe-4S] ferredoxins. Two sets of relatively broad axial-type EPR signals with different temperature dependence and power saturation profile were detected in the dithionite-reduced preparations at a low temperature range (8-18 K). Due to a negative shift (<600 mV) of the apparent redox midpoint potential of the iron-sulfur clusters in the soluble form of the truncated NQO9 subunit, the following two possible cases could not be discriminated: (i) two sets of EPR signals arise from two distinct species of tetranuclear iron-sulfur clusters with two intrinsically different spectral parameters g ʈ ʈ,Ќ Ќ ؍ 2.05, ϳ1.93, and g ʈ ʈ,Ќ Ќ ؍ 2.08, ϳ1.90, and respective slow (P1 ⁄2 ؍ 8 milliwatts) and fast (P1 ⁄2 ؍ 342 milliwatts) spin relaxation; (ii) two clusters exhibit similar intrinsic EPR spectra (g ʈ ʈ,Ќ Ќ ؍ 2.05, ϳ1.93) with slow spin relaxation. When both clusters in the same subunit are concomitantly paramagnetic, their spin-spin interactions cause a shift of spectra to g ʈ ʈ,Ќ Ќ ؍ 2.08, ϳ1.90, with enhanced spin relaxation. In either case, our EPR data provide the first experimental evidence for the presence of two [4Fe-4S] ironsulfur clusters in the NQO9 subunit. The proton-translocating NADH-quinone (Q) 1 oxidoreductase is one of five enzyme complexes in the oxidative phosphorylation system that is located in the inner membrane of mitochondria and in the cytoplasmic membrane of bacteria. The mitochondrial enzyme is termed complex I, and the bacterial enzyme is called NDH-1. Both enzyme complexes catalyze the Reaction 1. NADH ϩ Q ϩ H ϩ ϩ nH ϩ ϩ NAD ϩ in 3 NAD ϩ ϩ QH 2 ϩ nH ϩ out (n ϭ 3-5)
International Journal of Antimicrobial Agents, 2014
Clostridium difficile, a highly drug-resistant Gram-positive, spore-forming bacterium, remains a ... more Clostridium difficile, a highly drug-resistant Gram-positive, spore-forming bacterium, remains a leading cause of hospital-acquired diarrhoea and antibiotic-associated colitis. Clinically, only a handful of antibiotics are used for treating C. difficile infection (CDI), suggesting a necessity for the development of new treatment options. Here we performed a high-throughput screen of 2000 drug-like compounds for inhibition of C. difficile. From this screen, one compound, 5-nitro-1,10phenanthroline (5-NP), showed potent bactericidal effects in vitro. In addition, this compound displayed high potency towards other Clostridium spp. as well as Mycobacterium bovis but not towards other tested Gram-positive and Gram-negative bacteria. Furthermore, we show that this inhibition may proceed through a metal chelation-dependent mechanism. More importantly, preliminary evidence suggests moderate efficacy for this compound in treating CDI in a murine infection model. These results present a possible basis for the further development of this compound as an antibiotic treatment for CDI.
FEBS Letters, 1991
The amino acid sequence of rusticyanin, a copper protein, purified from the iron-oxidizing bacter... more The amino acid sequence of rusticyanin, a copper protein, purified from the iron-oxidizing bacterium Tiliobacillrrs~rrooxiduns was determined. Rusticyanin contained 154 amino acid residues in a single polypeptide chain and its molecular weight was calculated to be about 16400 based on the amino acid sequence. The N-terminal sequence up to the 20th residue of the protein apparently resembled those of Merhylobacteriurn extorquerrs AM 1 amicyanin and poplar leaf plastocyanin rather than those of azurin family proteins. In the C-terminal region of the sequence, rusticyanin had one cysteine, one histidine and one mcthionine which arc conserved through many copper proteins. In the middle region of the sequence, rusticyanin was not similar to any other copper protein. The sequence nearby NW of rusticyanin was similar to those of other copper proteins to some extent. However, Asn which follows HiP and is highly conserved in other copper proteins did not exist in rusticyanin. Therefore, it seemed difficult to conclude on the basis of the results obtained in the present study that Hiss4 in rusticyanin was the fourth ligand to the copper atom. TlrioOacillus fererrooxiduns; Amino acid sequence; Rusticyanin; Copper protein '~~ChllOiO~~, NJ&W& Yokohnmn, 227, hlMl1. ML The cluates which contained rusticyanin were combiacd, dialyzed brought to you by CORE View metadata, citation and similar papers at core.ac.uk
Biochemistry, 2014
Type II NADH-quinone oxidoreductase (NDH-2) catalyzes the transfer electrons from NADH to the qui... more 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 pingpong 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.
