Frankfurt am Main, Germany
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Kowalewski B.,Bielefeld University | Poppe J.,Max Planck Institute For Biophysik | Demmer U.,Max Planck Institute For Biophysik | Warkentin E.,Max Planck Institute For Biophysik | And 3 more authors.
Journal of the American Chemical Society | Year: 2012

Some N 2-fixing bacteria prolong the functionality of nitrogenase in molybdenum starvation by a special Mo storage protein (MoSto) that can store more than 100 Mo atoms. The presented 1.6 Å X-ray structure of MoSto from Azotobacter vinelandii reveals various discrete polyoxomolybdate clusters, three covalently and three noncovalently bound Mo 8, three Mo 5-7, and one Mo 3 clusters, and several low occupied, so far undefinable clusters, which are embedded in specific pockets inside a locked cage-shaped (αβ) 3 protein complex. The structurally identical Mo 8 clusters (three layers of two, four, and two MoO n octahedra) are distinguishable from the [Mo 8O 26] 4- cluster formed in acidic solutions by two displaced MoO n octahedra implicating three kinetically labile terminal ligands. Stabilization in the covalent Mo 8 cluster is achieved by Mo bonding to Hisα156-N ε2 and Gluα129-O ε1. The absence of covalent protein interactions in the noncovalent Mo 8 cluster is compensated by a more extended hydrogen-bond network involving three pronounced histidines. One displaced MoO n octahedron might serve as nucleation site for an inhomogeneous Mo 5-7 cluster largely surrounded by bulk solvent. In the Mo 3 cluster located on the 3-fold axis, the three accurately positioned His140-N ε2 atoms of the α subunits coordinate to the Mo atoms. The formed polyoxomolybdate clusters of MoSto, not detectable in bulk solvent, are the result of an interplay between self- and protein-driven assembly processes that unite inorganic supramolecular and protein chemistry in a host-guest system. Template, nucleation/protection, and catalyst functions of the polypeptide as well as perspectives for designing new clusters are discussed. © 2012 American Chemical Society.


Althoff T.,Max Planck Institute For Biophysik | Mills D.J.,Max Planck Institute For Biophysik | Popot J.-L.,French National Center for Scientific Research | Kuhlbrandt W.,Max Planck Institute For Biophysik
EMBO Journal | Year: 2011

The respiratory chain in the inner mitochondrial membrane contains three large multi-enzyme complexes that together establish the proton gradient for ATP synthesis, and assemble into a supercomplex. A 19-à.. 3D map of the 1.7-MDa amphipol-solubilized supercomplex I 1 III 2 IV 1 from bovine heart obtained by single-particle electron cryo-microscopy reveals an amphipol belt replacing the membrane lipid bilayer. A precise fit of the X-ray structures of complex I, the complex III dimer, and monomeric complex IV indicates distances of 13 nm between the ubiquinol-binding sites of complexes I and III, and of 10-11 nm between the cytochrome c binding sites of complexes III and IV. The arrangement of respiratory chain complexes suggests two possible pathways for efficient electron transfer through the supercomplex, of which the shorter branch through the complex III monomer proximal to complex I may be preferred. © 2011 European Molecular Biology Organization | All Rights Reserved.


Buschmann S.,Max Planck Institute For Biophysik | Warkentin E.,Max Planck Institute For Biophysik | Xie H.,Max Planck Institute For Biophysik | Langer J.D.,Max Planck Institute For Biophysik | And 2 more authors.
Science | Year: 2010

The heme-copper oxidases (HCOs) accomplish the key event of aerobic respiration; they couple O2 reduction and transmembrane proton pumping. To gain new insights into the still enigmatic process, we structurally characterized a C-family HCO-essential for the pathogenicity of many bacteria-that differs from the two other HCO families, A and B, that have been structurally analyzed. The x-ray structure of the C-family cbb3 oxidase from Pseudomonas stutzen at 3.2 angstrom resolution shows an electron supply system different from families A and B. Like family-B HCOs, C HCOs have only one pathway, which conducts protons via an alternative tyrosine-histidine cross-link. Structural differences around hemes b and b3 suggest a different redox-driven proton-pumping mechanism and provide clues to explain the higher activity of family-C HCOs at low oxygen concentrations.


