Hamid R. Nasiri

Interplay of ‘induced fit’ and preorganization in the ligand induced folding of the aptamer domain of the guanine binding riboswitch

Riboswitches are highly structured elements in the 5′-untranslated regions (5′-UTRs) of messenger RNA that control gene expression by specifically binding to small metabolite molecules. They consist of an aptamer domain responsible for ligand binding and an expression platform. Ligand binding in the aptamer domain leads to conformational changes in the expression platform that result in transcription termination or abolish ribosome binding. The guanine riboswitch binds with high-specificity to guanine and hypoxanthine and is among the smallest riboswitches described so far. The X-ray-structure of its aptamer domain in complex with guanine/hypoxanthine reveals an intricate RNA-fold consisting of a three-helix junction stabilized by long-range base pairing interactions. We analyzed the conformational transitions of the aptamer domain induced by binding of hypoxanthine using high-resolution NMR-spectroscopy in solution. We found that the long-range base pairing interactions are already present in the free RNA and preorganize its global fold. The ligand binding core region is lacking hydrogen bonding interactions and therefore likely to be unstructured in the absence of ligand. Mg2+-ions are not essential for ligand binding and do not change the structure of the RNA-ligand complex but stabilize the structure at elevated temperatures. We identified a mutant RNA where the long-range base pairing interactions are disrupted in the free form of the RNA but form upon ligand binding in an Mg2+-dependent fashion. The tertiary interaction motif is stable outside the riboswitch context.

Evidence for transmembrane proton transfer in a dihaem-containing membrane protein complex

Membrane protein complexes can support both the generation and utilisation of a transmembrane electrochemical proton potential (‘proton‐motive force’), either by transmembrane electron transfer coupled to protolytic reactions on opposite sides of the membrane or by transmembrane proton transfer. Here we provide the first evidence that both of these mechanisms are combined in the case of a specific respiratory membrane protein complex, the dihaem‐containing quinol:fumarate reductase (QFR) of Wolinella succinogenes, so as to facilitate transmembrane electron transfer by transmembrane proton transfer. We also demonstrate the non‐functionality of this novel transmembrane proton transfer pathway (‘E‐pathway’) in a variant QFR where a key glutamate residue has been replaced. The ‘E‐pathway’, discussed on the basis of the 1.78‐Å‐resolution crystal structure of QFR, can be concluded to be essential also for the viability of pathogenic ε‐proteobacteria such as Helicobacter pylori and is possibly relevant to proton transfer in other dihaem‐containing membrane proteins, performing very different physiological functions.

Design, synthesis, and biological testing of novel naphthoquinones as substrate-based inhibitors of the quinol/fumarate reductase from Wolinella succinogenes.

Novel naphthoquinones were designed, synthesized, and tested as substrate-based inhibitors against the membrane-embedded protein quinol/fumarate reductase (QFR) from Wolinella succinogenes, a target closely related to QFRs from the human pathogens Helicobacter pylori and Campylobacter jejuni. For a better understanding of the hitherto structurally unexplored substrate binding pocket, a structure-activity relationship (SAR) study was carried out. Analogues of lawsone (2-hydroxy-1,4-naphthoquinone 3a) were synthesized that vary in length and size of the alkyl side chains (3b-k). A combined study on the prototropic tautomerism of 2-hydroxy-1,4-naphthoquinones series indicated that the 1,4-tautomer is the more stable and biologically relevant isomer and that the presence of the hydroxyl group is crucial for inhibition. Furthermore, 2-bromine-1,4-naphthoquinone (4a-c) and 2-methoxy-1,4-naphthoquinone (5a-b) series were also discovered as novel and potent inhibitors. Compounds 4a and 4b showed IC50 values in low micromolar range in the primary assay and no activity in the counter DT-diaphorase assay.

Production, characterization and determination of the real catalytic properties of the putative ‘succinate dehydrogenase’ from Wolinella succinogenes

Both the genomes of the epsilonproteobacteria Wolinella succinogenes and Campylobacter jejuni contain operons (sdhABE) that encode for so far uncharacterized enzyme complexes annotated as ‘non‐classical’ succinate:quinone reductases (SQRs). However, the role of such an enzyme ostensibly involved in aerobic respiration in an anaerobic organism such as W. succinogenes has hitherto been unknown. We have established the first genetic system for the manipulation and production of a member of the non‐classical succinate:quinone oxidoreductase family. Biochemical characterization of the W. succinogenes enzyme reveals that the putative SQR is in fact a novel methylmenaquinol:fumarate reductase (MFR) with no detectable succinate oxidation activity, clearly indicative of its involvement in anaerobic metabolism. We demonstrate that the hydrophilic subunits of the MFR complex are, in contrast to all other previously characterized members of the superfamily, exported into the periplasm via the twin‐arginine translocation (tat)‐pathway. Furthermore we show that a single amino acid exchange (Ala86→His) in the flavoprotein of that enzyme complex is the only additional requirement for the covalent binding of the otherwise non‐covalently bound FAD. Our results provide an explanation for the previously published puzzling observation that the C. jejuni sdhABE operon is upregulated in an oxygen‐limited environment as compared with microaerophilic laboratory conditions.