Biochemistry, 2003
2 To date the 3D structure of succinate dehydrogenase is not available. However, high-resolution ... more 2 To date the 3D structure of succinate dehydrogenase is not available. However, high-resolution 3D structures of bacterial fumarate reductases, which are believed to be counterparts of succinate dehydrogenase, have been solved.
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2000
NADH-ubiquinone oxidoreductase (called complex I for mitochondrial enzyme and NDH-1 for bacterial... more NADH-ubiquinone oxidoreductase (called complex I for mitochondrial enzyme and NDH-1 for bacterial counterparts) is an energy transducer, which utilizes the redox energy derived from the oxidation of NADH with ubiquinone to generate an electrochemical proton gradient vW H across the membrane. The complex I/NDH-1 contain one non-covalently bound flavin mononucleotide and as many as eight iron-sulfur clusters as electron transfer components in common. In addition, electron paramagnetic resonance (EPR) spectroscopic studies have revealed that three ubisemiquinone (SQ) species with distinct spectroscopic and thermodynamic properties are detectable in complex I and function as electron/proton translocators. Thus, the understanding of molecular properties of the individual quinone species is prerequisite to elucidate the energy-coupling mechanism of complex I. We have investigated these SQ species using EPR spectroscopy and found that the three SQ species have strikingly different properties. We will report characteristics of these SQ species and discuss possible functional roles of individual quinone species in the electron/proton transfer reaction of complex I/NDH-1.
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1998
BIOCHIMICA ET BIOPHYSICA ACTA aaN Structure-function studies of iron-sulfur clusters and semiquin... more BIOCHIMICA ET BIOPHYSICA ACTA aaN Structure-function studies of iron-sulfur clusters and semiquinones in the NADH-Q oxidoreductase segment of the respiratory chain • .a~ la c
Journal of Biological Chemistry, 2010
FIGURE 1. Structures of clofazimine and a new, more soluble analog, KS6.
Developments in Geochemistry, 1991
Abstract From Thiobacillus ferrooxidans , an enzyme has been purified which catalyses oxidation o... more Abstract From Thiobacillus ferrooxidans , an enzyme has been purified which catalyses oxidation of Fe 2+ ions with T. ferrooxidans cytochrome c -552 as the electron acceptor. The enzyme shows absorption peaks at 282 and 382 nm and contains 18-20 mol of nonhaem iron and 6 mol of inorganic sulphide in 63,000 g. The enzyme does not use rusticyanin as the electron acceptor for oxidation of Fe 2+ ions. T. ferrooxidans cytochrome c -552 has also been purified to an electrophoretically homogeneous state. It shows an absorption peak at 411 nm in the oxidized form and peaks at 417, 523 and 552 nm in the reduced form. Its molecular weight is about 14,000 and its redox potential at pH 7.0 is approx. +0.37 V.
