Stefan Bartoschek

Three molecules of ubiquinone bind specifically to mitochondrial cytochrome bc1 complex.

Bifurcated electron flow to high potential “Rieske” iron-sulfur cluster and low potential hemebL is crucial for respiratory energy conservation by the cytochrome bc1 complex. The chemistry of ubiquinol oxidation has to ensure the thermodynamically unfavorable electron transfer to heme bL . To resolve a central controversy about the number of ubiquinol molecules involved in this reaction, we used high resolution magic-angle-spinning nuclear magnetic resonance experiments to show that two out of threen-decyl-ubiquinones bind at the ubiquinol oxidation center of the complex. This substantiates a proposed mechanism in which a charge transfer between a ubiquinol/ubiquinone pair explains the bifurcation of electron flow.

Re-Face Stereospecificity of Methylenetetrahydromethanopterin and Methylenetetrahydrofolate Dehydrogenases is Predetermined by Intrinsic Properties of the Substrate

Four different dehydrogenases are known that catalyse the reversible dehydrogenation of N5,N10‐methylenetetrahydromethanopterin (methylene‐H4MPT) or N5,N10‐methylenetetrahydrofolate (methylene‐H4F) to the respective N5,N10‐methenyl compounds. Sequence comparison indicates that the four enzymes are phylogenetically unrelated. They all catalyse the Re‐face‐stereospecific removal of the pro‐R hydrogen atom of the coenzymes methylene group. The Re‐face stereospecificity is in contrast to the finding that in solution the pro‐S hydrogen atom of methylene‐H4MPT and of methylene‐H4F is more reactive to heterolytic cleavage. For a better understanding we determined the conformations of methylene‐H4MPT in solution and when enzyme‐bound by using NMR spectroscopy and semiempirical quantum mechanical calculations. For the conformation free in solution we find an envelope conformation for the imidazolidine ring, with the flap at N10. The methylene pro‐S C−H bond is anticlinal and the methylene pro‐R C−H bond is synclinal to the lone electron pair of N10. Semiempirical quantum mechanical calculations of heats of formation of methylene‐H4MPT and methylene‐H4F indicate that changing this conformation into an activated one in which the pro‐S C−H bond is antiperiplanar, resulting in the preformation of the leaving hydride, would require a ΔΔH

An NMR-based scoring function improves the accuracy of binding pose predictions by docking by two orders of magnitude

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A Combination of Spin Diffusion Methods for the Determination of Protein-Ligand Complex Structural Ensembles.

of +53 kJ mol−1 for methylene‐H4MPT and of +51 kJ mol−1 for methylene‐H4F. This is almost twice the energy required to force the imidazolidine ring in the enzyme‐bound conformation of methylene‐H4MPT (+29 kJ mol−1) or of methylene‐H4F (+35 kJ mol−1) into an activated conformation in which the pro‐R hydrogen atom is antiperiplanar to the lone electron pair of N10. The much lower energy for pro‐R hydrogen activation thus probably predetermines the Re‐face stereospecificity of the four dehydrogenases. Results are also presented explaining why the chemical reduction of methenyl‐H4MPT+ and methenyl‐H4F+ with NaBD4 proceeds Si‐face‐specific, in contrast to the enzyme‐catalysed reaction.

Stereochemistry and conformation of skyllamycin, a non-ribosomally synthesized peptide from Streptomyces sp. Acta 2897.

Low-affinity ligands can be efficiently optimized into high-affinity drug leads by structure based drug design when atomic-resolution structural information on the protein/ligand complexes is available. In this work we show that the use of a few, easily obtainable, experimental restraints improves the accuracy of the docking experiments by two orders of magnitude. The experimental data are measured in nuclear magnetic resonance spectra and consist of protein-mediated NOEs between two competitively binding ligands. The methodology can be widely applied as the data are readily obtained for low-affinity ligands in the presence of non-labelled receptor at low concentration. The experimental inter-ligand NOEs are efficiently used to filter and rank complex model structures that have been pre-selected by docking protocols. This approach dramatically reduces the degeneracy and inaccuracy of the chosen model in docking experiments, is robust with respect to inaccuracy of the structural model used to represent the free receptor and is suitable for high-throughput docking campaigns.

Re-face stereospecificity of NADP dependent methylenetetrahydromethanopterin dehydrogenase from Methylobacterium extorquens AM1 as determined by NMR spectroscopy

Structure-based drug design (SBDD) is a powerful and widely used approach to optimize affinity of drug candidates. With the recently introduced INPHARMA method, the binding mode of small molecules to their protein target can be characterized even if no spectroscopic information about the protein is known. Here, we show that the combination of the spin-diffusion-based NMR methods INPHARMA, trNOE, and STD results in an accurate scoring function for docking modes and therefore determination of protein-ligand complex structures. Applications are shown on the model system protein kinase A and the drug targets glycogen phosphorylase and soluble epoxide hydrolase (sEH). Multiplexing of several ligands improves the reliability of the scoring function further. The new score allows in the case of sEH detecting two binding modes of the ligand in its binding site, which was corroborated by X-ray analysis.

Purification and catalytic properties of Ech hydrogenase from Methanosarcina barkeri.

Skyllamycin is a non-ribosomally synthesized cyclic depsipeptide from Streptomyces sp. Acta 2897 that inhibits PDGF-signaling. The peptide scaffold contains an N-terminal cinnamoyl moiety, a β-methylation of aspartic acid, three β-hydroxylated amino acids and one rarely occurring α-hydroxy glycine. With the exception of α-hydroxy glycine, the stereochemistry of the amino acids was assigned by comparison to synthetic reference amino acids applying chiral GC-MS and Marfey-HPLC analysis. The stereochemistry of α-hydroxy glycine, which is unstable under basic and acidic conditions, was determined by conformational analysis, employing a combination of data from NOESY-NMR spectroscopy, simulated annealing and free MD simulations. The simulation procedures were applied for both R- and S-configured α-hydroxy glycine of the skyllamycin structure and compared to the NOESY data. Both methods, simulated annealing and free MD simulations independently support S-configured α-hydroxy glycine thus enabling the assignment of all stereocenters in the structure of skyllamycin and devising the role of two-component flavin dependent monooxygenase (Sky39) as S-selective.

A Glutathione-dependent Formaldehyde-activating Enzyme (Gfa) from Paracoccus denitrificans Detected and Purified via Two-dimensional Proton Exchange NMR Spectroscopy

MtdA catalyzes the dehydrogenation of N 5,N 10‐methylenetetrahydromethanopterin (methylene‐H4MPT) with NADP+ as electron acceptor. In the reaction two prochiral centers are involved, C14a of methylene‐H4MPT and C4 of NADP+, between which a hydride is transferred. The two diastereotopic protons at C14a of methylene‐H4MPT and at C4 of NADPH can be seen separately in 1H‐NMR spectra. This fact was used to determine the stereospecificity of the enzyme. With (14aR)‐[14a‐2H1]‐[14a‐13C]methylene‐H4MPT as the substrate, it was found that the pro‐R hydrogen of methylene‐H4MPT is transferred by MtdA into the pro‐R position of NADPH.