The correlation of cathodic peak potentials of vitamin K3 derivatives and their calculated electron affinities: The role of hydrogen bonding and conformational changes

2-methyl-1,4-naphtoquinone 1 (vitamin K(3), menadione) derivatives with different substituents at the 3-position were synthesized to tune their electrochemical properties. The thermodynamic midpoint potential (E(1/2)) of the naphthoquinone derivatives yielding a semi radical naphthoquinone anion were measured by cyclic voltammetry in the aprotic solvent dimethoxyethane (DME). Using quantum chemical methods, a clear correlation was found between the thermodynamic midpoint potentials and the calculated electron affinities (E(A)). Comparison of calculated and experimental values allowed delineation of additional factors such as the conformational dependence of quinone substituents and hydrogen bonding which can influence the electron affinities (E(A)) of the quinone. This information can be used as a model to gain insight into enzyme-cofactor interactions, particularly for enzyme quinone binding modes and the electrochemical adjustment of the quinone motif.

Electrochemical and crystal structural analysis of α- and dehydro-α-lapachones

Lapachones are pharmaceutically active compounds generating reactive oxygen species. The crystal structure and redox behaviour of the title lapachones, derivates of quinones, were determined by X-ray diffraction and cyclovoltametric measurements. The observed results were compared with β-lapachone.

TAUTOMERISM OF 4-HYDROXY-4(1H)QUINOLON

The tautomerism of 4-Hydroxy-4(1H) quinolon I was studied using infrared spectroscopy, 1 H, 13 C NMR spectroscopy and X-ray crystallography. The keto-form of I is favored in the crystal form and at room temperature in polar solutions like water and dimethylsulfoxide. Introduction. Quinolons produced by different bacteria inhibit the respiratory (1) as well as photosynthetic electron transport chains (2). They were used as lead compounds for finding new strong inhibitors. One of the most important physical and chemical properties of this class of heteroaromatic compounds is their involvement in a prototropic tautomerism (Figure 1). In order to understand the biological activity of I we investigated its crystal structure and studied the tautomerism in water and DMSO solution by 1 H and 13 C NMR spectroscopy. To further investigate the tautomerism of compound I in solution we synthesized the O-methylated derivate II, which mimics the enol-tautomeric form (Figure 1). Single crystals of I for X-ray analysis were obtained from dichloromethane. The crystal structure exists as a dimer. The monomers are connected by an intermolecular hydrogen bond (O...HN distance of 1.795 A) between the imino hydrogen and the carbonyl group (Figure 2 and Figure 3). The carbon oxygen bonds length of 1.246 A for C1A-O1A and 1.262 A for C1-O1 are in the range of the C=O length. Also the C3N4 and C5N4 bonds of 1.352 A and 1.383 A indicate a nitrogen carbon single bond. This results show that the keto tautomer is favored in the crystal structure.

Investigation on the electrochemistry and cytotoxicity of the natural product marcanine A and its synthetic derivatives

The electrochemistry and cytotoxicity of marcanine A were investigated by electrochemical, computational and cellular studies. To enable a structure–toxicity-relationship of the natural product, eleven novel synthetic derivatives with different electrochemical properties were synthesized and tested. Derivative 5 revealed a GI50 in the low μM range, being more active than the actual natural product. A clear correlation was found between the experimental and the calculated data.

Creation of a gold nanoparticle based electrochemical assay for the detection of inhibitors of bacterial cytochrome bd oxidases

Cytochrome bd oxidases are membrane proteins expressed by bacteria including a number of pathogens, which make them an attractive target for the discovery of new antibiotics. An electrochemical assay is developed to study the activity of these proteins and inhibition by quinone binding site tool compounds. The setup relies on their immobilization at electrodes specifically modified with gold nanoparticles, which allows achieving a direct electron transfer to/from the heme cofactors of this large enzyme. After optimization of the protein coverages, the assay shows at pH7 a good reproducibility and readout stability over time, and it is thus suitable for further screening of small molecule collections.

2-Hydroxy-3-(3-oxobutyl)naphthalene-1,4-dione.

The title compound, C14H12O4, forms crystals which appear monoclinic but are actually twinned triclinic. The asymmetric unit consists of two similar molecules, which differ only in the conformation of the 3-oxobutyl side chain. The molecular conformation is characterized by an intramolecular O-H...O hydrogen bond between the hydroxy group and the adjacent carbonyl O atom. The crystal structure is stabilized by O-H...O hydrogen bonds connecting the molecules into zigzag chains running along the b axis.