Journal of Biological Chemistry, 1994
The 25-kDa subunit of the proton-translocating NADH-quinone oxidoreductase (NDH-1) of Paracoccus ... more The 25-kDa subunit of the proton-translocating NADH-quinone oxidoreductase (NDH-1) of Paracoccus denitrificans has been expressed in Escherichia coli and purified to homogeneity. EPR studies of the reduced recombinant protein indicated that the expressed subunit contains a single [2Fe-2S] cluster (Yano, T., Sled', V. D., Ohnishi, T., and Yagi, T. (1994) Biochemistry 33,494-499). In this report, the electronic, magnetic, and vibrational properties of the [2Fe-2SI2+,+ center have been investigated by the combination of absorption, circular dichroism, variable-temperature magnetic circular dichroism, electron paramagnetic resonance, and resonance Raman spectroscopies and compared with a range of simple [BFe-2S]-containing proteins. The results are consistent with coordination by two cysteinyl residues at both the reducible and nonreducible iron sites and reveal a striking similarity between the properties of the [2Fe-2S] cluster in the l? denitrificans NDH-1 25-kDa subunit and those of the subclass of ferredoxin-type [2Fe-2S] centers typified by Clostridium pasteurianum 2Fe ferredoxin. The four cyteines residues involved in cluster ligation in these proteins have been tentatively identified based on sequence homology considerations. Paracoccus denitrificans is a Gram-negative soil bacterium (1, 2). Aerobically grown P: denitrificans expresses a mammalian mitochondrial-type respiratory chain (3, 4) which appears to bear only the proton-translocating NADH-quinone (Q)' oxidoreductase (NDH-1) (5-7). I? denitrificans NDH-1 is probably composed of 14 unlike subunits and bears noncovalently bound FMN and at least 5 EPR-detectable iron-sulfur clusters as pros-Grants R01-GM33712 (to T. y.) and R01-GM51962 (to M. K. J.
Angewandte Chemie (International ed. in English), Jan 19, 2018
The generation of ATP through oxidative phosphorylation is an essential metabolic function for My... more The generation of ATP through oxidative phosphorylation is an essential metabolic function for Mycobaterium tuberculosis (Mtb), regardless of the growth environment. The type II NADH dehydrogenase (Ndh-2) is the conduit for electrons into the pathway, and is absent in the mammalian genome, thus making it a potential drug target. Herein, we report the identification of two types of small molecules as selective inhibitors for Ndh-2 through a multicomponent high-throughput screen. Both compounds block ATP synthesis, lead to effects consistent with loss of NADH turnover, and importantly, exert bactericidal activity against Mtb. Extensive medicinal chemistry optimization afforded the best analogue with an MIC of 90 nm against Mtb. Moreover, the two scaffolds have differential inhibitory activities against the two homologous Ndh-2 enzymes in Mtb, which will allow precise control over Ndh-2 function in Mtb to facilitate the assessment of this anti-TB drug target.
Journal of medicinal chemistry, Feb 11, 2017
The approval of bedaquiline to treat tuberculosis has validated adenosine triphosphate (ATP) synt... more The approval of bedaquiline to treat tuberculosis has validated adenosine triphosphate (ATP) synthase as an attractive target to kill Mycobacterium tuberculosis. Herein, we report the discovery of two diverse lead series imidazo[1,2-a]pyridine ethers (IPE) and squaramides (SQA) as inhibitors of mycobacterial ATP-synthesis. Through medicinal chemistry exploration, we established a robust structure activity relationship of these two scaffolds resulting in nanomolar potencies in an ATP-synthesis inhibition assay. A biochemical deconvolution cascade suggested cytochrome c oxidase as the potential target of IPE class of molecules, whereas characterization of spontaneous resistant mutants of SQAs unambiguously identified ATP synthase as its molecular target. Absence of cross resistance against bedaquiline resistant mutants suggested a different binding site for SQAs on ATP synthase. Furthermore, SQAs were found to be non-cytotoxic and demonstrated efficacy in a mouse model of tuberculosis...
MedChemComm, 2016
2,4-Diaminoquinazolines, 2,4-diaminoquinolines and aminopyrazolopyrimidines, inhibitors of mycoba... more 2,4-Diaminoquinazolines, 2,4-diaminoquinolines and aminopyrazolopyrimidines, inhibitors of mycobacterial ATP synthesis, are novel lead molecules towards discovery and development of new anti-tubercular agents.
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2014
Methicillin-resistant Staphylococcus aureus (MRSA) is currently one of the principal multiple dru... more Methicillin-resistant Staphylococcus aureus (MRSA) is currently one of the principal multiple drug resistant bacterial pathogens causing serious infections, many of which are life-threatening. Consequently, new therapeutic targets are required to combat such infections. In the current work, we explore the type 2 Nicotinamide adenine dinucleotide reduced form (NADH) dehydrogenases (NDH-2s) as possible drug targets and look at the effects of phenothiazines, known to inhibit NDH-2 from Mycobacterium tuberculosis. NDH-2s are monotopic membrane proteins that catalyze the transfer of electrons from NADH via flavin adenine dinucleotide (FAD) to the quinone pool. They are required for maintaining the NADH/Nicotinamide adenine dinucleotide (NAD +) redox balance and contribute indirectly to the generation of proton motive force. NDH-2s are not present in mammals, but are the only form of respiratory NADH dehydrogenase in several pathogens, including S. aureus. In this work, the two putative ndh genes present in the S. aureus genome were identified, cloned and expressed, and the proteins were purified and characterized. Phenothiazines were shown to inhibit both of the S. aureus NDH-2s with half maximal inhibitory concentration (IC 50) values as low as 8 μM. However, evaluating the effects of phenothiazines on whole cells of S. aureus was complicated by the fact that they are also acting as uncouplers of oxidative phosphorylation. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.
