VAAM-Jahrestagung 2012 18.–21. März in Tübingen

175PSV013Localization and regulation of PHB granules in Synechocystissp. PCC 6803M. Schlebusch*, W. Hauf, K. ForchhammerUniversity of Tübingen, Institute of Microbiology and Infection Medicine ,Tübingen, GermanyPolyhydroxyalkanoates (PHA) are organic polyesters composed of (R)-3-hydroxy fatty acids which are synthesized by many bacteria as a carbonand energy storage material under unbalanced nutrient and energyavailability. PHAs are deposited intracellularly as insoluble sphericalinclusion called PHA granules, which consist of a polyester coresurrounded by a phospholipid layer with attached proteins. One of theseproteins is the PHA synthase, the key enzyme of PHA biosynthesis, whichcatalyses the formation from (R)-3-hydroxyacyl-CoA precursors. Onlylittle is known about the regulation and biogenesis of PHA accumulation incyanobacteria. We investigate the granule self-assembly process, thefunction of granule-associated proteins and the regulation of the PHBaccumulation in Synechocytis PCC 6803. We applied differentfluorescence dyes, as well as GFP-PHA synthase fusion proteins to studythe early PHA granule formation. With these tools we investigate, whetherthis process is located at the cytoplasmatic membrane. We analyzed theproteome of purified PHA granules and identified new putative granuleassociatedproteins. To gain insight into the regulation of PHA synthesis,we investigate mutants, which are impaired in PHA accumulation. Herewe show that the NADPH pool is crucial for the PHA accumulation.PSV014Biosynthesis of (Bacterio)chlorophylls: ATP-DependentTransient Subunit Interaction and Electron Transfer of DarkoperativeProtochlorophyllide OxidoreductaseJ. Moser* 1 , C. Lange 1 , M. Bröcker 1 , M. Saggu 2 , F. Lendzian 2 , H. Scheer 3 ,D. Jahn 11 Technische Universität Braunschweig, Inst. f. Mikrobiologie, Braunschweig,Germany2 Technische Universität Berlin, Inst. f. Chemie, Berlin, Germany3 Ludwig-Maximilians-Universität München, Biologie 1, München, GermanyThe biosynthesis of (bacterio)chlorophylls is fundamental for the primaryproduction on earth. Reduction of the fully conjugated ring system ofprotochlorophyllide results in the common core ring architecture which ischaracteristic for all (bacterio)chlorophylls. Dark-operativeprotochlorophyllide oxidoreductase (DPOR) is a multi-subunit enzymeemploying nitrogenase-like catalysis for the chemically difficult twoelectron reduction of ring D.During ATP-dependent DPOR catalysis the homodimeric ChlL 2 subunitcarrying a [4Fe-4S] cluster, transfers electrons to the correspondingheterotetrameric catalytic subunit (ChlN/ChlB) 2 which also possesses aredox active [4Fe-4S] cluster. To investigate the transient interaction ofboth subcomplexes and the resulting electron transfer reactions, the ternaryDPOR enzyme holocomplex comprising subunits ChlN, ChlB and ChlLwas trapped as an octameric (ChlN/ChlB) 2(ChlL 2) 2 complex afterincubation with non hydrolyzable ATP analogs. A nucleotide-dependentswitch mechanism triggering ternary complex formation and electrontransfer was concluded.The crystal structure of the catalytic (ChlN/ChlB) 2 complex of DPOR fromthe cyanobacterium Thermosynechococcus elongatus was solved at aresolution of 2.4 Å. Subunits ChlN and ChlB exhibit a related architectureof three subdomains built around a central, parallel ß-sheet surrounded by-helices. The (ChlN/ChlB) 2 protein revealed a [4Fe-4S] clustercoordinated by an oxygen atom of an aspartate residue alongside threecommon cysteine ligands. Two substrate binding sites enriched witharomatic residues for coordination of the protochlorophyllide substratemolecules are located at the interface of each ChlN/ChlB half-tetramer.The complete octameric (ChlN/ChlB) 2(ChlL 2) 2 complex of DPOR wasmodeled based on the obtained structure and earlier functional studies. Theelectron transfer pathway via the various redox centers of DPOR to thesubstrate was reconstructed.Bröcker, M. J., Schomburg, S., Heinz, D. W., Jahn, D., Schubert, W. D., and Moser, J. (2010).Crystal structure of the nitrogenase-like dark operative protochlorophyllide oxidoreductase catalyticcomplex (ChlN/ChlB)2. J. Biol. Chem.285, 27336-27345.Wätzlich, D., Bröcker, M., Uliczka, F., Ribbe, M., Virus, S.; Jahn, D. & Moser, J. (2009). ChimericNitrogenase-like Enzymes of (Bacterio)Chlorophyll Biosynthesis.J. Biol. Chem.284:15530-40.Bröcker, M.J.; Virus, S.; Ganskow, S.; Heathcote, P.; Heinz, D.W.; Schubert, W-D.; Jahn, D. &Moser, J. (2008). ATP-Driven Reduction by Dark-Operative Protochlorophyllide OxidoreductasefromChlorobium tepidumMechanistically Resembles Nitrogenase Catalysis.J. Biol.Chem.283:10559-67.PSV015Carbon disulfide hydrolase: a new enzyme for CS 2 conversionin acidothermophilic microorganismsM. Jetten* 1 , M. Smeulders 1 , H. Op den Camp 1 , T. Barends 2 , I. Schlichting 21 Radboud University Nijmegen, Microbiology, Nijmegen, Netherlands2 