Methanosarcina barkeri has recently been shown to produce a multisubunit membrane-bound [NiFe] hydrogenase designated Ech (Escherichia coli hydrogenase 3) hydrogenase. In the present study Ech hydrogenase was purified to apparent homogeneity in a high yield. The enzyme preparation obtained only contained the six polypeptides which had previously been shown to be encoded by the ech operon. The purified enzyme was found to contain 0.9 mol of Ni, 11.3 mol of nonheme-iron and 10.8 mol of acid-labile sulfur per mol of enzyme. Using the purified enzyme the kinetic parameters were determined. The enzyme catalyzed the H 2 dependent reduction of a M. barkeri 2[4Fe-4S] ferredoxin with a specific activity of 50 U´mg protein 21 at pH 7.0 and exhibited an apparent K m for the ferredoxin of 1 mm. The enzyme also catalyzed hydrogen formation with the reduced ferredoxin as electron donor at a rate of 90 U´mg protein 21 at pH 7.0. The apparent K m for the reduced ferredoxin was 7.5 mm. Reduction or oxidation of the ferredoxin proceeded at similar rates as the reduction or oxidation of oxidized or reduced methylviologen, respectively. The apparent K m for H 2 was 5 mm. The kinetic data strongly indicate that the ferredoxin is the physiological electron donor or acceptor of Ech hydrogenase. Ech hydrogenase amounts to about 3% of the total cell protein in acetate-grown, methanol-grown or H 2 /CO 2 -grown cells of M. barkeri, as calculated from quantitative Western blot experiments. The function of Ech hydrogenase is ascribed to ferredoxin-linked H 2 production coupled to the oxidation of the carbonyl-group of acetyl-CoA to CO 2 during growth on acetate, and to ferredoxin-linked H 2 uptake coupled to the reduction of CO 2 to the redox state of CO during growth on H 2 /CO 2 or methanol.Keywords: Methanosarcina barkeri; hydrogenase; complex I; energy conservation; iron±sulfur protein; ferredoxin.Methanosarcina barkeri is a methanogenic archaeon that can utilize H 2 /CO 2 , methanol or methylamines as energy substrates (reviewed in [1±3]). Three different [NiFe] hydrogenases have been characterized from Methanosarcina species: F 420 -reducing hydrogenase, F 420 -nonreducing hydrogenase and Ech hydrogenase.F 420 -reducing hydrogenase has been purified and characterized from M. barkeri [4,5]. The enzyme catalyzes the reduction of coenzyme F 420 which plays an important role as redox carrier in methanogenic archaea. The genome of M. barkeri contains two gene clusters ( frh and fre) encoding two related F 420 -reducing hydrogenases indicating the presence of two isoenzymes. Both operons were transcribed during growth on H 2 /CO 2 , methanol or trimethylamine [6]. In acetategrown cells no transcripts of these operons were detectable although these cells contain small amounts of F 420 -reducing hydrogenase activity (about 5% of the activity detectable in methanol-grown cells; Meuer and Hedderich, unpublished results).F 420 -nonreducing hydrogenase has been purified from M. barkeri and from Methanosarcina mazei. The purified enzyme was fo...

Lantibiotics are peptides, produced by bacteria, that contain the noncanonical amino acid lanthionine and many of them exhibit antibacterial activities. The labyrinthopeptin A1 (LabyA1) is a prototype peptide of a novel class of carbacyclic lantibiotics. Here, we extensively evaluated its broad-spectrum activity against HIV and HSV in vitro, studied its mechanism of action and evaluated potential microbicidal applications. LabyA1 exhibited a consistent and broad anti-HIV activity (EC50s: 0.70–3.3 µM) and anti-HSV activity (EC50s: 0.29–2.8 µM) in cell cultures. LabyA1 also inhibited viral cell-cell transmission between persistently HIV-infected T cells and uninfected CD4+ T cells (EC50∶2.5 µM) and inhibited the transmission of HIV captured by DC-SIGN+-cells to uninfected CD4+ T cells (EC50∶4.1 µM). Time-of-drug addition studies revealed that LabyA1 acts as an entry inhibitor against HIV and HSV. Cellular and virus binding studies combined with SPR/FLIPR technology showed that LabyA1 interacted with the HIV envelope protein gp120, but not with the HIV cellular receptors. LabyA1 also demonstrated additive to synergistic effects in its anti-HIV-1 and anti-HSV-2 activity with anti(retro)viral drugs in dual combinations such as tenofovir, acyclovir, saquinavir, raltegravir and enfuvirtide. LabyA1 can be considered as a novel lead peptide as it had profound antiviral activity against HIV and HSV. Pre-treatment of PBMCs with LabyA1 neither increased the expression of the activation markers CD69 and CD25, nor enhanced HIV replication, nor significantly induced various inflammatory cytokines/chemokines. LabyA1 also did not affect the growth of vaginal Lactobacilli populations. Based on the lack of toxicity on the vaginal Lactobacillus strains and its synergistic/additive profile in combination with clinically approved anti(retro)virals, it deserves further attention as a potential microbicide candidate in the prevention of sexual transmitted diseases.