Structure, 2000
The mitochondrial bioenergetics field has experienced an exciting breakthrough with the recent st... more The mitochondrial bioenergetics field has experienced an exciting breakthrough with the recent structure determination of several key membrane complexes. The latest addition to this line of structures, that of quinolfumarate reductase, provides new insights into the mechanism of energy transduction.
Proceedings of the National Academy of Sciences, 1999
The proton-translocating NADH-quinone oxidoreductase (EC 1.6.99.3 ) is the largest and least unde... more The proton-translocating NADH-quinone oxidoreductase (EC 1.6.99.3 ) is the largest and least understood enzyme complex of the respiratory chain. The mammalian mitochondrial enzyme (also called complex I) contains more than 40 subunits, whereas its structurally simpler bacterial counterpart (NDH-1) in Paracoccus denitrificans and Thermus thermophilus HB-8 consists of 14 subunits. A major unsolved question is the location and mechanism of the terminal electron transfer step from iron–sulfur cluster N2 to quinone. Potent inhibitors acting at this key region are candidate photoaffinity probes to dissect NADH-quinone oxidoreductases. Complex I and NDH-1 are very sensitive to inhibition by a variety of structurally diverse toxicants, including rotenone, piericidin A, bullatacin, and pyridaben. We designed (trifluoromethyl)diazirinyl[ 3 H]pyridaben ([ 3 H]TDP) as our photoaffinity ligand because it combines outstanding inhibitor potency, a suitable photoreactive group, and tritium at high ...
Nature Structural & Molecular Biology, 2008
Bacterial polysulfide reductase (PsrABC) is an integral membrane protein complex responsible for ... more Bacterial polysulfide reductase (PsrABC) is an integral membrane protein complex responsible for quinone coupled reduction of polysulfide, a process important in extreme environments such as deep-sea vents and hot springs. We determined the structure of polysulfide reductase from Thermus thermophilus at 2.4 Å resolution, revealing how the PsrA subunit recognizes and reduces its unique poly anionic substrate. The integral membrane subunit PsrC was characterized using the natural substrate menaquinone-7 and inhibitors, providing a comprehensive representation of a quinone binding site and revealing the presence of a water filled cavity connecting the quinone binding site on the periplasmic side to the cytoplasm. These results suggest that polysulfide reductase could be a key energy-conserving enzyme of the T. thermophilus respiratory chain,
Journal of Biological Chemistry, 2004
The proton-pumping NADH-quinone oxidoreductase from Escherichia coli houses nine iron-sulfur clus... more The proton-pumping NADH-quinone oxidoreductase from Escherichia coli houses nine iron-sulfur clusters, eight of which are found in its mitochondrial counterpart, complex I. The extra putative iron-sulfur cluster binding site with a CXXCXXXCX 27 C motif in the NuoG subunit has been assigned to ligate a [2Fe-2S] (N1c). However, we have shown previously that the Thermus thermophilus N1c fragment containing this motif ligates a [4Fe-4S] (Nakamaru-Ogiso, E., Yano, T., Ohnishi, T., and Yagi, T. (2002) J. Biol. Chem. 277, 1680-1688). In the current study, we individually inactivated four sets of the iron-sulfur binding motifs in the E. coli NuoG subunit by replacing all four ligands with Ala. Each mutant subunit, designated ⌬N1b, ⌬N1c, ⌬N4, and ⌬N5, was expressed as maltose-binding protein fusion proteins. After in vitro reconstitution, all mutant subunits were characterized by EPR. Although EPR signals from cluster N1b were not detected in any preparations, we detected two [4Fe-4S] EPR signals with g values of g x,y,z ؍ 1.89, 1.94, and 2.06, and g x,y,z ؍ 1.91, 1.94, and 2.05 at 6-20 K in wild type, ⌬N1b, and ⌬N5. The former signal was assigned to cluster N4, and the latter signal was assigned to cluster N1c because of their disappearance in ⌬N4 and ⌬N1c. Confirming that a [4Fe-4S] cluster ligates to the N1c motif, we propose to replace its misleading [2Fe-2S] name, N1c, with "cluster N7." In addition, because these mutations differently affected the assembly of peripheral subunits by in trans complementation analysis with the nuoG knockout strain, the implicated structural importance of the iron-sulfur binding domains is discussed.
