Role of the Na(+)-translocating NADH:quinone oxidoreductase in voltage generation and Na(+) extrusion in Vibrio cholerae.

For Vibrio cholerae, the coordinated import and export of Na(+) is crucial for adaptation to habitats with different osmolarities. We investigated the Na(+)-extruding branch of the sodium cycle in this human pathogen by in vivo (23)Na-NMR spectroscopy. The Na(+) extrusion activity of cells was monitored after adding glucose which stimulated respiration via the Na(+)-translocating NADH:quinone oxidoreductase (Na(+)-NQR). In a V. cholerae deletion mutant devoid of the Na(+)-NQR encoding genes (nqrA-F), rates of respiratory Na(+) extrusion were decreased by a factor of four, but the cytoplasmic Na(+) concentration was essentially unchanged. Furthermore, the mutant was impaired in formation of transmembrane voltage (ΔΨ, inside negative) and did not grow under hypoosmotic conditions at pH8.2 or above. This growth defect could be complemented by transformation with the plasmid encoded nqr operon. In an alkaline environment, Na(+)/H(+) antiporters acidify the cytoplasm at the expense of the transmembrane voltage. It is proposed that, at alkaline pH and limiting Na(+) concentrations, the Na(+)-NQR is crucial for generation of a transmembrane voltage to drive the import of H(+) by electrogenic Na(+)/H(+) antiporters. Our study provides the basis to understand the role of the Na(+)-NQR in pathogenicity of V. cholerae and other pathogens relying on this primary Na(+) pump for respiration.

NMR reveals double occupancy of quinone-type ligands in the catalytic quinone binding site of the Na+-translocating NADH:Quinone oxidoreductase from Vibrio cholerae.

The sodium ion-translocating NADH:quinone oxidoreductase (Na(+)-NQR) from the pathogen Vibrio cholerae exploits the free energy liberated during oxidation of NADH with ubiquinone to pump sodium ions across the cytoplasmic membrane. The Na(+)-NQR consists of four membrane-bound subunits NqrBCDE and the peripheral NqrF and NqrA subunits. NqrA binds ubiquinone-8 as well as quinones with shorter prenyl chains (ubiquinone-1 and ubiquinone-2). Here we show that the quinone derivative 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), a known inhibitor of the bc1 and b6f complexes found in mitochondria and chloroplasts, also inhibits quinone reduction by the Na(+)-NQR in a mixed inhibition mode. Tryptophan fluorescence quenching and saturation transfer difference NMR experiments in the presence of Na(+)-NQR inhibitor (DBMIB or 2-n-heptyl-4-hydroxyquinoline N-oxide) indicate that two quinone analog ligands are bound simultaneously by the NqrA subunit with very similar interaction constants as observed with the holoenzyme complex. We conclude that the catalytic site of quinone reduction is located on NqrA. The two ligands bind to an extended binding pocket in direct vicinity to each other as demonstrated by interligand Overhauser effects between ubiquinone-1 and DBMIB or 2-n-heptyl-4-hydroxyquinoline N-oxide, respectively. We propose that a similar spatially close arrangement of the native quinone substrates is also operational in vivo, enhancing the catalytic efficiency during the final electron transfer steps in the Na(+)-NQR.

Girard derivatization for LC-MS/MS profiling of endogenous ecdysteroids in Drosophila.

Ecdysteroids are potent developmental regulators that control molting, reproduction, and stress response in arthropods. In developing larvae, picogram quantities of individual ecdysteroids and their conjugated forms are present along with milligrams of structural and energy storage lipids. To enhance the specificity and sensitivity of ecdysteroid detection, we targeted the 6-ketone group, which is common to all ecdysteroids, with Girard reagents. Unlike other ketosteroids, during the reaction, Girard hydrazones of ecdysteroids eliminated the C14-hydroxyl group, creating an additional C14-C15 double bond. Dehydrated hydrazones of endogenous ecdysteroids were detected by LC-MS/MS in the multiple reaction monitoring (MRM) mode using two mass transitions: one relied upon neutral loss of a quaternary amine from the Girard T moiety; another complementary transition followed neutral loss of the hydrocarbon chain upon C20-C27 cleavage. We further demonstrated that a combination of Girard derivatization and LC-MS/MS enabled unequivocal detection of three major endogenous hormones at the picogram level in an extract from a single Drosophila pupa.