Rekittke I.,Justus Liebig University | Jomaa H.,Justus Liebig University | Ermler U.,Max Planck Institute For Biophysik
FEBS Letters | Year: 2012

Isoprenoid precursor biosynthesis occurs through the mevalonate or the methylerythritol phosphate (MEP) pathway, used i.e.; by humans and by many human pathogens, respectively. In the MEP pathway, 2-C-methyl-d-erythritol-2,4-cyclo- diphosphate (MEcPP) is converted to (E)-1-hydroxy-2-methyl-but-2-enyl-4- diphosphate (HMBPP) by the iron-sulfur cluster enzyme HMBPP synthase (GcpE). The presented X-ray structure of the GcpE-MEcPP complex from Thermus thermophilus at 1.55 Å resolution provides valuable information about the catalytic mechanism and for rational inhibitor design. MEcPP binding inside the TIM-barrel funnel induces a 60° rotation of the [4Fe-4S] cluster containing domain onto the TIM-barrel entrance. The apical iron of the [4Fe-4S] cluster ligates with the C3 oxygen atom of MEcPP. © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.


Steinbach A.,University of Konstanz | Fraas S.,University of Konstanz | Harder J.,Max Planck Institute for Marine Microbiology | Warkentin E.,Max Planck Institute For Biophysik | And 2 more authors.
FEBS Journal | Year: 2012

The thiamine diphosphate (ThDP) dependent flavoenzyme cyclohexane-1,2-dione hydrolase (CDH) () catalyses a key step of a novel anaerobic degradation pathway for alicyclic alcohols by converting cyclohexane-1,2-dione (CDO) to 6-oxohexanoate and further to adipate using NAD + as electron acceptor. To gain insights into the molecular basis of these reactions CDH from denitrifying anaerobe Azoarcus sp. strain 22Lin was structurally characterized at 1.26 Å resolution. Notably, the active site funnel is rearranged in an unprecedented manner providing the structural basis for the specific binding and cleavage of an alicyclic compound. Crucial features include a decreased and displaced funnel entrance, a semi-circularly shaped loop segment preceding the C-terminal arm and the attachment of the C-terminal arm to other subunits of the CDH tetramer. Its structural scaffold and the ThDP activation is related to that observed for other members of the ThDP enzyme family. The selective binding of the competitive inhibitor 2-methyl-2,4-pentane-diol (MPD) to the open funnel of CDH reveals an asymmetry of the two active sites found also in the dimer of several other ThDP dependent enzymes. The substrate binding site is characterized by polar and non-polar moieties reflected in the structures of MPD and CDO and by three prominent histidine residues (His28, His31 and His76) that most probably play a crucial role in substrate activation. The NAD + dependent oxidation of 6-oxohexanoate remains enigmatic as the redox-active cofactor FAD seems not to participate in catalysis, and no obvious NAD + binding site is found. Based on the structural data both reactions are discussed. © 2012 FEBS.


Ardevol A.,University of Barcelona | Ardevol A.,Max Planck Institute For Biophysik | Rovira C.,University of Barcelona | Rovira C.,Catalan Institution for Research and Advanced Studies
Journal of the American Chemical Society | Year: 2015

Carbohydrate-active enzymes such as glycoside hydrolases (GHs) and glycosyltransferases (GTs) are of growing importance as drug targets. The development of efficient competitive inhibitors and chaperones to treat diseases related to these enzymes requires a detailed knowledge of their mechanisms of action. In recent years, sophisticated first-principles modeling approaches have significantly advanced in our understanding of the catalytic mechanisms of GHs and GTs, not only the molecular details of chemical reactions but also the significant implications that just the conformational dynamics of a sugar ring can have on these mechanisms. Here we provide an overview of the progress that has been made in the past decade, combining molecular dynamics simulations with density functional theory to solve these sweet mysteries of nature. © 2015 American Chemical Society.


Tamura H.,Max Planck Institute For Terrestrische Mikrobiologie | Tamura H.,Research Institute for Biological science RIBS Okayama | Salomone-Stagni M.,EMBL Hamburg | Fujishiro T.,Max Planck Institute For Terrestrische Mikrobiologie | And 6 more authors.
Angewandte Chemie - International Edition | Year: 2013

Inhibition mechanism: Isocyanides strongly inhibit [Fe]-hydrogenase. X-ray crystallography and X-ray absorption spectroscopy revealed that the isocyanide binds to the trans position, versus the acyl carbon of the Fe center, and is covalently bound to the pyridinol hydroxy oxygen. These results also indicated that the hydroxy group is essential for H2 activation. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.