Journal of Biological Chemistry, 1999
The proton-translocating NADH-quinone oxidoreductase (NDH-1) of Paracoccus denitrificans is compo... more The proton-translocating NADH-quinone oxidoreductase (NDH-1) of Paracoccus denitrificans is composed of at least 14 subunits (NQO1-14) and is located in the cytoplasmic membrane. In the present study, topological properties and stoichiometry of the 7 subunits (NQO1-6 and NQO9) of the P. denitrificans NDH-1 in the membranes were investigated using immunological techniques. Treatments with chaotropic reagents (urea, NaI, or NaBr) or with alkaline buffer (pH 10-12) resulted in partial or complete extraction of all the subunits from the membranes. Of interest is that when NaBr or urea were used, the NQO6 and NQO9 subunits remained in the membranes, whereas the other subunits were completely extracted, suggesting their direct association with the membrane part of the enzyme complex. Both deletion study and homologous expression study of the NQO9 subunit provided a clue that its hydrophobic Nterminal stretch plays an important role in such an association. In light of this observation and others, topological properties of the subunits in the NDH-1 enzyme complex are discussed. In addition, determination of stoichiometry of the peripheral subunits of the P. denitrificans NDH-1 was completed by radioimmunological methods. All the peripheral subunits are present as one molecule each in the enzyme complex. These results estimated the total number of cofactors in the P. denitrificans NDH-1; the enzyme complex contains one molecule of FMN and up to eight iron-sulfur clusters, 2؋[2Fe-2S] and 6؋[4Fe-4S], provided that the NQO6 subunit bears one [4Fe-4S] cluster.
Journal of Biological Chemistry, 2001
The proton-translocating NADH-quinone oxidoreductase (NDH-1) of Thermus thermophilus HB-8 is comp... more The proton-translocating NADH-quinone oxidoreductase (NDH-1) of Thermus thermophilus HB-8 is composed of 14 subunits (designated Nqo1-14). This NDH-1 houses nine putative iron-sulfur binding sites, eight of which are generally found in bacterial NDH-1 and its mitochondrial counterpart (complex I). The extra site contains a CXXCXXXCX 27 C motif and is located in the Nqo3 subunit. This motif was originally found in Escherichia coli NDH-1 and was assigned to a binuclear cluster (g z, y, x ؍ 2.00, 1.95, 1.92) and named N1c. In this report, the Thermus Nqo3 fragment containing this motif was heterologously overexpressed, using a glutathione S-transferase fusion system. This fragment contained a small amount of iron-sulfur cluster, whose content was significantly increased by in vitro reconstitution. The UV-visible and EPR spectroscopic properties of this fragment indicate that the ligated iron-sulfur cluster is tetranuclear with nearly axial symmetry (g ʈ,Ќ ؍ 2.045, ϳ1.94). Site-directed mutants show that all four cysteines participate in the ligation of a [4Fe-4S] cluster. Considering the fact that the same motif coordinates only tetranuclear clusters in other enzymes so far known, we propose that the CXXCXXXCX 27 C motif in the Nqo3 subunit most likely ligates the [4Fe-4S] cluster. Bacterial proton-translocating NADH-quinone (Q) oxidoreductase (NDH-1) 1 and its mitochondrial counterpart (com
Journal of Biological Chemistry, 1999