Detecting and Characterizing Interactions of Metabolites with Proteins by Saturation Transfer Difference Nuclear Magnetic Resonance (STD NMR) Spectroscopy.

Saturation transfer difference (STD) nuclear magnetic resonance (NMR) spectroscopy is an established technique for detecting and characterizing the binding of small molecules, such as metabolites, to biological macromolecules like proteins and nucleic acids. STD NMR allows detection of binding in complex mixtures of potential ligands, which is often used for library screening in the pharmaceutical industry but may also be beneficial for binding studies with metabolite mixtures. The nature of the ligand is normally restricted to small molecules in terms of NMR spectroscopy, and the size of the macromolecule on the other side should be larger than 10-15 kDa. This technique is especially applicable to detecting binders of intermediate to low affinity with the dissociation constant (KD) above 1 µM. In this chapter, we focus on recent developments and the applications of STD NMR to studying interactions of natural products and metabolites, in particular. The reader is also referred to excellent reviews of the field and the literature cited therein. This chapter also provides a detailed experimental protocol for performing the STD NMR measurement based on the example of the subunit A of the Na+-transporting NADH/ubiquinone oxidoreductase (Na+-NQR) from V. cholerae interacting with its natural quinone substrate and inhibitors.

Thiol-Mediated Uptake of a Cysteine-Containing Nanobody for Anticancer Drug Delivery.

The identification of tumor-specific biomarkers is one of the bottlenecks in the development of cancer therapies. Previous work revealed altered surface levels of reduced/oxidized cysteines in many cancers due to overexpression of redox-controlling proteins such as protein disulfide isomerases on the cell surface. Alterations in surface thiols can promote cell adhesion and metastasis, making thiols attractive targets for treatment. Few tools are available to study surface thiols on cancer cells and exploit them for theranostics. Here, we describe a nanobody (CB2) that specifically recognizes B cell lymphoma and breast cancer in a thiol-dependent manner. CB2 binding strictly requires the presence of a nonconserved cysteine in the antigen-binding region and correlates with elevated surface levels of free thiols on B cell lymphoma compared to healthy lymphocytes. Nanobody CB2 can induce complement-dependent cytotoxicity against lymphoma cells when functionalized with synthetic rhamnose trimers. Lymphoma cells internalize CB2 via thiol-mediated endocytosis which can be exploited to deliver cytotoxic agents. CB2 internalization combined with functionalization forms the basis for a wide range of diagnostic and therapeutic applications, rendering thiol-reactive nanobodies promising tools for targeting cancer.

Localization of ubiquinone-8 in the Na+-pumping NADH:quinone oxidoreductase from Vibrio cholerae.

Na(+) is the second major coupling ion at membranes after protons, and many pathogenic bacteria use the sodium-motive force to their advantage. A prominent example is Vibrio cholerae, which relies on the Na(+)-pumping NADH:quinone oxidoreductase (Na(+)-NQR) as the first complex in its respiratory chain. The Na(+)-NQR is a multisubunit, membrane-embedded NADH dehydrogenase that oxidizes NADH and reduces quinone to quinol. Existing models describing redox-driven Na(+) translocation by the Na(+)-NQR are based on the assumption that the pump contains four flavins and one FeS cluster. Here we show that the large, peripheral NqrA subunit of the Na(+)-NQR binds one molecule of ubiquinone-8. Investigations of the dynamic interaction of NqrA with quinones by surface plasmon resonance and saturation transfer difference NMR reveal a high affinity, which is determined by the methoxy groups at the C-2 and C-3 positions of the quinone headgroup. Using photoactivatable quinone derivatives, it is demonstrated that ubiquinone-8 bound to NqrA occupies a functional site. A novel scheme of electron transfer in Na(+)-NQR is proposed that is initiated by NADH oxidation on subunit NqrF and leads to quinol formation on subunit NqrA.