Parey K.,Max Planck Institute For Biophysik | Parey K.,Max Planck Institute For Terrestrische Mikrobiologie | Warkentin E.,Max Planck Institute For Biophysik | Kroneck P.M.H.,University of Konstanz | Ermler U.,Max Planck Institute For Biophysik
Biochemistry | Year: 2010

A vital process in the biogeochemical sulfur cycle is the dissimilatory sulfate reduction pathway in which sulfate (SO42-) is converted to hydrogen sulfide (H2S). Dissimilatory sulfite reductase (dSir), its key enzyme, hosts a unique siroheme-[4Fe-4S] cofactor and catalyzes the six-electron reduction of sulfite (SO32-) to H 2S. To explore this reaction, we determined the X-ray structures of dSir from the archaeon Archaeoglobus fulgidus in complex with sulfite, sulfide (S2-), carbon monoxide (CO), cyanide (CN-), nitrite (NO2-), nitrate (NO3-), and phosphate (PO43-). Activity measurements indicated that dSir of A. fulgidus reduces, besides sulfite and nitrite, thiosulfate (S 2O32-) and trithionate (S3O 62-) and produces the latter two compounds besides sulfide. On this basis, a three-step mechanism was proposed, each step consisting of a two-electron transfer, a two-proton uptake, and a dehydration event. In comparison, the related active site structures of the assimilatory sulfite reductase (aSir)- and dSir-SO32- complexes reveal different conformations of Argα170 and Lysα211 both interacting with the sulfite oxygens (its sulfur atom coordinates the siroheme iron), a sulfite rotation of ∼60° relative to each other, and different access of solvent molecules to the sulfite oxygens from the active site cleft. Therefore, solely in dSir a further sulfite molecule can be placed in van der Waals contact with the siroheme-ligated sulfite or sulfur-oxygen intermediates necessary for forming thiosulfate and trithionate. Although reported for dSir from several sulfate-reducing bacteria, the in vivo relevance of their formation is questionable. © 2010 American Chemical Society.


Celsi F.,International School for Advanced Studies | D'errico A.,International School for Advanced Studies | D'errico A.,Max Planck Institute For Biophysik | Menini A.,International School for Advanced Studies
Chemical Senses | Year: 2012

The rodent vomeronasal organ plays an important role in many social behaviors. Using the calcium imaging technique with the dye fluo-4 we measured intracellular calcium concentration changes induced by the application of sulfated steroids to neurons isolated from the vomeronasal organ of female mice. We found that a mix of 10 sulfated steroids from the androgen, estrogen, pregnanolone, and glucocorticoid families induced a calcium response in 71% of neurons. Moreover, 31% of the neurons responded to a mix composed of 3 glucocorticoid-derived compounds, and 28% responded to a mix composed of 3 pregnanolone-derived compounds. Immunohistochemistry showed that neurons responding to sulfated steroids expressed phosphodiesterase 4A, a marker specific for apical neurons expressing V1R receptors. None of the neuron that responded to 1 mix responded also to the other, indicating a specificity of the responses. Some neurons responded to more than 1 individual component of the glucocorticoid-derived mix tested at high concentration, suggesting that these neurons are broadly tuned, although they still displayed strong specificity, remaining unresponsive to high concentrations of the ineffective compounds. © The Author 2012. Published by Oxford University Press. All rights reserved.


Mager T.,Max Planck Institute For Biophysik | Rimon A.,Hebrew University of Jerusalem | Padan E.,Hebrew University of Jerusalem | Fendler K.,Max Planck Institute For Biophysik
Journal of Biological Chemistry | Year: 2011

Using an electrophysiological assay the activity of NhaA was tested in a wide pH range from pH 5.0 to 9.5. Forward and reverse transport directions were investigated at zero membrane potential using preparations with inside-out and right side-out-oriented transporters with Na+ or H+ gradients as the driving force. Under symmetrical pH conditions with a Na + gradient for activation, both the wt and the pH-shifted G338S variant exhibit highly symmetrical transport activity with bell-shaped pH dependences, but the optimal pH was shifted 1.8 pH units to the acidic range in the variant. In both strains the pH dependence was associated with a systematic increase of the Km for Na+ at acidic pH. Under symmetrical Na+ concentration with a pH gradient for NhaA activation, an unexpected novel characteristic of the antiporter was revealed; rather than being down-regulated, it remained active even at pH as low as 5. These data allowed a transport mechanism to advance based on competing Na+ and H+ binding to a common transport site and a kinetic model to develop quantitatively explaining the experimental results. In support of these results, both alkaline pH and Na+ induced the conformational change of NhaA associated with NhaA cation translocation as demonstrated here by trypsin digestion. Furthermore, Na+ translocation was found to be associated with the displacement of a negative charge. In conclusion, the electrophysiological assay allows the revelation of the mechanism of NhaA antiport and sheds new light on the concept of NhaA pH regulation. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

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