Molecular properties of the NQO9 subunit of Paracoccus denitrificans NDH-1, which is predicted to... more Molecular properties of the NQO9 subunit of Paracoccus denitrificans NDH-1, which is predicted to contain 2؋[4Fe-4S] clusters, were investigated using recombinant expression techniques and EPR spectroscopy. The full-length form of NQO9 subunit co-expressed with thioredoxin in Escherichia coli at ambient temperature was found dominantly in the cytoplasmic membrane with low amplification. Genetic deletion of relatively hydrophobic and less conserved N-terminal stretches (30 or 40 amino acid residues long) of the NQO9 subunit resulted in the overexpression of the truncated soluble form of the subunit in a high yield in the cytoplasm. The purified soluble form of the NQO9 subunit contained only a small quantity of Fe and S 2؊ (2.0-2.2 mol each per mol of subunit). However, the iron-sulfur content was considerably increased by in vitro reconstitution. The reconstituted NQO9 subunit contained 7.6-7.7 mol each of Fe and S 2؊ per molecule and exhibited optical absorption spectra similar to those of 2؋[4Fe-4S] ferredoxins. Two sets of relatively broad axial-type EPR signals with different temperature dependence and power saturation profile were detected in the dithionite-reduced preparations at a low temperature range (8-18 K). Due to a negative shift (<600 mV) of the apparent redox midpoint potential of the iron-sulfur clusters in the soluble form of the truncated NQO9 subunit, the following two possible cases could not be discriminated: (i) two sets of EPR signals arise from two distinct species of tetranuclear iron-sulfur clusters with two intrinsically different spectral parameters g ʈ ʈ,Ќ Ќ ؍ 2.05, ϳ1.93, and g ʈ ʈ,Ќ Ќ ؍ 2.08, ϳ1.90, and respective slow (P1 ⁄2 ؍ 8 milliwatts) and fast (P1 ⁄2 ؍ 342 milliwatts) spin relaxation; (ii) two clusters exhibit similar intrinsic EPR spectra (g ʈ ʈ,Ќ Ќ ؍ 2.05, ϳ1.93) with slow spin relaxation. When both clusters in the same subunit are concomitantly paramagnetic, their spin-spin interactions cause a shift of spectra to g ʈ ʈ,Ќ Ќ ؍ 2.08, ϳ1.90, with enhanced spin relaxation. In either case, our EPR data provide the first experimental evidence for the presence of two [4Fe-4S] ironsulfur clusters in the NQO9 subunit. The proton-translocating NADH-quinone (Q) 1 oxidoreductase is one of five enzyme complexes in the oxidative phosphorylation system that is located in the inner membrane of mitochondria and in the cytoplasmic membrane of bacteria. The mitochondrial enzyme is termed complex I, and the bacterial enzyme is called NDH-1. Both enzyme complexes catalyze the Reaction 1. NADH ϩ Q ϩ H ϩ ϩ nH ϩ ϩ NAD ϩ in 3 NAD ϩ ϩ QH 2 ϩ nH ϩ out (n ϭ 3-5)
International Journal of Antimicrobial Agents, 2014
Clostridium difficile, a highly drug-resistant Gram-positive, spore-forming bacterium, remains a ... more Clostridium difficile, a highly drug-resistant Gram-positive, spore-forming bacterium, remains a leading cause of hospital-acquired diarrhoea and antibiotic-associated colitis. Clinically, only a handful of antibiotics are used for treating C. difficile infection (CDI), suggesting a necessity for the development of new treatment options. Here we performed a high-throughput screen of 2000 drug-like compounds for inhibition of C. difficile. From this screen, one compound, 5-nitro-1,10phenanthroline (5-NP), showed potent bactericidal effects in vitro. In addition, this compound displayed high potency towards other Clostridium spp. as well as Mycobacterium bovis but not towards other tested Gram-positive and Gram-negative bacteria. Furthermore, we show that this inhibition may proceed through a metal chelation-dependent mechanism. More importantly, preliminary evidence suggests moderate efficacy for this compound in treating CDI in a murine infection model. These results present a possible basis for the further development of this compound as an antibiotic treatment for CDI.
