Characterization of the Type 2 NADH: Menaquinone oxidoreductases from Staphylococcus aureus and the bactericidal action of phenothiazines.
ABSTRACT Methicillin-resistant Staphylococcus aureus (MRSA) is currently one of the principal multiply 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 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 FAD to the quinone pool. They are required for maintaining the NADH/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 purified and characterized. Phenothiazines were shown to inhibit both of the S. aureus NDH-2s with IC50 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.
Full-textDOI: · Available from: Lici Ariane Schurig-Briccio, Jun 17, 2014
SourceAvailable from: Christopher J Lagace[Show abstract] [Hide abstract]
ABSTRACT: A series of typical (chlorpromazine, haloperidol and thioridazine) and atypical (risperidone, quetiapine, clozapine and olanzapine) antipsychotics were tested for effects on integrated bioenergetic functions of isolated rat liver mitochondria. Polarographic measurement of oxygen consumption in freshly isolated mitochondria showed that electron transfer activity at respiratory complex I is inhibited by chlorpromazine, haloperidol, risperidone, and quetiapine, but not by clozapine, olanzapine, or thioridazine. Chlorpromazine and thioridazine act as modest uncouplers of oxidative phosphorylation. The typical neuroleptics inhibited NADH-coenzyme Q reductase in freeze-thawed mitochondria, which is a direct measure of complex I enzyme activity. The inhibition of NADH-coenzyme Q reductase activity by the atypicals risperidone and quetiapine was 2-4 fold less than that for the typical neuroleptics. Clozapine and olanzapine had only slight effects on NADH-coenzyme Q reductase activity, even at 200 microM. The relative potencies of these neuroleptic drugs as inhibitors of mitochondrial bioenergetic function is similar to their relative potencies as risk factors in the reported incidence of extrapyramidal symptoms, including tardive dyskinesia (TD). This suggests that compromised bioenergetic function may be involved in the cellular pathology underlying TD.Archives of Pharmacal Research 12/2003; 26(11):951-9. DOI:10.1007/BF02980205 · 1.75 Impact Factor
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ABSTRACT: The majority of bacterial multidrug resistance transporters belong to the class of secondary transporters. LmrP is a proton/drug antiporter of Lactococcus lactis that extrudes positively charged lipophilic substrates from the inner leaflet of the membrane to the external medium. This study shows that LmrP is a true secondary transporter. In the absence of a proton motive force, LmrP facilitates downhill fluxes of ethidium in both directions. These fluxes are inhibited by other substrates of LmrP. The cysteine-reactive agent p-chloromercuri-benzene sulfonate inhibits these fluxes in wild type LmrP but not in the cysteine-less LmrP C270A mutant. Cysteine mutagenesis of LmrP resulted in three mutants, D68C/C270A, D128C/C270A, and E327C/C270A, with an energy-uncoupled phenotype. Asp68 is located in the conserved motif GXXX(D/E)(R/K)XGRK for the major facilitator superfamily of secondary transporters and was found to play an important role in energy coupling, whereas the negatively charged residues Asp128 and Glu327 have indirect effects on the transport process. L. lactis strains expressing these uncoupled mutants of LmrP show an increased rate of ethidium influx and an increased drug susceptibility compared with cells harboring an empty vector. The rate of influx in these mutants is enhanced by a transmembrane electrical potential, inside negative. These observations suggest a new strategy for eliminating drug-resistant microbial pathogens, i.e. the design and use of modulators of secondary multidrug resistance transporters that uncouple drug efflux from proton influx, thereby allowing transmembrane electrical potential-driven influx of cationic drugs.Journal of Biological Chemistry 02/2004; 279(1):103-8. DOI:10.1074/jbc.M306579200 · 4.60 Impact Factor
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ABSTRACT: In a previous paper, we introduced MUSCLE, a new program for creating multiple alignments of protein sequences, giving a brief summary of the algorithm and showing MUSCLE to achieve the highest scores reported to date on four alignment accuracy benchmarks. Here we present a more complete discussion of the algorithm, describing several previously unpublished techniques that improve biological accuracy and / or computational complexity. We introduce a new option, MUSCLE-fast, designed for high-throughput applications. We also describe a new protocol for evaluating objective functions that align two profiles. We compare the speed and accuracy of MUSCLE with CLUSTALW, Progressive POA and the MAFFT script FFTNS1, the fastest previously published program known to the author. Accuracy is measured using four benchmarks: BAliBASE, PREFAB, SABmark and SMART. We test three variants that offer highest accuracy (MUSCLE with default settings), highest speed (MUSCLE-fast), and a carefully chosen compromise between the two (MUSCLE-prog). We find MUSCLE-fast to be the fastest algorithm on all test sets, achieving average alignment accuracy similar to CLUSTALW in times that are typically two to three orders of magnitude less. MUSCLE-fast is able to align 1,000 sequences of average length 282 in 21 seconds on a current desktop computer. MUSCLE offers a range of options that provide improved speed and / or alignment accuracy compared with currently available programs. MUSCLE is freely available at http://www.drive5.com/muscle.BMC Bioinformatics 09/2004; 5:113. DOI:10.1186/1471-2105-5-113 · 2.67 Impact Factor