MPI Heidelberg, Heidelberg, GermanyAcidophilic, thermophilic Archaea that live in mudpots of volcaniceocsystems obtain their energy from the oxidation of sulfur compoundssuch as carbon disulfide and hydrogen sulfide, thereby creating anextremely acidic environment with pH values as low as 1. Ahyperthermophilic Acidianus strain A1-3 was isolated from the fumarolic,ancient sauna building at the Solfatara volcano (Naples, Italy). It wasshown to rapidly convert CS 2 into H 2S and carbon dioxide (CO 2), but sofar little was known about the modes of action and the evolution of theenzyme(s) involved. In this study we elucidated the structure, the proposedmechanism and evolution of the isolated CS 2 hydrolase from AcidianusA1-3. The enzyme monomer displayed a typical b-carbonic anhydrase foldand active site, yet CO 2 was not one of the typical substrates. Largecarboxy- and amino-terminal arm extensions, and an unusualhexadecameric catenane oligomer were apparent in the enzyme. Thesestructure features resulted in the blocking of the usual entrance to carbonicanhydrase active sites, and the formation of a single 1,5 nm long, highlyhydrophobic tunnel that functions as a specificity filter. The tunneldetermines the enzyme's substrate specificity for CS 2. The transposonsequences that surround the gene encoding this CS 2 hydrolase point tohorizontal gene transfer as a mechanism for its acquisition duringevolution. Our results show how the ancient b-carbonic anhydrase, whichis central to global carbon metabolism, was transformed by divergentevolution into a crucial enzyme in CS 2 metabolism.Smeulders MJ, Barends T, et al (2011) Evolution of a new enzyme for carbon disulphideconversion by an acidothermophilic archaeon. Nature 478(7369):412-416 doi:10.1038/nature10464PSV016A promiscuous archaeal ATP synthase concurrently coupledto Na + and H + translocationK. Schlegel* 1 , V. Leone 2 , J. Faraldo-Gómez 2 , V. Müller 11 Molecular Microbiology & Bioenergetics, Institute for MolecularBiosciences, Goethe University, Frankfurt/Main, Germany, Germany2 Theoretical Molecular Biophysics Group, Max Planck Institute ofBiophysics, Frankfurt/Main, Germany, GermanyCytochrome-containing methanogenic archaea are one of the very feworganisms that generate a primary proton- as well as a primary sodium iongradient during their metabolism (1). Thus, the critical question is howboth ion gradients are used for the synthesis of ATP. Since only one ATPsynthase (A 1A O type) is expressed, the enzyme may use both ions ascoupling ion, or the sodium ion gradient is converted to a secondary protongradient via Na + /H + antiporter or vice versa (2). We have addressed thislong standing question using energetically intact inside out membranevesicles of Methanosarcina acetivorans. Our results show that the A 1A OATP synthase translocates both ions, H + and Na + , simultaneously underphysiological conditions. To further elucidate this phenomenon, we usedfree-energy molecular simulations to analyse the ion-selectivity of the ionbindingsite of the subunit c. This appears to have been tuned via aminoacidsubstitutions allowing the usage of H + and Na + under physiologicalconditions. The adaptation of the binding site could be an adaptation to usethe heterogeneous ion gradient established during methanogensis.1. Deppenmeier U, & Müller V (2008) Life close to the thermodynamic limit: how methanogenicarchaea conserve energy. Results Probl. Cell. Differ.45: 123-152.2. Pisa KY, Weidner C, Maischak H, Kavermann H, & Müller V (2007) The coupling ion inmethanoarchaeal ATP synthases: H + versus Na + in the A1AO ATP synthase from the archaeonMethanosarcina mazei Gö1. FEMS Microbiol. Lett.277:56-63.PSP001A tool for online measurement of the intracellular pH inCorynebacterium glutamicumK.M. Kirsch*, S. Mayr, R. Krämer, K. MarinUniversität Köln, Institut für Biochemie, Köln, GermanyThe soil bacterium C. glutamicum is a well established organism forindustrial amino acid production. In its natural habitat as well as duringlarge scale fermentation, C. glutamicum is exposed to significant changesof the external pH. The limited mixing capacity, pH regulation by additionof acids or NH 3 as well as elevated CO 2 concentrations at high celldensities contribute to transient fluctuations of the external pH. It isconsidered that most bacteria perform efficient pH homeostasis in order tomaintain the structural and functional integrity of cellular macromoleculesbut, it is unknown for almost all bacteria to what extent the internal pH canvary and how fast the adjustment of the internal pH is achieved uponexternal shifts. Additionally, the major players of pH regulation and theenergetic costs of pH homeostasis are unknown, although their impact onthe efficiency of production processes seems to be obvious. In order toaddress these questions we established a method for online measurementBIOspektrum | Tagungsband 2012