Strategies for Enzymatic Inactivation of the Veterinary Antibiotic Florfenicol.

Large quantities of the antibiotic florfenicol are used in animal farming and aquaculture, contaminating the ecosystem with antibiotic residues and promoting antimicrobial resistance, ultimately leading to untreatable multidrug-resistant pathogens. Florfenicol-resistant bacteria often activate export mechanisms that result in resistance to various structurally unrelated antibiotics. We devised novel strategies for the enzymatic inactivation of florfenicol in different media, such as saltwater or milk. Using a combinatorial approach and selection, we optimized a hydrolase (EstDL136) for florfenicol cleavage. Reaction kinetics were followed by time-resolved NMR spectroscopy. Importantly, the hydrolase remained active in different media, such as saltwater or cow milk. Various environmentally-friendly application strategies for florfenicol inactivation were developed using the optimized hydrolase. As a potential filter device for cost-effective treatment of waste milk or aquacultural wastewater, the hydrolase was immobilized on Ni-NTA agarose or silica as carrier materials. In two further application examples, the hydrolase was used as cell extract or encapsulated with a semi-permeable membrane. This facilitated, for example, florfenicol inactivation in whole milk, which can help to treat waste milk from medicated cows, to be fed to calves without the risk of inducing antibiotic resistance. Enzymatic inactivation of antibiotics, in general, enables therapeutic intervention without promoting antibiotic resistance.

Investigations on the Degradation of the Bile Salt Cholate via the 9,10-Seco-Pathway Reveals the Formation of a Novel Recalcitrant Steroid Compound by a Side Reaction in Sphingobium sp. Strain Chol11.

Bile salts such as cholate are steroid compounds from the digestive tracts of vertebrates, which enter the environment upon excretion, e.g., in manure. Environmental bacteria degrade bile salts aerobically via two pathway variants involving intermediates with Δ1,4- or Δ4,6-3-keto-structures of the steroid skeleton. Recent studies indicated that degradation of bile salts via Δ4,6-3-keto intermediates in Sphingobium sp. strain Chol11 proceeds via 9,10-seco cleavage of the steroid skeleton. For further elucidation, the presumptive product of this cleavage, 3,12ß-dihydroxy-9,10-seco-androsta-1,3,5(10),6-tetraene-9,17-dione (DHSATD), was provided to strain Chol11 in a co-culture approach with Pseudomonas stutzeri Chol1 and as purified substrate. Strain Chol11 converted DHSATD to the so far unknown compound 4-methyl-3-deoxy-1,9,12-trihydroxyestra-1,3,5(10)7-tetraene-6,17-dione (MDTETD), presumably in a side reaction involving an unusual ring closure. MDTETD was neither degraded by strains Chol1 and Chol11 nor in enrichment cultures. Functional transcriptome profiling of zebrafish embryos after exposure to MDTETD identified a significant overrepresentation of genes linked to hormone responses. In both pathway variants, steroid degradation intermediates transiently accumulate in supernatants of laboratory cultures. Soil slurry experiments indicated that bacteria using both pathway variants were active and also released their respective intermediates into the environment. This instance could enable the formation of recalcitrant steroid metabolites by interspecies cross-feeding in agricultural soils.

Crystallization and preliminary analysis of the NqrA and NqrC subunits of the Na+-translocating NADH:ubiquinone oxidoreductase from Vibrio cholerae.

The Na+-translocating NADH:ubiquinone oxidoreductase (Na+-NQR) from Vibrio cholerae is a membrane protein complex consisting of six different subunits NqrA-NqrF. The major domains of the NqrA and NqrC subunits were heterologously expressed in Escherichia coli and crystallized. The structure of NqrA1-377 was solved in space groups C2221 and P21 by SAD phasing and molecular replacement at 1.9 and 2.1 Šresolution, respectively. NqrC devoid of the transmembrane helix was co-expressed with ApbE to insert the flavin mononucleotide group covalently attached to Thr225. The structure was determined by molecular replacement using apo-NqrC of Parabacteroides distasonis as search model at 1.8 Šresolution.