FEBS Letters, 1991
The amino acid sequence of rusticyanin, a copper protein, purified from the iron-oxidizing bacter... more The amino acid sequence of rusticyanin, a copper protein, purified from the iron-oxidizing bacterium Tiliobacillrrs~rrooxiduns was determined. Rusticyanin contained 154 amino acid residues in a single polypeptide chain and its molecular weight was calculated to be about 16400 based on the amino acid sequence. The N-terminal sequence up to the 20th residue of the protein apparently resembled those of Merhylobacteriurn extorquerrs AM 1 amicyanin and poplar leaf plastocyanin rather than those of azurin family proteins. In the C-terminal region of the sequence, rusticyanin had one cysteine, one histidine and one mcthionine which arc conserved through many copper proteins. In the middle region of the sequence, rusticyanin was not similar to any other copper protein. The sequence nearby NW of rusticyanin was similar to those of other copper proteins to some extent. However, Asn which follows HiP and is highly conserved in other copper proteins did not exist in rusticyanin. Therefore, it seemed difficult to conclude on the basis of the results obtained in the present study that Hiss4 in rusticyanin was the fourth ligand to the copper atom. TlrioOacillus fererrooxiduns; Amino acid sequence; Rusticyanin; Copper protein '~~ChllOiO~~, NJ&W& Yokohnmn, 227, hlMl1. ML The cluates which contained rusticyanin were combiacd, dialyzed brought to you by CORE View metadata, citation and similar papers at core.ac.uk
Biochemistry, 2014
Type II NADH-quinone oxidoreductase (NDH-2) catalyzes the transfer electrons from NADH to the qui... more 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 pingpong 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.
Biochemistry, 2003
2 To date the 3D structure of succinate dehydrogenase is not available. However, high-resolution ... more 2 To date the 3D structure of succinate dehydrogenase is not available. However, high-resolution 3D structures of bacterial fumarate reductases, which are believed to be counterparts of succinate dehydrogenase, have been solved.
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2000
NADH-ubiquinone oxidoreductase (called complex I for mitochondrial enzyme and NDH-1 for bacterial... more NADH-ubiquinone oxidoreductase (called complex I for mitochondrial enzyme and NDH-1 for bacterial counterparts) is an energy transducer, which utilizes the redox energy derived from the oxidation of NADH with ubiquinone to generate an electrochemical proton gradient vW H across the membrane. The complex I/NDH-1 contain one non-covalently bound flavin mononucleotide and as many as eight iron-sulfur clusters as electron transfer components in common. In addition, electron paramagnetic resonance (EPR) spectroscopic studies have revealed that three ubisemiquinone (SQ) species with distinct spectroscopic and thermodynamic properties are detectable in complex I and function as electron/proton translocators. Thus, the understanding of molecular properties of the individual quinone species is prerequisite to elucidate the energy-coupling mechanism of complex I. We have investigated these SQ species using EPR spectroscopy and found that the three SQ species have strikingly different properties. We will report characteristics of these SQ species and discuss possible functional roles of individual quinone species in the electron/proton transfer reaction of complex I/NDH-1.
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1998
BIOCHIMICA ET BIOPHYSICA ACTA aaN Structure-function studies of iron-sulfur clusters and semiquin... more BIOCHIMICA ET BIOPHYSICA ACTA aaN Structure-function studies of iron-sulfur clusters and semiquinones in the NADH-Q oxidoreductase segment of the respiratory chain • .a~ la c
Journal of Biological Chemistry, 2010
FIGURE 1. Structures of clofazimine and a new, more soluble analog, KS6.
Developments in Geochemistry, 1991
Abstract From Thiobacillus ferrooxidans , an enzyme has been purified which catalyses oxidation o... more Abstract From Thiobacillus ferrooxidans , an enzyme has been purified which catalyses oxidation of Fe 2+ ions with T. ferrooxidans cytochrome c -552 as the electron acceptor. The enzyme shows absorption peaks at 282 and 382 nm and contains 18-20 mol of nonhaem iron and 6 mol of inorganic sulphide in 63,000 g. The enzyme does not use rusticyanin as the electron acceptor for oxidation of Fe 2+ ions. T. ferrooxidans cytochrome c -552 has also been purified to an electrophoretically homogeneous state. It shows an absorption peak at 411 nm in the oxidized form and peaks at 417, 523 and 552 nm in the reduced form. Its molecular weight is about 14,000 and its redox potential at pH 7.0 is approx. +0.37 V.