The formation of S-hydroxymethylglutathione from formaldehyde and glutathione is a central reaction in the consumption of the cytotoxin formaldehyde in some methylotrophic bacteria as well as in many other organisms. We describe here the discovery of an enzyme from Paracoccus denitrificans that accelerates this spontaneous condensation reaction. The rates of S-hydroxymethylglutathione formation and cleavage were determined under equilibrium conditions via two-dimensional proton exchange NMR spectroscopy. The pseudo first order rate constants k 1 * were estimated from the temperature dependence of the reaction and the signal to noise ratio of the uncatalyzed reaction. At 303 K and pH 6.0 k 1 * was found to be 0.02 s ؊1 for the spontaneous reaction. A 10-fold increase of the rate constant was observed upon addition of cell extract from P. denitrificans grown in the presence of methanol corresponding to a specific activity of 35 units mg ؊1 . Extracts of cells grown in the presence of succinate revealed a lower specific activity of 11 units mg ؊1 . The enzyme catalyzing the conversion of formaldehyde and glutathione was purified and named glutathione-dependent formaldehyde-activating enzyme (Gfa). The gene gfa is located directly upstream of the gene for glutathione-dependent formaldehyde dehydrogenase, which catalyzes the subsequent oxidation of S-hydroxymethylglutathione. Putative proteins with sequence identity to Gfa from P. denitrificans are present also in Rhodobacter sphaeroides, Sinorhizobium meliloti, and Mesorhizobium loti.

Bifurcated electron flow to high potential "Rieske" iron-sulfur cluster and low potential heme b L is crucial for respiratory energy conservation by the cytochrome bc 1 complex. The chemistry of ubiquinol oxidation has to ensure the thermodynamically unfavorable electron transfer to heme b L . 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 three n-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.

Within the last few decades, structure-based drug design (SBDD) has evolved into a powerful tool for the optimization of many low-molecular-weight lead compounds into highly potent drugs.[1] The principle of SBDD lies in the combination of different chemical moieties with the aim of obtaining a molecule that, while possessing the pharmacological properties necessary for a drug, is complementary in shape to the receptor binding pocket. This process requires knowledge of the exact structure of the protein/ligand complex. At present, structural genomics initiatives provide protein structures of biomedically relevant targets at an increasing rate [2] and recent structures of ion channels [3] and G-protein-coupled receptors (GPCRs) [4][5][6] bring these protein classes within reach for SBDD. Despite these successes, the daily work of pharmaceutical discovery is often limited by the ability to obtain high-resolution crystal structures of the target proteins in complex with the lower affinity ligands (lead structures) that are commonly identified by high-throughput screening or by fragment-based lead discovery.[1] In view of this limitation, SBDD would benefit from methods providing the relative orientations of different chemical fragments binding competitively to a receptor site. Such an approach would provide protein/ligand structures of novel ligands or fragments in relation to the known cocrystal structure of a reference ligand.Recently, we reported the observation by NMR spectroscopy of interligand NOE peaks occurring between two small ligands binding weakly and competitively to the same binding pocket of a common macromolecular receptor (Figure 1). [7,8] The measured mixture in solution contained two ligands (L A and L B ) in a 10-to 50-fold excess relative to the target receptor (T). As the ligands were competitive binders, these NOEs did not originate from a direct transfer of magnetization between the two ligands, [9] but rather from a spindiffusion process mediated by the protons of the receptor binding pocket. We proposed that such interligand NOEs can be used to define the relative orientation of the two ligands in the receptor binding pocket (the relative binding mode) and we termed the novel effect INPHARMA (internuclear NOEs for pharmacophore mapping; [8] Figure 1). INPHARMA can be observed for complexes with a dissociation constant (K d ) in the low micromolar to millimolar range.The scope of this work is to demonstrate for the first time that the INPHARMA method allows the determination of the relative, and in favorable cases even the absolute, binding mode of two low-affinity ligands binding competitively to a common receptor site and that it can thus be applied in the context of SBDD. In accordance with existing SBDD workflows, the experimental information derived from INPHARMA was used to select the correct binding mode